National Industrial Chemicals Notification and
Assessment Scheme
Benzene
________________________________________
Priority Existing Chemical
Assessment Report No. 21
September 2001
Commonwealth of Australia 2001
ISBN 0 642-51896-3
This work is copyright. Apart from any use as permitted under the Copyright Act 1968
(Cwlth), no part may be reproduced by any process without written permission from
AusInfo. Requests and inquiries concerning reproduction and rights should be directed to
the Manager, Legislative Services, AusInfo, GPO Box 84, Canberra ACT 2601.
Priority Existing Chemical Number 21
ii
Preface
This assessment was carried out under the National Industrial Chemicals Notification and
Assessment Scheme (NICNAS). This Scheme was established by the Industrial Chemicals
(Notification and Assessment) Act 1989 (the Act), which came into operation on 17 July
1990.
The principal aim of NICNAS is to aid in the protection of people at work, the public and
the environment from the harmful effects of industrial chemicals.
NICNAS assessments are carried out in conjunction with Environment Australia (EA) and
the Therapeutic Goods Administration (TGA), which carry out the environmental and
public health assessments, respectively.
NICNAS has two major programs: the assessment of the health and environmental effects
of new industrial chemicals prior to importation or manufacture; and the other focussing on
the assessment of chemicals already in use in Australia in response to specific concerns
about their health/or environmental effects.
There is an established mechanism within NICNAS for prioritising and assessing the many
thousands of existing chemicals in use in Australia. Chemicals selected for assessment are
referred to as Priority Existing Chemicals.
This Priority Existing Chemical report has been prepared by the Director (Chemicals
Notification and Assessment) in accordance with the Act. Under the Act manufacturers and
importers of Priority Existing Chemicals are required to apply for assessment. Applicants
for assessment are given a draft copy of the report and 28 days to advise the Director of any
errors. Following the correction of any errors, the Director provides applicants and other
interested parties with a copy of the draft assessment report for consideration. This is a
period of public comment lasting for 28 days during which requests for variation of the
report may be made. Where variations are requested the Director's decision concerning
each request is made available to each respondent and to other interested parties (for a
further period of 28 days). Notices in relation to public comment and decisions made
appear in the Commonwealth Chemical Gazette.
In accordance with the Act, publication of this report revokes the declaration of this
chemical as a Priority Existing Chemical, therefore manufacturers and importers wishing to
introduce this chemical in the future need not apply for assessment. However,
manufacturers and importers need to be aware of their duty to provide any new information
to NICNAS, as required under section 64 of the Act.
Copies of this and other Priority Existing Chemical reports are available from NICNAS
either by using the prescribed application form at the back of this report, the website
www.nicnas.gov.au or ordering directly from the following address:
GPO Box 58
Sydney
NSW 2001
AUSTRALIA
Benzene iii
Priority Existing Chemical Number 21
iv
Overview
Benzene (CAS No. 71-43-2) was declared a Priority Existing Chemical on 7 April 1998 in
response to occupational and public health concerns.
Benzene occurs naturally in fossil fuels and is produced incidentally in the course of natural
processes and human activities that involve the combustion of organic matter such as wood,
coal and petroleum products. The main industrial use of benzene is as a starting material for
the synthesis of other chemicals. Most benzene feedstock is imported, but some is
manufactured at an Australian steelworks as a by-product of coal coking. Large quantities
of benzene are produced during the refining of petroleum and retained as a component of
petrol. Petrol vehicle emissions are the predominant source of benzene in the environment.
Benzene is volatile and water-soluble and is considered biodegradable. Its major release is
to the atmosphere, where it will break down in a matter of weeks. Direct release to the
aquatic compartment is expected to be minor and significant removal will occur from
volatilisation. Benzene release to soil is likely to be marginal. Concentrations in aquatic
systems are expected to be far lower than of concern and a low aquatic risk is predicted.
Due to the low expected exposure, a low environmental risk to terrestrial organisms is
predicted. The short atmospheric lifetime of benzene indicates concentrations will not occur
at levels harmful to the atmosphere. While widespread transport within the troposphere is
possible, the chemical is not expected to reach the stratosphere and therefore would not
have an influence on global warming or ozone depletion.
In animals and humans, benzene is absorbed by all routes of exposure, although dermal
absorption is limited by its rapid evaporation from the skin. It is metabolised in the liver
and several other organs, including the bone marrow. The parent molecule is eliminated
with exhaled air. The metabolites are excreted in the urine.
In animals, benzene is not highly acutely toxic. Chronic exposure can result in central
nervous system depression, immunosuppression, bone marrow depression, degenerative
lesions of the gonads, foetal growth retardation, damage to genetic material and solid
tumours in several organs.
In humans, acute exposure to high concentrations of benzene vapours can result in irritation
of the skin, eyes and respiratory system and in central nervous system depression. Chronic
exposure can result in bone marrow depression and leukaemia, particularly acute myeloid
leukaemia, and possibly an increased risk of non-Hodgkin's lymphoma and multiple
myeloma. Structural and numerical chromosome aberrations have been detected in
peripheral blood cells of workers exposed to high levels of benzene. For bone marrow
depression, the lowest observed adverse effect level in humans is 7.6 parts per million
(ppm), based on minimal blood count changes in otherwise healthy workers. No threshold
has been established for the genotoxic and carcinogenic effects of benzene.
Epidemiological evidence indicates that the risk of leukaemia increases with exposure and
is significantly elevated at cumulative exposures above 50 ppm-years, corresponding to an
8-hour time-weighted average exposure above 1.25 ppm over a working life of 40 years.
Chronic benzene toxicity has been attributed to the formation of reactive metabolites that
appear to exert their toxic effect in combination, with no one metabolite accounting for all
of the observed effects.
Benzene v
Benzene is currently listed in the NOHSC List of Designated Hazardous Substances with
the following classification: `Flammable', `Carcinogen, Category 1' and `Toxic: Danger of
serious damage to health by prolonged exposure through inhalation, in contact with skin
and if swallowed'. Category 1 carcinogens are those substances known to be carcinogenic
to humans. Based on the assessment of health effects, this report has concluded that
benzene also meets the NOHSC Approved Criteria for Classifying Hazardous Substances
for classification as a skin, eye and respiratory system irritant and as a mutagenic substance
in Category 3.
The public is exposed to benzene through the inhalation of indoor, in-vehicle and outdoor
air contaminated with the chemical through releases that predominantly derive from vehicle
exhaust, petrol evaporation and tobacco smoke. The 24-hour average lifetime exposure in
the Australian urban population is estimated at 5.2 parts per billion (ppb). It is one-fifth
higher in passive smokers exposed to tobacco smoke at home, at work and in their cars (6.1
ppb) and almost three times as high (15.2 ppb) in the average smoker.
Benzene-induced bone marrow depression is not expected to present a significant public
health risk. Based upon low-dose extrapolation of relevant quantitative risk estimates and
the above-mentioned exposure estimate, the excess lifetime risk of benzene-induced
leukaemia in the Australian urban population is estimated to be in the order of one case per
10,000 with increased risk in sensitive subpopulations or at higher exposure levels.
However, the estimated excess risk is based on substantial uncertainties in the exposure
assessment which should be validated through collection of monitoring data.
As benzene is an established human carcinogen for which no safe level of exposure has
been established, it is recommended that any increase in public exposure be avoided and
that measures be taken to reduce exposure where this is practicable. The establishment of a
national ambient air benzene level would facilitate these objectives.
Occupational exposure to benzene is predominantly by the inhalation route. It occurs
primarily in the petroleum, steel, chemical and associated industries and in laboratories
using the chemical for research or analysis. Occupational exposure to benzene can also
result from the contamination of workplace environments with petrol vapours, engine
exhaust or tobacco smoke, for example, in vehicle mechanics, professional drivers and
hospitality workers. It is estimated that current long-term occupational exposures to
benzene are less than or equal to 0.7 ppm in the steel and associated industries and during
maintenance of phenol plants; less than 0.1 ppm in the upstream petroleum industry (oil
and gas production); less than 0.5 ppm in the chemical industry and in laboratory workers;
less than 0.2 ppm in vehicle mechanics; less than 0.7 ppm in the downstream petroleum
industry (refining, distribution and marketing of petroleum products); and less than 0.05
ppm in people who work in roadside or in-vehicle environments contaminated with vehicle
exhaust or in indoor environments contaminated with tobacco smoke.
The occupational risk characterisation found no cause for concern about acute health effects
or bone marrow depression, given the control measures which are already in place in
Australian workplaces. However, there is cause for concern about the risk for leukaemia in
all workers with repeated occupational exposure to benzene. There is no known threshold
for the carcinogenic effects of benzene, but because the risk for leukaemia increases with
exposure, it can be reduced by controlling exposure to the highest practicable standard.
With regard to occupational health and safety, it is recommended that the national exposure
standard for benzene be revised. It is recommended that an eight-hour time-weighted
average of 0.5 ppm be adopted. It is further recommended that the current hazard
classification be amended to include classification as `Irritating to eyes, respiratory system
and skin' (risk phrase R36/37/38) and as a mutagenic substance in Category 3 (risk phrase
Priority Existing Chemical Number 21
vi
R40: `Possible risks of irreversible effects, Mutagen Category 3'). Occupational exposures
to benzene should be minimised by improving workplace control measures and by using
the best available technology.
This report has identified the need to reduce public exposure to air benzene levels as much
as practicable. Public health recommendations include measures to reduce indoor benzene
levels, such as proper sealing of attached garages and minimising environmental tobacco
smoke. In order to better characterise the risk to the public from benzene exposure, personal
and ambient air monitoring is recommended and a national ambient air standard should be
set.
Benzene vii
Priority Existing Chemical Number 21
viii
Contents
PREFACE iii
OVERVIEW iv
ABBREVIATIONS AND ACRONYMS xv
INTRODUCTION 1
1.
1.1 Declaration 1
1.2 Objectives 1
1.3 Sources of information 1
1.4 Peer review 2
2. BACKGROUND 3
2.1 Introduction 3
2.2 International perspective 3
2.3 Australian perspective 3
2.4 Assessments by other national or international bodies 5
3. APPLICANTS 6
4. CHEMICAL IDENTITY AND COMPOSITION 7
4.1 Chemical name (IUPAC) 7
4.2 Registry numbers 7
4.3 Other names 7
4.4 Molecular formula 7
4.5 Structural formula 7
4.6 Molecular weight 8
4.7 Composition of commercial grade product 8
5. PHYSICAL AND CHEMICAL PROPERTIES 9
5.1 Physical state 9
5.2 Physical properties 9
5.3 Chemical properties 9
6. METHODS OF DETECTION AND ANALYSIS 11
6.1 Characterisation 11
6.2 Detection and analysis 11
6.3 Atmospheric monitoring methods 11
Benzene ix
6.4 Biological monitoring methods 13
7. MANUFACTURE, IMPORTATION AND USE 14
7.1 Manufacture and importation 14
7.2 Manufacturing processes and end use 15
7.2.1 Petroleum industry 15
7.2.2 Steel and associated industries 19
7.2.3 Chemical industry 20
7.2.4 Laboratory uses 22
7.2.5 Coincidental production 23
7.3 Summary 23
8. ENVIRONMENTAL RELEASE, FATE AND EFFECTS 24
8.1 Environmental release 24
8.2 Environmental fate 24
8.2.1 Atmospheric fate 24
8.2.2 Aquatic fate 27
8.2.3 Terrestrial fate 28
8.2.4 Biodegradation 29
8.2.5 Bioaccumulation 30
8.3 Effects on organisms in the environment 31
8.3.1 Aquatic organisms 31
8.3.2 Terrestrial organisms 33
8.4 Summary 34
9. KINETICS AND METABOLISM 35
9.1 Absorption 35
9.1.1 Animal studies 35
9.1.2 Human studies 36
9.2 Distribution 38
9.2.1 Animal studies 38
9.2.2 Human studies 39
9.3 Metabolism 40
9.3.1 General metabolic pathways 40
9.3.2 Formation of phenolic metabolites 42
9.3.3 Formation of trans,trans-muconaldehyde 43
9.4 Elimination and excretion 44
9.4.1 Animal data 44
9.4.2 Human data 46
9.5 Comparative kinetics and metabolism 47
9.5.1 Oral studies 47
Priority Existing Chemical Number 21
x
9.5.2 Inhalation studies 48
9.5.3 Dermal studies 49
9.5.4 In vitro studies 49
9.6 Summary 50
10. EFFECTS ON LABORATORY MAMMALS AND OTHER TEST SYSTEMS 51
10.1 Acute toxicity 51
10.2 Irritation and corrosivity 52
10.3 Sensitisation 52
10.4 Repeated dose toxicity (other than carcinogenicity) 52
10.4.1 Short-term exposure 52
10.4.2 Long-term exposure 55
10.5 Reproductive toxicity 56
10.5.1 Effects on fertility and lactation 56
10.5.2 Developmental toxicity 58
10.6 Genotoxicity 63
10.7 Carcinogenicity 64
10.8 Summary and conclusions 69
11. HUMAN HEALTH EFFECTS 71
11.1 Acute toxicity 71
11.2 Irritation 72
11.3 Sensitisation 72
11.4 Repeated dose toxicity (other than carcinogenicity) 72
11.4.1 Neurological effects 72
11.4.2 Effects on the immune system 72
11.4.3 Cardiovascular effects 73
11.4.4 Haematological effects 73
11.4.5 Reproductive effects 78
11.4.6 Other health effects 81
11.5 Genotoxic effects 81
11.6 Carcinogenicity 83
11.6.1 Cohort studies 83
11.6.2 Case-control studies 96
11.6.3 Ecological studies 101
11.7 The Illawarra leukaemia cluster 102
11.8 Summary and conclusions 102
12. MODES OF ACTION 104
12.1 Activation of benzene metabolites 104
12.1.1 Activation of phenol 105
Benzene xi
12.1.2 Activation of hydroquinone and catechol 105
12.1.3 Role of cyclooxygenase 106
12.1.4 Formation of reactive oxygen species 106
12.2 Reactivity of benzene metabolites 108
12.2.1 Genotoxicity 108
12.2.2 Oxidative stress 110
12.2.3 Modulation of cellular function 111
12.3 Critical biological effects 112
12.3.1 Bone marrow toxicity 112
12.3.2 Leukaemia 112
12.3.3 Tumours in Zymbal, Harderian, lacrimal and mammary
glands 113
12.4 Interindividual variations in susceptibility 114
12.4.1 Gender effects 114
12.4.2 Genetic polymorphisms 115
12.4.3 Environmental influences 117
12.5 Summary 118
13. HEALTH HAZARD ASSESSMENT 120
13.1 Acute effects 120
13.1.1 CNS effects 120
13.1.2 Skin, eye and respiratory tract irritation 120
13.2 Repeated dose effects (other than carcinogenicity) 120
13.2.1 CNS effects 120
13.2.2 Immunosuppression 121
13.2.3 Bone marrow depression 121
13.2.4 Fertility effects 122
13.2.5 Developmental effects 123
13.2.6 Other non-neoplastic effects 123
13.3 Genotoxicity 123
13.4 Carcinogenicity 124
13.4.1 Leukaemia 124
13.4.2 Solid tumours 125
13.5 Summary and conclusions 126
14. CLASSIFICATION FOR OCCUPATIONAL HEALTH AND SAFETY 128
14.1 Physicochemical hazards 128
14.2 Health hazards 128
14.2.1 Acute toxicity 128
14.2.2 Irritant and corrosive effects 129
14.2.3 Sensitising effects 129
Priority Existing Chemical Number 21
xii
14.2.4 Effects from repeated or prolonged exposure 129
14.2.5 Reproductive effects 130
14.2.6 Mutagenic effects 131
14.2.7 Carcinogenicity 132
14.3 Summary 132
15. ENVIRONMENTAL EXPOSURE 133
15.1 Point source releases to air 133
15.1.1 Petroleum industry 133
15.1.2 Steel and associated industries 136
15.1.3 Aluminium industry 137
15.1.4 Chemical industry 139
15.1.5 Fossil fuel burning for power generation 141
15.1.6 Other point sources 142
15.1.7 Summary 143
15.2 Diffuse releases to urban air 144
15.2.1 Emissions estimation 144
15.2.2 Predicted environmental concentration in urban air 147
15.3 Indoor air concentrations 149
15.3.1 Homes 149
15.3.2 Non-residential buildings 152
15.3.3 Motor vehicles and other means of transportation 153
15.4 Concentrations in water and soil 154
15.4.1 Water 154
15.4.2 Soil 156
15.5 Summary 156
16. PUBLIC EXPOSURE 157
16.1 Direct exposure 157
16.2 Indirect exposure via the environment 157
16.3 Exposure assessment 159
16.4 Summary and conclusions 162
17. OCCUPATIONAL EXPOSURE 164
17.1 Petroleum industry 164
17.1.1 Petroleum production and refining 164
17.1.2 Petrol distribution and marketing 166
17.1.3 Petroleum and petrol cleaning operations 168
17.1.4 Conclusions 168
17.2 Steel and coal tar distillation industries 168
17.2.1 Coke ovens 168
Benzene xiii
17.2.2 Coal gas by-product plants 169
17.2.3 Coal tar distillation 169
17.2.4 Conclusions 170
17.3 Chemical industry 170
17.3.1 Ethane and naphtha (gas oil) cracking 170
17.3.2 Bulk distribution 171
17.3.3 Butadiene rubber manufacture 172
17.3.4 Styrene and phenol manufacture 172
17.3.5 Conclusions 173
17.4 Laboratory use for research or analysis 174
17.5 Contaminated workplace environments 174
17.5.1 Petrol vapours and vehicle exhaust 174
17.5.2 Environmental tobacco smoke 175
17.5.3 Conclusions 176
17.6 Aluminium industry 176
17.7 Summary 177
18. RISK CHARACTERISATION 178
18.1 Environmental risks 178
18.1.1 Atmospheric risk 178
18.1.2 Aquatic risk 179
18.1.3 Terrestrial risk 179
18.2 Occupational health risks 180
18.2.1 Acute effects 180
18.2.2 Effects from repeated exposure 180
18.2.3 Uncertainties involved 183
18.2.4 Areas of concern 184
18.3 Public health risks 184
18.3.1 Bone marrow depression 184
18.3.2 Leukaemia 185
18.3.3 Uncertainties involved 186
18.3.4 Conclusions 186
18.4 Risk assessments by other national or international bodies 187
19. RISK MANAGEMENT 190
19.1 Environmental and public health controls 190
19.2 Occupational health and safety controls 191
19.2.1 Regulatory controls 191
19.2.2 Current control measures 195
19.3 National transport regulation (ADG Code) 198
Priority Existing Chemical Number 21
xiv
20. DISCUSSION AND CONCLUSIONS 199
20.1 Environmental exposure and risks 199
20.2 Health effects 199
20.3 Public exposure and health risks 201
20.4 Occupational exposure and health risks 202
20.5 Data gaps 205
21. RECOMMENDATIONS 206
22. SECONDARY NOTIFICATION 210
APPENDIX 1 211
REFERENCES 218
Benzene xv
Abbreviations and Acronyms
American Conference of Governmental Industrial Hygienists
ACGIH
Australian Dangerous Goods
ADG
Australian Institute of Petroleum
AIP
absolute lymphocyte count
ALC
acute lymphatic leukaemia
ALL
acute myeloid leukaemia
AML
acute non-lymphocytic leukaemia
ANLL
atmosphere
atm
light aircraft gasoline
Avgas
bioconcentration factor
BCF
benzene poisoning
BP
benzene/toluene/xylenes
BTX
body weight
BW
centigrade
C
Chemical Abstracts Service
CAS
colony forming units of erythrocyte progenitor cells
CFU-E
colony forming units of granulocyte/macrophage progenitor cells
CFU-M
confidence interval
CI
chronic lymphatic leukaemia
CLL
centimetre
cm
cm2 square centimetre
cm3 cubic centimetre
Chemical Manufacturers Association
CMA
chronic myeloid leukaemia
CML
central nervous system
CNS
colony stimulating factor
CSF
cytochrome-P450
CYP
deoxyribonucleic acid
DNA
Environment Australia
EA
median effective concentration
EC50
European Inventory of Existing Chemical Substances
EINECS
Environment Protection Authority
EPA
environmental tobacco smoke
ETS
gram
g
gas chromatography
GC
gas chromatography-mass spectrometry
GC-MS
gestation day
GD
Good Laboratory Practices
GLP
granulocyte/macrophage colony stimulating factor
GM-CSF
glutathione
GSH
glutathione-S-transferase
GST
hour
h
hectare
ha
haemoglobin
Hb
haematocrit
Hct
Priority Existing Chemical Number 21
xvi
International Agency for Research on Cancer
IARC
interleukin-1
IL-1
International Programme on Chemical Safety
IPCS
International Uniform Chemical Information Database
IUCLID
International Union for Pure and Applied Chemistry
IUPAC
Kelvin
K
kilogram
kg
Michaelis-Menten constant
Km
kilometre
km
km2 square kilometre
sorption coefficient
Koc
kilopascal
kPa
kilotonne
kt
litre
L
lymphocyte
LC
median lethal concentration
LC50
median lethal dose
LD50
lowest observed adverse effect level
LOAEL
leaded petrol
LP
liquid pressurised gas
LPG
m3 cubic meter
mean corpuscular volume
MCV
myelodysplastic syndrome
MDS
milligram
mg
millilitre
mL
megalitre
ML
millimolar
mM
multiple myeloma
MM
micronucleus
MN
mole
mol
material safety data sheet
MSDS
nicotinamide adenine dinucleotide phosphate
NADPH
National Environment Protection Council
NEPC
National Environment Protection Measures
NEPM
nanogram
ng
National Health Interview Survey
NHIS
non-Hodgkin's lymphoma
NHL
National Industrial Chemicals Notification and Assessment Scheme
NICNAS
National Institute of Occupational Safety and Health
NIOSH
nanometre
nm
nanomole
nmol
nanomolar
nM
no observed adverse effect level
NOAEL
no observed effect concentration
NOEC
National Occupational Health and Safety Commission
NOHSC
National Pollutant Inventory
NPI
NAD(P)H:quinone oxidoreductase
NQO1
non-steroidal anti-inflammatory drug
NSAID
Organisation for Economic Co-Operation and Development
OECD
odds ratio
OR
Benzene xvii
Occupational Safety and Health Administration
OSHA
polycyclic aromatic hydrocarbon
PAH
prostaglandin E2
PGE2
protein kinase C
PKC
blood platelet
Plt
predicted no-effect concentration
PNEC
octanol/water partition coefficient
Po/w
persistent organic pollutant
POP
parts per billion
ppb
personal protective equipment
PPE
parts per million
ppm
premium unleaded petrol
PULP
red blood cell (erythrocyte)
RBC
ribonucleic acid
RNA
relative risk
RR
second
s
spontaneous abortion
SAb
secondary acute myeloid leukaemia
s-AML
subcutaneous
SC
sister chromatid exchange
SCE
small-for-gestational age
SGA
standardised incidence rate
SIR
State marketing area
SMA
standardised mortality rate
SMR
sewage treatment plant
STP
Standard for the Uniform Scheduling of Drugs and Poisons
SUSDP
tonne
t
Technical Guidance Document
TGD
time-weighted average
TWA
8-h time-weighted average
TWA8
unleaded petrol
ULP
United Nations
UN
United States Environmental Protection Agency
USEPA
upper tolerance limit of a distribution's 95th percentile
UTL95%,95%
volume/volume
v/v
volatile organic chemical
VOC
weight/weight
w/w
white blood cell (leukocyte)
WBC
World Health Organization
WHO
year
y
microgram
µg
microlitre
µL
micromolar
µM
micromole
µm o l
8-hydroxydeoxyguanosine
8-OHdG
Priority Existing Chemical Number 21
xviii
1. Introduction
1.1 Declaration
The chemical benzene (CAS No. 71-43-2) was declared a Priority Existing
Chemical for full assessment under the Industrial Chemicals (Notification and
Assessment) Act 1989 on 7 April 1998. It was nominated by the public because of
concerns about its human health effects and the adequacy of the current Australian
occupational exposure standard.
1.2 Objectives
The objectives of this assessment were to:
· characterise the properties of benzene;
· determine the uses of benzene in Australia;
· determine the extent of occupational, public and environmental exposure to
benzene;
· characterise the intrinsic capacity of benzene to cause adverse effects on
persons or the environment;
· characterise the risks to humans and the environment resulting from exposure
to benzene; and
· determine the extent to which any risk is capable of being reduced.
1.3 Sources of information
Consistent with the objectives, this report presents a summary and critical
evaluation of relevant information relating to the potential health and
environmental hazards from exposure to benzene. Relevant scientific data were
submitted by the applicants listed in Section 3, obtained from published papers
identified in a comprehensive literature search of several online databases up to
December 2000, or retrieved from other sources such as the reports and resource
documents prepared for the health surveillance program of the Australian Institute
of Petroleum (AIP) and the Illawarra leukaemia cluster investigation. Due to the
availability of several peer-reviewed overseas assessment reports, not all primary
sources of data were evaluated. However, relevant studies published since the cited
reviews were assessed on an individual basis.
The characterisation of health and environmental risks in Australia was based upon
information on use patterns, product specifications, occupational exposure and
emissions to the environment made available by applicants and relevant State
authorities. Information to assist in the assessment was also obtained through site
visits and telephone interviews. The site visits included two petroleum refineries,
two petrol terminals, a steelworks, a coal tar distillery, a bulk liquid storage facility
and three chemical plants.
Benzene 1
1.4 Peer review
During all stages of preparation, the report has been subject to internal peer review
by NICNAS, Environment Australia (EA) and the Therapeutic Goods
Administration (TGA). Selected parts of the report were also peer reviewed by
Professor Tom Beer, CSIRO Atmospheric Research (Sections 8 and 15); Dr
Stephen Corbett, New South Wales Department of Health (Section 11); Dr Andrea
Hinwood, Department of Environmental Protection, Western Australia (Sections 9,
11 and 13); and Professor Martyn T. Smith, University of California (Sections 9
and 12).
Priority Existing Chemical Number 21
2
2. Background
2.1 Introduction
Benzene is a naturally occurring, hazardous, volatile organic compound which is
ubiquitous in the environment. It is formed from biomass under the impact of heat,
pressure and geological time. As such, it is present in fossil fuels which may release
it to air when unearthed and, in particular, when heated to combustion. Benzene is
also a product of natural processes and human activities that involve the
instantaneous thermal degradation of organic matter. These sources of entry include
bush fires, crop residue and forest management burning, petroleum refining, petrol
combustion, wood and charcoal fires, fumes from heated cooking oils, tobacco
smoke, incense, and waste incineration. In addition, benzene enters the
environment in emissions and waste streams from industrial processes and waste
disposal facilities.
2.2 International perspective
Benzene was first isolated in 1825 and gradually became widely used as a solvent
and starting material for the synthesis of a number of organic chemicals (Folkins,
1984). Benzene also became recognised as a valuable constituent of petrol because
of its antiknock properties and ability to increase the octane rating of automotive
fuels.
Until World War II, benzene was isolated from light oil, which is a by-product of
the carbonisation of coal to produce gas for heating or coke for the blast furnaces of
the steel industry. Beginning in the 1930s, new catalytic and thermal processes for
the production of aromatic hydrocarbons from crude oil were discovered and
commercialised in the petroleum industry. With the advent of natural gas in the
1960s, worldwide coal gas production started to diminish. Simultaneously, the
introduction of modern steel processing methods decreased coke production and
made it attractive to burn the light oil as fuel rather than segregate it into benzene
and other products. In consequence, the petroleum industry is now the predominant
source of benzene.
In recent years, the use of benzene-containing solvents has been practically
eliminated because of the toxicity of the chemical. Current worldwide consumption
of benzene is 30-35 million metric tonnes (t) per annum, primarily as chemical
feedstock in the production of large-scale intermediates such as ethyl benzene,
cumene and cyclohexane (Chemistry & Industry News, 1996). This figure does not
include benzene produced by the petroleum industry and retained as a petrol
component.
Commercial low-grade qualities are sometimes referred to as benzol. Benzene is
not to be confused with benzine, which is a mixture of several low-boiling
hydrocarbons obtained in the distillation of petroleum.
2.3 Australian perspective
Developments in Australia have followed the general pattern outlined above, albeit
with a delay of 1-2 decades. The recovery of benzene from coal gas is now limited
Benzene 3
to the steelworks at Port Kembla in New South Wales and Whyalla in South
Australia.
There are eight petroleum refineries in Australia: two in Brisbane and Sydney and
one in Adelaide, Geelong, Melbourne and Perth. Since the 1970s, close to 100% of
local demand for petrol has been met from crude which is low in aromatic fractions
(Tresider, 1998). As such, all Australian petroleum refineries have processes in
place to increase the content of aromatic hydrocarbons including benzene in their
petrol blendstock. Petroleum-derived benzene feedstock for the chemical industry
is not produced in Australia.
As of 1986, new petrol-driven cars had to be fitted with catalytic converters and use
unleaded fuel. An Australian Standard for petrol for motor vehicles was established
in 1990 and limited the benzene content to a maximum of 5% v/v (Standards
Australia, 1990). In 1998, the average benzene content in Australian petrol was 2.9,
2.6 and 3.3% v/v in leaded petrol (LP), unleaded petrol (ULP) and premium
unleaded petrol (PULP) respectively (AIP, 1998b). The Fuel Quality Standards Act
2000 enables the Commonwealth to make mandatory national quality standards for
fuel supplied in Australia. Among others, these will include a maximum content of
benzene in petrol of 1% v/v from January 1 2006. Meanwhile, Western Australia
and Queensland have introduced regulations limiting the benzene content in petrol
to 1% and 3.5% respectively (EA, 2000b).
In 1980, AIP contracted The University of Melbourne to set up an epidemiological
health surveillance program called Health Watch. The program covers about 95%
of the industry's 18,000 employees in refineries, natural gas plants, distribution
terminals and production sites. It consists of a prospective cohort study of all-cause
mortality and cancer incidence, in addition to a case-control study of lympho-
haematopoietic cancers and benzene exposure established in 1988 (Glass et al.,
1998, 2000; Health Watch, 1998).
Air pollution became a major concern in the 1990s and prompted environment and
health authorities from the Commonwealth, States and Territories to initiate several
research projects into ambient air quality. Early results of this research resulted in
the inclusion of benzene in the National Pollutant Inventory (NPI), which was
established by the National Environment Protection Council (NEPC) in 1998. The
NPI currently comprises 36 chemicals of health and environmental concern which
must be reported to EA if the quantity used or handled per site exceeds a threshold
limit, which for benzene is 10 t per year (EA, 1999b). More recently, the Australian
and New Zealand Environment and Conservation Council contracted the Victorian
Environment Protection Authority (EPA) to assess the available air level data and
derive a risk-based rank order of hazardous air pollutants according to their
priorities for further research (EPA Victoria, 1999). Based on a scoring system as
well as on professional judgement, benzene came first among 15 chemicals
recommended for general urban air monitoring. Benzene is also the subject of a
publication in the series of National Environmental Health Forum Monographs,
which are intended to provide plain language information about important, topical
environmental health matters (Wadge & Salisbury, 1997). Current EA initiatives
such as the Fuel Quality Review and Living Cities Air Toxics Program both
address a number of environmental aspects relating to benzene (EA, 2000a, 2000b).
Public concern about exposure to benzene reached a peak in 1996, when a cluster
of leukaemia cases was identified in people living in the suburbs adjacent to the
coke ovens and coal gas by-product plant at the Port Kembla steelworks. A
committee reporting to the New South Wales Department of Health was set up to
Priority Existing Chemical Number 21
4
investigate the matter. It concluded that based on the available data, it was not
possible to ascribe the cluster to a particular exposure (including benzene). The
investigation produced several useful publications relating to benzene and the risk
of leukaemia (ILISC, 1997; Westley-Wise et al, 1999).
2.4 Assessments by other national or international bodies
Although there have been restrictions on the manufacture, handling, storage and
use of benzene in Australia since 1978, this report represents the first
comprehensive risk assessment by a national agency.
Benzene has been assessed by several overseas or international bodies involved in
the review or evaluation of data pertaining to the health and/or environmental
hazards posed by chemicals. Of these, the most noteworthy are:
· The Advisory Committee to the German Chemical Society on Existing
Chemicals of Environmental Relevance (GDCh, 1988);
· The Agency for Toxic Substances and Disease Registry under the US
Department of Health and Human Services (ATSDR, 1997);
· The Commission of the European Communities (EC, 1989, 2000);
· Environment and Health Canada (Government of Canada, 1993);
· The International Agency for Research on Cancer (IARC, 1982a, 1987);
· The International Programme on Chemical Safety (IPCS, 1993);
· The UK Department of the Environment (DoE, 1994);
· The US Environmental Protection Agency (USEPA, 1985, 1998a, 1998c); and
· The OECD SIDS International Assessment Report (draft) (OECD, 2000).
Benzene 5
3. Applicants
Following the declaration of benzene as a Priority Existing Chemical, 21
companies or organisations applied for assessment of the chemical. The applicants
supplied information on the properties, import and manufacturing quantities and
uses of benzene and, in some cases, on occupational exposures and releases to the
environment. In accordance with the Industrial Chemicals (Notification and
Assessment) Act 1989, NICNAS provided the applicants with a draft copy of the
report for comments during the corrections/variation phase of the assessment. The
applicants were as follows:
Koppers Coal Tar Products Pty Ltd
Alltech Associates (Australia) Pty Ltd
PO Box 23
PO Box 6005
Mayfield NSW 2304
Baulkham Hills NSW 2153
Australian Institute of Petroleum Merck Pty Ltd
207 Colchester Rd
GPO Box 279
Kilsyth VIC 3137
Canberra ACT 2601
Mobil Oil Australia Pty Ltd
Australian Council of Trade Unions
417 St Kilda Rd
393 Swanston Street
Melbourne VIC 3004
Melbourne VIC 3000
BHP Steel Flat Products
Australian Manufactures Workers Union
PO Box 1854
3/440 Elizabeth Street
Wollongong NSW 2505
Melbourne VIC 3000
Bio-Scientific Pty Ltd Qenos Pty Ltd
PO Box 78 Private Bag 3
Gymea NSW 2227 Altona VIC 3018
BP Australia Holding Limited Selby-Biolab
360 Elizabeth St Private Bag 24
Melbourne VIC 3000 Mulgrave North VIC 3170
Caltex Petroleum Australia Pty Ltd Sigma-Aldrich Pty Ltd
19-29 Martin Pl PO Box 970
Sydney NSW 2000 Castle Hill NSW 2154
Terminals Pty Ltd
Crown Scientific Pty Ltd
PO Box 268
Private Mail Bag 4
Footscray VIC 3011
Moorebank NSW 2170
3M Australia Pty Ltd
Huntsman Chemical Company Australia
PO Box 144
Pty Ltd
St Marys NSW 2760
PO Box 62
West Footscray VIC 3012
Trafigura Fuels Australia Pty Ltd
ICN Biomedicals Australasia
Unit 2, 47 Epping Rd
PO Box 187
North Ryde NSW 2113
Seven Hills NSW 2147
Whyalla Steelworks (OneSteel
Manufacturing)
PO Box 21
Whyalla SA 5600
Priority Existing Chemical Number 21
6
4. Chemical Identity and
Composition
4.1 Chemical name (IUPAC)
Benzene
4.2 Registry numbers
Benzene is listed on the Australian Inventory of Chemical Substances (AICS) as
benzene.
CAS number 71-43-2
EINECS number 200-753-7
UN number 1144
4.3 Other names
Annulene
Benzol(e)
Bicarburet of hydrogen
Carbon oil
Coal naphtha
Cyclohexatriene
Mineral naphtha
Motor benzol
Phenyl hydride
Pyrobenzol(e)
4.4 Molecular formula
C6H6
4.5 Structural formula
or
Benzene 7
4.6 Molecular weight
78.11
4.7 Composition of commercial grade product
Several different grades of benzene are commercially available. The principal
impurities are toluene, xylenes and other hydrocarbons with boiling points similar
to that of benzene. The higher the grade, the lower the content of thiophene
(thiofuran) and other sulfur compounds, which foul many catalysts used in
reactions of benzene (Fruscella, 1992). The specifications for two typical import
grades and the benzene/toluene/xylenes (BTX) mixture produced at the Port
Kembla steelworks are shown in Table 4.1.
Table 4.1: Raw material specifications for some commercially available
benzene grades
Test Pure benzene Crude benzene BTX
Benzene (% v/v) >99 95 80
C9 & higher (% v/v) - <1.5 <1.6
Carbon disulfide (ppm) - <50 <4000
H2S & SO2 None - -
Non-aromatic C5-C6 (% v/v) <0.15 <0.7 <1.5
Styrene (% v/v) - - <1.8
Thiophene (ppm) <1 <6000 <6000
Toluene (% v/v) - - <12.5
Total sulfur (ppm) - - <6000
Xylenes & styrene (% v/v) - - <3.8
Priority Existing Chemical Number 21
8
5. Physical and Chemical
Properties
5.1 Physical state
Benzene is a volatile, colourless and flammable liquid with a characteristic, sweet
aromatic odour (Budavari, 1996). The odour threshold ranges from 0.8-160 ppm
(AIHA, 1989); 50% of the population can identify the odour at 2 ppm and 100% at
5ppm (Verscheuren, 1996). The physical properties of benzene are summarised in
Table 5.1.
Conversion factors (at 25°C):
1 mg/m3 = 0.31 ppm and 1 ppm = 3.2 mg/m3 (Cavender, 1994).
5.2 Physical properties
Table 5.1: Physical properties
Property Value Reference
Melting point 5.53ºC Folkins (1984)
Boiling point 80.1ºC Folkins (1984)
Density
0.885 kg/L Fruscella (1992)
· at 15ºC
0.879 kg/L
· at 20ºC
0.874 kg/L
· at 25ºC
Vapour density 2.8 (relative to air = 1) Cavender (1994)
Vapour pressure
3.47 kPa Folkins (1984), Fruscella
· at 0ºC
9.97 kPa (1992)
· at 20ºC
12.6 kPa
· at 25ºC
24.2 kPa
· at 40ºC
35.8 kPa
· at 50ºC
Water solubility (at 25ºC) 1.80 g/L IPCS (1993)
3
Henry's Law constant (at 20ºC) 0.56 kPa.m /mol Mackay & Leinonen (1975)
Partition coefficient (log Po/w) 1.56-2.15 IPCS (1993)
Sorption coefficient (log Koc) 1.8-1.9 IPCS (1993)
Flash point (closed cup) 11ºC Fruscella (1992)
Autoignition temperature 560ºC Fruscella (1992)
Explosive limits
3
1.4% v/v (45 g/m ) Cavender (1994)
· lower
3
7.9% v/v (250 g/m )
· upper
5.3 Chemical properties
The six carbon atoms of benzene form a regular hexagon and all 12 atoms lie in a
single plane, with all bond angles being exactly 120° (Fruscella, 1992). The
molecule is traditionally depicted as having alternating single and double bonds
(see structure (1) in Section 4). However, as the six carbon-carbon bonds are
Benzene 9
physically and chemically identical and intermediate in length between single and
double bonds (as indicated by structure (2) in Section 4), benzene does not react as
a typical unsaturated compound.
Benzene has great thermal stability and elevated temperatures are required for its
decomposition. It undergoes substitution and addition reactions and ring cleavage.
For industrial applications, the most important reactions are alkylation with
ethylene or propylene to produce ethyl benzene or cumene, hydrogenation to
cyclohexane, nitration and sulfonation to form nitrobenzene and benzenesulfonic
acid, and halogenations. Benzene cannot be hydrolysed.
Benzene is miscible with numerous other organic solvents including alcohol,
acetone, diethyl ether, ethyl acetate, chloroform, carbon disulfide, glacial acetic
acid and oils (Budavari, 1996). Its solubility in water ranges from 1.13% v/v at
25°C to 5.07% v/v at 107°C (Folkins, 1984). Benzene forms binary and tertiary
azeotropes with water and a large number of organic substances (for examples, see
Folkins (1984)).
Benzene is highly flammable and potentially explosive. Combustion products
include carbon dioxide, water vapour and carbon monoxide. With a deficiency of
air or oxygen, partial decomposition and soot deposition occur (Folkins, 1984).
Vapours burn with a sooty flame.
Priority Existing Chemical Number 21
10
6. Methods of Detection and
Analysis
6.1 Characterisation
Benzene can be characterised by infrared, ultraviolet and mass spectrometry and
nuclear magnetic resonance techniques (Fruscella, 1992).
6.2 Detection and analysis
A time-honoured spot test for benzene in the workplace or surroundings involves
the treatment of a sample with nitric acid followed by ether extraction and
dissolution in a mixture of alcohol and methyl ethyl ketone. Benzene is converted
to m-dinitrobenzene which imparts a persistent red colour to the solution (Dolin,
1943, cited in Fruscella, 1992).
Standard analytical methods for benzene in air, water, soil, foods, smoke,
biological samples, petroleum products etc. rely on gas chromatography (GC) with
flame or photo ionisation detection, or on gas chromatography-mass spectrometry
(GC-MS) (Fruscella, 1992; IPCS, 1993). Benzene in water, soil and food is usually
measured by a purge and trap method by bubbling an inert gas through the sample
and collecting the chemical on an absorbent. Benzene is then desorbed and
determined. The best available GC methods are able to detect benzene at 0.1 ppb in
air or 1 ng/kg in liquid or solid media, although 3 ppb in air and 1µg/L in water are
the limits of detection in routine analysis (IPCS, 1993; NHMRC, 1996). The GC-
MS method is not quite as sensitive, but more reliable in the case of samples with
multiple components with retention times similar to that of benzene (IPCS, 1993).
6.3 Atmospheric monitoring methods
In the environment
The methods commonly used for measuring the concentration of benzene in
ambient air fall into the following two categories (EPA Victoria, 1999):
(1) discrete air sampling with subsequent laboratory analysis; or
(2) continuous or semi-continuous in-field analysis.
Among the former, the most widely used method involves the collection of air into
a stainless steel canister over a predetermined period of time such as 24 h, followed
by analysis of a concentrate of the air sample by GC or GC-MS. This method is
described in more detail by DEP Western Australia (2000).
A commonly used continuous method for in-field analysis utilises an optical remote
sensing system to determine the concentration of the chemical by means of the
differential absorption of transmitted light by gaseous compounds along the light
path. The system consists of a light transmitter and sensor placed at a given
distance apart at the monitoring site.
Alternatively, the concentration in air can be analysed by semi-continuous gas
chromatography. Samples of air are collected directly onto solid absorbents,
Benzene 11
desorbed thermally onto the GC column and analysed while the next sample is
collected.
The analytical limit of detection of the above methods typically ranges from 0.003-
0.1 ppb. All of the methods allow for the simultaneous determination of several
other gaseous air pollutants in the same sample. Discrete sampling methods
determine average pollutant levels over the sample collection time. Continuous or
semi-continuous methods enable more detailed information about concentration
variations to be obtained.
In the workplace
This section summarises the methods commonly used for the measurement of
benzene in the workplace. Other past and present techniques are described in a
recent review by Verma & des Tombe (1999a, 1999b).
For personal monitoring during full shifts or tasks, workers are equipped with a
charcoal tube or badge placed in the breathing zone. For area monitoring, the tube
or badge is placed at a fixed location in the workplace environment. Tubes are
connected with a portable metering pump, whereas badges sample the air by
diffusion. At the end of the sampling period, the tube or badge is sealed and
transferred to a laboratory, where the chemical is liberated from the absorbent by
elution or thermal desorption and quantified by GC (NIOSH, 1994). The result is
expressed as a time-weighted average (TWA) concentration in ppm or mg/m3 over
the duration of the sampling period. The analytical detection limit depends on the
airflow across the absorbent and the duration of the sampling period. The detection
limit using charcoal tube sampling and analysis according to the NIOSH method is
0.02 ppm.L, or 0.004 ppm for a sample collected over 60 min at a pump speed of
80 mL/min (IPCS, 1993). The agreement between the tube and badge methods is
not perfect, but the differences are generally of little importance (Hotz et al., 1997;
Purdham et al., 1994).
`Grab sampling' or instantaneous measurement of the concentration of airborne
benzene is conducted with colorimetric detector tubes. These are glass tubes sealed
at both ends with a graduated concentration scale etched into the outer surface. The
tubes contain a carrier material covered with chemical reagents that react with
benzene to produce a colour change whose end-point is read against the scale. Prior
to use, the seals are broken, the tube is connected to a hand pump and the pump is
operated to draw a defined amount of air through the tube. A colorimetric detector
has become available which can measure benzene at 0.2 ppm in the presence of
other hydrocarbons, with a measuring time of 8 minutes. Non-selective
photoionisation detectors can be used for instantaneous measurement of benzene
concentrations with a detection limit of approximately 0.1 ppm but, because the
detector also responds to volatile organic chemicals, they are limited to situations
where the vapour is known to be pure benzene. Recently, a benzene-selective
photoionisation detector has become available which is claimed to be able to
measure benzene at concentrations of 0.1 ppm 200 ppm in the presence of other
hydrocarbons within approximately 1 minute. The detector uses a single use pre-
treat tube to filter out interfering hydrocarbons except for C3 alkanes and must be
calibrated against 5 ppm benzene prior to use.
A less widely used method is continuous area monitoring, which is performed by
pumping air collected at one or more fixed locations through an auto analyser
equipped with an ultraviolet spectrometer. This method delivers readings for TWA
Priority Existing Chemical Number 21
12
as well as peak concentrations and has a limit of detection of approximately 0.2
ppm (IPCS, 1993).
6.4 Biological monitoring methods
Biological monitoring for benzene exposure involves the measurement of
unmetabolised benzene in blood, urine or breath samples, of benzene metabolites in
the urine, or of protein adducts with the benzene oxide metabolite.
The concentration of benzene in venous blood and urine can be determined by GC,
with a detection limit of around 0.5 µg/L (IPCS, 1993). For the determination of
benzene in breath air, an end-exhaled sample is collected and analysed by GC-MS,
with a detection limit of 3-6 ppb (Money & Gray, 1989). However, these methods
are only suitable for research purposes, as great care must be exercised to avoid
contamination of the samples with ambient benzene.
Various metabolites are excreted in the urine, including phenol, hydroquinone and
catechol conjugates, S-phenylmercapturic acid and trans,trans-muconic acid (see
Section 9.3), although none of them is formed exclusively from benzene. These
metabolites can be quantified by GC, GC-MS or high-performance liquid
chromatography as described by Ducos et al. (1990), Hotz et al. (1997), Lee et al.
(1993), Popp et al. (1994) and others. Whereas the urinary concentration of phenol
has been widely used as an index of benzene exposure, the background levels make
it unreliable at exposures <5-10 ppm. The concentration of S-phenylmercapturic
acid or muconic acid relative to creatinine in an end-of-shift urine sample has been
shown to be a fairly good indicator of exposure in the 0.25-1 ppm range, even in
smokers (Ghittori et al, 1995; Hotz et al, 1997; Ong et al, 1996).
The metabolite benzene oxide binds to nucleophilic sites and forms phenyl cysteine
residues with proteins such as haemoglobin and albumin (see Section 9.3.1). The
concentration of such adducts in blood correlates with benzene exposure. However,
high background levels severely limit the practical use of the S-phenyl cysteine
adduct as a biological marker for benzene uptake (Yeowell-O'Connell et al, 1998).
Benzene 13
7. Manufacture, Importation and
Use
7.1 Manufacture and importation
Benzene is introduced into Australia through extraction, importation and
manufacture.
The total Australian production of crude in 1994-98 is shown in Figure 7.1. Crude
includes unrefined oil as well as condensate, which is a liquid mixture of
hydrocarbons recovered from gas wells. The mean annual and 1998 volumes are
both approximately 31,000 ML. Australian crude is reported to have a low benzene
content, estimated at about 0.1% v/v (Glass et al, 1998). As such, the annual
extraction of benzene from Australian oil and gas fields can be estimated at
approximately 31 ML. As the density of benzene is around 0.88 kg/L, this
corresponds to a quantity of 27 kilotonnes (kt) pure benzene.
Figure 7.1: Australian production of crude oil and condensate 1994-98 (AIP,
1999b)
34
33
32
ML x 1000
31
30
29
28
27
1994 1995 1996 1997 1998 M ean
The throughput of crude at Australian refineries in 1998 was 44,678 ML, of which
62% was of non-Australian origin (AIP, 1999a). Most of the imports come from oil
fields in the Pacific basin and contain approximately the same concentration of
benzene as Australian crude, that is, about 0.1% v/v (Glass et al, 1998). This
corresponds to a total input of 45 ML or 39 kt pure benzene.
From these figures, it can be concluded that Australia is a net importer of benzene
in crude to the tune of approximately 12 kt per annum.
Table 7.1 shows the throughput of benzene-containing gasoline products, that is,
leaded petrol (LP), unleaded petrol (ULP), premium unleaded petrol (PULP) and
light aircraft gasoline (Avgas), at Australian refineries in 1998. The table also gives
the average content of benzene and the corresponding quantities of pure benzene.
Data for Avgas are estimates, but have little impact on the total. Other petroleum
products such as liquefied petroleum gas, kerosene, civil aviation jet fuel, diesel oil,
fuel and heating oils and lubricants contain no or practically no (<0.02% v/v)
benzene (AIP, 1999a; IARC, 1989; Potter & Simmons, 1998).
The volume of pure benzene in petrol produced at Australian refineries in 1998 was
484 ML, corresponding to 426 kt pure benzene. As the throughput crude contained
Priority Existing Chemical Number 21
14
approximately 39 kt pure benzene, it can be concluded that the production of
benzene in the Australian petroleum industry amounted to 387 kt in 1998.
Table 7.1: Throughput of LP, ULP, PULP and Avgas at Australian refineries in
1998 (AIP, 1998b, 1999b)
LP ULP PULP Avgas Total
Petrol (ML) 4965 12,218 640 100 17,923
Benzene (% v/v) 2.9 2.6 3.3 1.0 -
Pure benzene (ML) 144 318 21 1 484
Petrol is also imported in finished form. From January 1994 through August 1999,
petrol imports averaged 680 ML per annum (DISR, 1999). There is no information
on the benzene content of imported petrol, but it is unlikely to differ much from
that of Australian ULP, which averages 2.6% v/v. As such, annual imports of
benzene as an ingredient in petrol purchased overseas is estimated at 18 ML
corresponding to approximately 15 kt pure benzene.
The Port Kembla steelworks produces 20-22 ML per annum of a commercial low-
grade benzene product called BTX. As the specifications stipulate a benzene
content of 80% v/v (Table 4.1), this corresponds to approximately 14.0-15.5 kt
pure benzene per annum. The Whyalla steelworks no longer produce BTX,
however, 0.12kt of benzene per annum, from the naphthalene still, is reinjected into
the fuel gas stream.
Benzene is also produced as a by-product stream component at two olefins
(pyrolysis) plants belonging to Qenos Pty Ltd. The total quantity amounts to
approximately 15 kt per annum, all of which is exported for use or further
processing overseas.
The only importer of benzene feedstock is Huntsman Chemical Company, whose
annual imports are stable at about 50 kt pure benzene and 30 kt crude benzene
(95% v/v), that is, approximately 80 kt pure benzene per annum.
Minor quantities not exceeding 1 t in the aggregate are imported for laboratory and
other small-scale uses, as described below.
7.2 Manufacturing processes and end use
7.2.1 Petroleum industry
Table 7.2 provides an overview of the location and ownership of the currently
operating oil refineries in Australia, as well as a summary of the processes
employed to produce benzene, their capacity, and the benzene content of locally
produced petrol. The latter is taken from a 1994 survey, which reported the
concentration in % w/w (Tresider, 1998). As such, it is not directly comparable to
the data provided in Table 7.11. The process technologies and end use of the
benzene produced are described below.
1
The factor needed to convert % w/w to % v/v varies with the density of the petrol. Based on the
average density of Australian petrol in 1999, multiplication of the concentration in % w/w with 0.84
will give an approximate concentration in % v/v (Exxonmobile personal communication, 2001).
Benzene 15
Table 7.2: Benzene processes at Australian oil refineries (AIP, 1997; Mobil,
2000; Tresider, 1998)
Benzene in
Benzene Capacity
LP/ULP/PULP (% w/w)
State Location Owner technology (kt/y)*
NSW Clyde Shell Reforming 890 2.7/ 2.4/3.6
Cracking 1558
Kurnell Caltex Reforming 1344 2.3/2.3/4.7
Cracking 2024
QLD Bulwer Island BP Reforming 588 2.5/3.7/5.0
Cracking 912
Lytton Caltex Reforming 1157 2.4/2.5/4.4
Cracking 1469
Reforming
SA Port Stanvac Mobil 1157 2.3/2.0/2.5
VIC Altona Mobil Reforming 1380 4.8/4.5/5.5
Cracking 1246
Geelong Shell Reforming 1380 3.6/3.6/5.4
Cracking 1780
WA Kwinana BP Reforming 979 1.9/2.0/2.2
Cracking 1456
* The capacity is calculated on the basis of 350 stream days per year and refers to the quantity of raw
material processed, not benzene produced.
Based on more recent information (AIP average data for 1999), ULP contains
approximately 3.01% (w/w) benzene, PULP contains 4.02% (w/w) and LP contains 3.46
(w/w) (Exxonmobile Personal Communication, 2001).
Petroleum refining
Petroleum refining involves a series of continuous, enclosed processes designed to
convert crude oil and condensate into end products such as liquefied petroleum gas,
Avgas, petrol, jet fuel, diesel, heating oil, lubricants and bitumen. The main
processes designed to augment the content of aromatics such as benzene in petrol
are shown in the flowchart in Figure 7.2 and summarised below.
Figure 7.2: Benzene production in petroleum refineries
Isobutane
Distillation
CRUDE Straight run gasoline
30-105°C
PETROL AND
Catalytic AVGAS
Naphtha reforming BLEND-
STOCKS
105-155°C
Vacuum
Catalytic
distillation Alkylation
cracking
Heavy
gas oil
340-425°C
Priority Existing Chemical Number 21
16
At all refineries, crude oil is first separated into a number of fractions by
atmospheric and vacuum distillation. Petrol is a blend of butane, refined naphthas,
isomerate, reformate, cracked gasoline and alkylate. Avgas is primarily made from
alkylate although reformate can also be used.
The straight run gasoline fraction contains a 5- to 10-fold concentrate of all the
benzene that was present in the crude, corresponding to a benzene concentration of
0.5-1% v/v.
The naphtha fraction, which contains many cyclic, saturated hydrocarbons,
undergoes catalytic reforming in a process using heat, pressure and a platinum
catalyst to convert a portion of the feedstock to aromatic compounds. The resulting
reformate typically contains 4-8% v/v of benzene (Audrey, 1994).
At the Mobil Altona refinery, part of the heavy gas oil fraction is piped to a nearby
petrochemical plant for steam cracking, as described below. This process gives rise
to a by-product known as steam cracked naphtha or pyrolysis gasoline, which
contains 6-8% benzene. This by-product stream is piped back to the oil refinery
where it is stored in floating-roof tanks and eventually exported to overseas
customers by shipping tanker.
The heavy gas oil fraction, which contains large, high boiling hydrocarbon
molecules, is cracked to a mixture of lower molecular weight compounds by means
of heat, pressure and a silica/aluminium oxide or zeolite catalyst. The benzene
content of cracked gasoline rarely exceeds 1-2% v/v, but varies depending on the
composition of the feedstock, the nature of the catalyst and the temperature and
pressure conditions. As shown in Figure 7.2, some of the output from the cracking
process is reacted with isobutane to form larger branched-chain molecules
(isoparaffins) that increase the octane rating of the final petrol blend. The alkylation
process does not augment benzene content.
Eventually, the various petrol feedstock qualities are blended to produce end
products with the desired specifications. These vary according to the likely ambient
temperatures in the area and season in question and generally require higher
concentrations of aromatic components such as benzene in colder climates.
Feedstock and end product are stored in tanks equipped with floating roofs or
connected to vapour recovery systems. The end products are distributed to larger
terminals by pipeline, in coastal tankers or bottom-loaded rail tankers and/or to
local depots and service stations in road tankers, the majority of which are bottom
loaded. In rural areas not all road tankers are bottom loaded. Terminals in Sydney,
Melbourne and Perth have vapour recovery systems to minimise vapour emissions
during truck filling operations. There were 8233 petrol retail outlets in Australia at
the end of 1998 (AIP, 2000).
End use
In Australia, all benzene produced by petroleum refiners is retained as one of several
aromatic components in automotive petrol and Avgas; most of this benzene is burnt
during normal engine operation. Figure 7.3 shows the total demand for benzene-
containing petroleum products in 1996, and its breakdown by State marketing area2.
2
The State marketing area (SMA) of Queensland includes the Murwillumbah district of NSW, which
is supplied from the refineries in Brisbane. The SMA of South Australia includes the Broken Hill-
Wilcannia district of NSW and the Murrayville district of Victoria, which are supplied from the
refinery at Port Stanvac. The SMA of Victoria includes the Riverina district of New South Wales,
Benzene 17
Figure 7.3: Demand for petrol in 1996 (AIP, 1997)
NT
NT
TAS
TAS
WA
WA
NSW
SA NSW
SA
QLD
QLD
VIC
VIC
LP: 6782 ML ULP: 10,847 ML
TAS
NT
NT
WA NSW
TAS
SA
WA
NSW
VIC
QLD
SA
QLD
VIC
PULP: 338 ML Avgas: 102 ML
As expected, the demand for petrol is highest in the most populous States of New
South Wales and Victoria. Queensland, Western Australia and the Northern
Territory account for more than one-half of the total demand for Avgas.
Since catalytic converters became mandatory on new cars in 1986, there has been a
steady increase in the demand for ULP and a corresponding decline in the demand
for LP. However, although LP contains more benzene than ULP, the corresponding
fall in benzene consumption has been counterbalanced by an overall growth in
petrol demand. PULP, which was first produced in significant quantities in 1989
and has the highest concentration of benzene (Table 7.1), accounted for only 2% of
total petrol sales in 1996, almost half of which was generated in New South Wales.
However, PULP demand is expected to grow as LP is phased out nationally by
2002 and pre-1986 cars will need to run on either PULP or lead replacement petrol,
that is, PULP pre-blended with an anti-valve seat recession additive.
The likely impact of the predicted changes in demand on the use of benzene in
petrol can be estimated on the basis of AIP's petrol sales forecasts for the decade
1998-2007 (AIP, 1998a). If current benzene concentrations are assumed to remain
which is supplied from the refineries at Altona and Geelong. The SMA of New South Wales includes
the Australian Commonwealth Territory.
Priority Existing Chemical Number 21
18
unchanged throughout the period, total benzene use in petrol is estimated to
increase from 434 kt/y in 1998 to 461 kt/y in 2007. However, if a nationwide
standard is introduced limiting the maximum content in petrol to 1%, total benzene
use in petrol is estimated to fall to 176 kt/y in 2007.
Independent petrol retailers
The main imports of petrol are marketed by independent chains or supermarkets
such as Trafigura (formerly Burmah) Fuels, Liberty and Woolworths, who had 564
service stations between them at the end of 1998 (AIP, 2000). Their imports pass
through the terminals of Vopak (formerly Van Ommeren) at Port Botany in Sydney
and Hastings Point near Melbourne, Gull at Kwinana in Western Australia, and
Fletcher Challenge in Brisbane. These terminals have a petrol storage capacity of
95, 70, 53 and 13 ML respectively (DISR, 1999; Vopak, 2000).
7.2.2 Steel and associated industries
BTX
In the steel industry, BTX is a by-product from volatile fractions produced in the
coking ovens. It contains 80% v/v benzene (see Table 4.1) and is recovered in an
enclosed process which yields from 3-5 kg pure benzene per t coke produced.
The coking ovens are arranged in batteries, each of which may contain in the region
of 60 units. The ovens are sequentially charged by means of mechanical hopper
systems through special lidded holes that are closed and sealed to keep out air
during the coking cycle. This cycle takes place at 900-1100°C for 12-24 h. On
completion of the cycle, the hot coke is removed mechanically through doors on
the sides of the oven and sprayed with flushing liquor (a dilute solution of ammonia
in water) to quench combustion upon exposure to air.
The coke oven gas contains hydrogen, methane, carbon monoxide and light oil,
which is a mixture of various aliphatic and aromatic hydrocarbons, including
benzene. The overhead gas is collected into a pipe that runs along the length of
each battery and propelled to the by-product plant. At emission, it has a
temperature of approximately 600°C which is brought down to 80°C by spraying
with flushing liquor. It is further cooled to 38°C, passed through an electrostatic
precipitator and an acid scrubber where tar and ammonia are removed and cooled
to a final temperature of 20°C. The gas then passes to a system of light oil
scrubbers, where most of the C5 and higher hydrocarbons are recovered by counter-
current absorption using a high-boiling (300-400°C) petroleum fraction. BTX is
recovered from the absorbent oil by steam stripping and separated from the water
by distillation. At Port Kembla, the distillation process is continuous and BTX is
collected and piped to a storage tank. At the smaller Whyalla steelworks, BTX is
separated by batch distillation and returned directly to the gas system. At both sites,
the refined coke oven gas is stored in a gasholder and used for heating.
Process waste water, which is mainly from the flushing liquor circuit and contains a
number of contaminants, including benzene, is passed through a water/oil separator
and discharged to a biological treatment plant. All storage tanks are eventually
vented to the atmosphere, but may be connected to a vent header with a sealpot
arrangement to prevent emission unless there is a build-up of pressure. Excess gas
is flared off.
Benzene 19
All BTX produced at Port Kembla is transported by road to Huntsman Chemical
Company in Melbourne for use as chemical feedstock.
Coal tar
Tar condensed from the coke oven gas and flushing liquor circuit is collected in a
system of decanting tanks and pumped to a wet tar storage tank. This tank decants
excess liquor back to the liquor circuit and transfers the tar sediment to a dry tar
tank farm. The residue from the BTX distillation, which is known as naphthalene
oil, is also pumped to the dry tar tank farm. The dry tar, which contains 0.16%
residual benzene, is shipped to Koppers Coal and Tar Products at Mayfield,
Newcastle, New South Wales, in splash top loaded rail or road tankers, or by sea
tanker.
The Newcastle plant receives in the order of 125 kt crude tar per annum containing
about 145 t residual benzene. The plant comprises two interconnected, fully
enclosed systems which separate the tar in a series of continuous distillation
processes. The distillation products include solvent naphtha (4% benzene), distilled
tar (0.5% benzene), creosote oil (0.2% benzene), naphthalene (no measurable
benzene) and coal tar pitch (no measurable benzene). Most of the solvent naphtha
representing about 80% of the benzene received is burnt as fuel. The remainder is
blended into creosote oil which is used in solvent-based industrial timber
preservatives or as feedstock in the manufacture of carbon black. Distilled tar is
used in the coatings industry. Naphthalene is exported and coal tar pitch onsold to
the aluminium industry for the manufacture of carbon electrodes.
Process waste water from the Koppers coal tar plant is passed through a water/oil
separator and discharged to a biological treatment plant. All storage tanks are
connected to fume scrubbing systems. Off-gases from the stills are burnt as fuel.
7.2.3 Chemical industry
Qenos
Ethane and naphtha (gas oil) cracking
The olefins plant at the Qenos site at Altona in Melbourne produces 10-12 kt
benzene per year as a by-product of the steam cracking of ethane and naphtha (gas
oil) to ethylene, propylene and butadiene, which are then converted into plastics
and rubbers. The Qenos (formerly Orica) olefins plant at Botany in Sydney
produces 2-3 kt benzene per year as a by-product of the steam cracking of ethane to
ethylene.
Steam cracking is a continuous, fully enclosed process which produces a variety of
products by free radical reactions. Steam and hydrocarbon feedstock are mixed and
subjected to a brief surge of extreme heat (750-900°C). The effluent is rapidly
cooled, compressed, purified in a caustic washer, dried, chilled and fractionated in
a train of distillation columns.
At the Altona plant, the by-product streams from the ethane and naphtha steam
cracking processes are combined and purified by distillation to produce a pyrolysis
gasoline containing 6-8% benzene, which is piped to the Mobil Altona refinery and
eventually exported for use overseas (Section 7.2.1).
Priority Existing Chemical Number 21
20
At the Botany plant, the heavier molecules produced in the cracking process are
collected in a feed tank and further processed in a fully enclosed system which is
on stream for approximately 60 days per annum. In the process, the by-product
stream is hydrogenated and then distilled to remove light ends, which are returned
to the ethane cracking system. The end product is a pyrolysis gasoline containing
approximately 55% aromatics including 35-36% benzene. This is stored on site in a
floating-roof tank. At intervals of 5-6 months, it is piped to the bulk liquid terminal
at Port Botany and shipped overseas for further processing.
Butadiene rubber manufacture
Qenos' Altona facility also uses about 40 t benzene per year as a solvent
component in the manufacture of butadiene rubber. The benzene is purchased from
Huntsman (see below) and supplied by dedicated road tanker. It is stored in a
nitrogen-blanketed tank and pumped to the butadiene rubber plant via sealed pipes.
In the plant, butadiene is polymerised in solution in a fully enclosed batch process.
The solvent contains cyclohexane and benzene in a ratio of about 2:1 and is not
consumed in the reaction. The polymerisation process is strongly exothermic and
the reactor temperature is kept at approximately 20°C by ammonia cooling. The
reaction is stopped with an antioxidant. The solvent is removed from the rubber-
solvent solution by steam stripping and is then condensed, purified and recycled to
the beginning of the process for feedstock blending. Waste water is steam stripped
to remove dissolved benzene prior to discharge to sewer. Off-gases containing
benzene are sent to a thermal oxidiser for destruction.
Huntsman Chemical Company
The Huntsman (formerly Chemplex) plant at West Footscray in Melbourne
converts about 80 kt benzene per annum to ethyl benzene and 10-15 kt to cumene
(isopropyl benzene). Ethyl benzene is further processed to styrene, which is used in
the production of polystyrene polymers and unsaturated polyester and vinyl ester
resin solutions. Cumene is oxidised to acetone and phenol. The phenol is used on
site in the production of phenol-formaldehyde resins. The acetone is onsold in bulk
to other manufacturers.
Huntsman purchases about 15% of their requirements for benzene in the form of
BTX produced at the Port Kembla steelworks. The remainder is imported from
Indonesia, Japan, Korea and Singapore in chemical tankers. The bulk chemical is
unloaded at Terminals Pty Ltd on Coode Island on Melbourne's waterfront where it
is kept in storage before being transported to Huntsman by dedicated road tanker. A
small part is trucked to Qenos' Altona facility for use as a solvent component in the
manufacture of butadiene rubber.
Styrene manufacture
The styrene plant was commissioned in 1977 and operates a series of four
continuous, fully enclosed processes, namely, ethylene, Litol, alkylation and
dehydrogenation. The principal feedstocks are ethane, pure benzene and BTX.
In the Litol plant, BTX is vaporised with hot hydrogen and passed through fixed
bed catalytic reactors to hydro-dealkylate toluene and xylenes to benzene and
destroy heterocyclic compounds. Pure benzene is recovered by fractional
distillation. By-product hydrocarbon gases, excess hydrogen and heavy distillation
residues are used as fuel. A waste stream rich in hydrogen sulfide is incinerated.
Benzene 21
The alkylation plant makes ethyl benzene from benzene and ethylene produced on
site by the cracking of ethane. In the first of two reactors, the alkylation is carried
out in the presence of a homogenous acidic catalyst prepared separately from
aluminium chloride. In the second reactor, recycled polyethyl benzene is trans-
alkylated to ethyl benzene. The remaining undesired components from the dilute
ethylene benzene stream are recovered, neutralised and used as fuel. Catalyst is
removed from the alkylated liquor in a 3-stage wash system. The aqueous wash
liquors containing aluminium and sodium chlorides and some hydrocarbon
contaminants are treated aerobically at the site effluent treatment plant before
discharge to sewer. The alkylation liquor is then refined in a 3-column distillation
train. Pure ethyl benzene is recovered for subsequent use in the dehydrogenation
plant. Excess benzene and the polyethyl benzene are recovered and recycled to the
reactors. The heavy distillation residue is utilised elsewhere in the complex to
lower the viscosity of other residue streams and ultimately utilised as fuel.
In the dehydrogenation plant, ethyl benzene is dehydrogenated to styrene at high
temperature and low pressure in the presence of steam. The dehydrogenated
mixture is condensed and cooled, the water is separated out, and the stream is
refined in a 3-column distillation train. Hydrogen and other gases produced in the
reactor are used as fuel. By-product benzene and toluene are recovered and sent to
the Litol plant for conversion to pure benzene. Unreacted ethyl benzene is
recovered and returned to the dehydrogenation reactor. Pure styrene is distilled and
dosed with a polymerisation inhibitor prior to storage.
Tanks containing benzene are vented to a carbon bed vapour emission control
system that recovers about 60 t of benzene per annum and returns it to the styrene
plant.
Phenol manufacture
The phenol plant was commissioned in 1968. It produces phenol and acetone in a
continuous, fully enclosed process. Cumene is formed by the reaction of pure
benzene and propylene in a fixed-bed reactor using a phosphoric acid catalyst on a
solid support. The pure benzene feedstock is either imported or produced in the
styrene plant and piped to the phenol plant. A 3-column refining section recovers
gaseous components from the cumene stream. Unreacted benzene is recycled into
the process, and cumene is sent on to the oxidation reactor. Heavy distillation
residue is utilised as fuel or as an aromatic feedstock in the Litol plant. The purified
cumene stream is partially oxidised with air to cumene hydroperoxide, which is
cleaved by acid to a mixture of crude phenol and acetone. The mixture is split into
phenol and acetone, which are purified by distillation in a 7-column refining train.
Heavy distillation residue is subjected to a 2-column system, where some
additional phenol is recovered via pyrolysis and distillation for recycling to the
refining train. Residue from this system is utilised as fuel. Spent air from the
oxidation reactor is chilled to remove most of the organic substances and then
passed through activated carbon beds before release to the atmosphere. A combined
aqueous waste stream is treated in the site effluent treatment plant prior to
discharge to sewer.
7.2.4 Laboratory uses
Seven of the applicants listed in Section 3 identified themselves as occasional
importers of reagent grade benzene. Between them, they imported approximately
500 kg benzene in 1999, which was onsold to a total of 55 end users. Of these, 27
Priority Existing Chemical Number 21
22
were commercial enterprises such as contract and company in-house analytical
laboratories. Twenty-one belonged to the science or medical faculties of 15
different universities. Seven were State or Commonwealth laboratories. The
quantities purchased by individual laboratories in 1999 ranged from 0.1-150 L
(0.88-130 kg), with a mean of 10 L (8.8 kg) and a median of 2.5 L (2.2 kg).
Benzene is also present in some ready-made liquid or gaseous standards for the
calibration of gas chromatographs and other analytical instruments. The quantity of
benzene consumed through the use of such standards is estimated at less than 1 kg
per annum.
7.2.5 Coincidental production
Benzene is formed coincidentally during the burning of aromatic and non-aromatic
organic compounds contained in biomass such as crops, wood and humus, in fossil
fuels such as black and brown coal, and in petroleum products including diesel and
jet engine fuel which have a negligible benzene content prior to combustion. These
processes are important sources of entry into the environment and will be
considered in detail in subsequent sections.
7.3 Summary
Table 7.3 summarises the industrial mass balance of benzene in Australia and the
available information on its major manufacturers, importers and users, and most
significant end uses. These figures are approximate and give a general indication of
industrial use of benzene in Australia. Benzene produced coincidentally in the
course of human activities or natural processes and products containing benzene as
an impurity are not accounted for.
Table 7.3: Benzene mass balance and major manufacturers, importers and
end users in Australia in 1998-99
Kilotonnes/year (rounded)
Industry or Extrac- Manu- Con-
company End use
Import Total Export
tion facture sumption
Petroleum 30 385 25 440 - 440 Petrol
Huntsman - - 80 80 - 95 Feedstock
Steel 15 - - 15 - 0.2 Fuel
Qenos - 15 - 15 15 0.040 Solvent
Others - - 0.0005 0.0005 - 0.0005 Reagent
TOTAL 45 400 105 550 15 535 -
In 1998-99, total benzene consumption in Australia was in the order of 535 kt per
year. Of this quantity, 105 kt were imported, 45 kt were extracted from crude oil
and coal gas, and the remainder produced at eight oil refineries. Petrol accounted
for approximately 82%, chemical synthesis for 18% and all other uses combined
for less than 1% of total consumption.
Benzene 23
8. Environmental Release, Fate
and Effects
As no environmental fate and toxicity studies were submitted for assessment, this
section is based on international, peer-reviewed reports such as GDCh (1988),
Government of Canada (1993) and IPCS (1993), the International Uniform
Chemical Information Database (IUCLID) and the USEPA's ECOTOX database
(USEPA, 2000). Data within these three reports are largely the same and also
appear in the databases.
IUCLID contains non-confidential data supplied by industry to the European
Commission. They have not undergone peer review and are therefore only reported
where they are not described elsewhere but nonetheless give guidance to the fate
and effects of benzene in the environment. Results in the ECOTOX database have
been published and are generally considered reliable.
8.1 Environmental release
Benzene is ubiquitous in the environment, with numerous sources of entry
including bush fires, crop residue and forest management burning, petrol
combustion, wood fires, tobacco smoking and emissions and waste streams from
various industries. Due to the nature of benzene being produced incidentally during
natural processes and human activities, it is not possible to obtain accurate figures
in estimating national releases. However, several point source releases provided in
NPI reports for the first reporting year of 1998/99 are described in Section 15,
which also gives an estimation of diffuse releases in a model urban environment.
Overall, release of benzene will primarily be to the atmosphere through emissions
in exhaust during petrol combustion in motor vehicles, followed by releases to air
from point sources in the petroleum, steel, aluminium, chemical and other
industries. By contrast, releases to water and soil are expected to be relatively
minor, as borne out in NPI reports where the highest annual release from a single
point source to water and soil was 1100 kg and 45 kg respectively, compared with
130,000 kg to air from an oil and gas extraction plant.
8.2 Environmental fate
The Trent University (1999) Level 1 Fugacity Based Environmental Equilibrium
Model indicates that in the order of 99% of benzene will partition to air, with
0.88% and 0.05% partitioning to water and soil respectively. Negligible amounts
are expected to partition to sediments, suspended sediments, biota and aerosols.
8.2.1 Atmospheric fate
The water solubility of benzene suggests that one removal mechanism from the
atmosphere is through returning to the terrestrial and aquatic compartments in
rainwater. However, the Henry's Law constant and volatility of benzene indicate
that the chemical would rapidly volatilise back into the atmosphere where it would
be available for abiotic breakdown.
Priority Existing Chemical Number 21
24
Direct photolysis
IPCS (1993) reports that direct photolysis of benzene in the troposphere is unlikely
since the UV-visible spectrum of benzene shows no appreciable absorbance at
wavelengths >260 nm. According to GDCh (1988), direct photolysis is of minor
importance for the same reason.
IUCLID provides test results for a smog chamber experiment in which light with a
wavelength >290 nm corresponding to tropospheric sunlight was used with
benzene at a concentration of 100 ppm (0.32 mg/L). Although the validity of this
test cannot be judged due to insufficient documentation, the outcome showed no
evidence of benzene degradation. After the addition of chemicals producing active
species, benzene half-lives were between 4-5 h. Using light with a higher intensity
(wavelengths >230 nm), a half-life of 6.5 h was detected. These findings indicate
that direct photolysis is minimal at environmentally significant wavelengths, stated
by Howard et al. (1991) to be >290 nm, and thus confirm that this process will not
be a major removal process for benzene in the troposphere.
Indirect photolysis
IUCLID provides details for several studies on indirect photolysis. The results of
those where a half-life was determined are presented in Table 8.1. In all tests,
hydroxyl radicals were used as the reactant, with air as the medium. Not all studies
had temperature reported. However, where available, it was 25ºC. While the
validity of these studies is uncertain, it is well accepted that indirect photolysis
through reaction with hydroxyl radicals is the major degradation pathway for
benzene in air (GDCh, 1988; Government of Canada, 1993; IPCS, 1993).
Table 8.1: Half-life of benzene in the atmosphere where degraded by hydroxyl
radicals
Hydroxyl concentration Rate constant
3 3
Light source (radicals/cm ) (cm /(molecule.s) Half-life (days)
5 -12
Sun light 5 x 10 1.2 x 10 13.4
5 -12
Other* 5 x 10 1.2 x 10 13.4
6 -12
1.3 x 10 19
Sun light 7.5 x 10
6 -12
Sun light 1.1 x 10 1.3 x 10 5.6
6 -12
1.2-1.6 x 10 5.3
Sun light 1.2 x 10
* Hydroxyl radicals produced by flash photolysis and using a resonance fluorescence method.
The Dutch Environment Ministry calculated a half-life of benzene in the
atmosphere of 5.3 days assuming an average hydroxyl radical concentration of 1.25
x 106 molecules/cm3 over the Netherlands with a rate constant of 1.3 x 10-12
cm3/(molecule.s). This is reported in both IPCS (1993) and GDCh (1988), although
neither report describes the basis for the assumed hydroxyl radical concentration.
The global 24-h average hydroxyl radical concentration has been reported to be
around 5 x 105 molecules/cm3 (Calamari, 1993; GDCh, 1988). Additionally, using
the OECD Environment Monograph No. 61 (OECD, 1993), a rate constant for
benzene can be calculated at 2 x 10-12 cm3/(molecule.s) (contrary to the range of
0.8-1.4 x 10-12 cm3/(molecule.s) quoted in GDCh (1988)). Applying the global
average hydroxyl radical concentration and rate constant from the OECD
monograph and following the methodology in this monograph, gives an estimated
half-life of 8 days. This is more in agreement with the Canadian authorities where a
half-life attributable to reactions with hydroxyl radicals was calculated to be 9 days
Benzene 25
under typical urban atmospheric conditions, although the hydroxyl radical
concentration and rate constant were not reported (Government of Canada, 1993).
The global concentration used above applies to the average for the whole
troposphere. In the lower troposphere where benzene and hydroxyl radicals occur
at higher concentrations, the benzene half-life would be expected to be lower, and
is reported as 3-10 days (GDCh, 1988). Additionally, in districts with high traffic
density, where there is a higher concentration of hydroxyl radicals because of
higher concentrations of precursors, a lower atmospheric half-life can be expected,
and again 3-10 days is reported (GDCh, 1988).
For the purposes of this assessment, an atmospheric half-life of 8 days will be used
based on the globally accepted tropospheric average for the concentration of
hydroxyl radicals and the methodology and rate constant prescribed in the OECD
monograph.
These results given above are all within the range predicted in Howard et al. (1991)
where the photooxidation half-life in air has been calculated to fall between 50.1 h
(2.09 days) and 501 h (20.9 days).
The proposed degradation pathway through reaction with hydroxyl radicals is
shown in Figure 8.1 (Verscheuren, 1996).
Figure 8.1: Proposed degradation pathway of benzene in the atmosphere
OH
HO2
+ HO
O
O2
NO HO2
O
O
O
glyoxal
NO2
O2
+
OH
Endoperoxide O
O
butenedial
In the IUCLID database one study is described where ozone was used as a reactant.
In this test, air was the medium and the light source was chemiluminescence. A
sensitiser concentration of 3 x 1012 molecules/cm3 and a rate constant of 1 x 10-22
cm3/(molecule.s) were used. In an urban atmosphere, the half-life for the reaction
of benzene with ozone was calculated to be 105 years. In a rural atmosphere, the
half-life would be 327 years, using an atmospheric concentration for ozone of 9.6 x
1011 molecules/cm3. Therefore, photolysis through reaction with ozone is not
expected to be a major removal process for benzene in the atmosphere.
One test is described where atomic oxygen was used as the reactant at a
concentration of 7.2 x 104 molecules/cm3 with a rate constant of 2.8 x 10-14
cm3/(molecule.s). The half-life for this reaction between benzene and atomic
Priority Existing Chemical Number 21
26
oxygen was calculated to be 10.9 years, indicating that this reaction will not be a
major removal process of benzene from the atmosphere.
One test describes the photodegradation using sulphur dioxide as the reactant. The
test was performed in air with sunlight as the light source (light spectrum >290
nm). Benzene was present at a concentration of 100 ppm (0.32 mg/L). No further
information is available on the method, so the validity of this test is unknown.
However, sulphur dioxide was present at a concentration of 10-110 ppm (0.026-
0.288 mg/L). Photodegradation was observed. IUCLID states that approximately 2
days was required for 50% degradation to CO2, although the half-life for
photodegradation of benzene is stated as 6 h.
8.2.2 Aquatic fate
Photolysis in water
Direct photolysis of benzene in aqueous solution was investigated with half-lives
observed varying from 9-673 days. However, the authors concluded that the test
method was not suitable for poorly soluble, volatile substances (GDCh, 1988).
Two direct photolysis studies are reported in IUCLID where benzene was tested
adsorbed on silica gel. In both tests the concentration of benzene was 0.32 mg/L
and the light source was not stated. Few details are reported and the validity of the
tests is unknown. However, one was irradiated at >230 nm (not environmentally
significant), and resulted in a half-life of 6.5 h. The other was irradiated at
tropospheric wavelengths (>290 nm) and showed that 5% had photomineralised to
CO2 after 17 h.
Hydrolysis
IUCLID provides two reports for abiotic degradation of benzene, both concluding
that hydrolysis is not expected to be a significant process for removing benzene.
Few details are available for these tests and validity cannot be assumed. However,
degradation by this route is not expected, as benzene has no hydrolysable groups.
Volatilisation
Volatility from water to air is summarised in several reports in IUCLID.
Based on a reported Henry's Law constant of 0.0053 atm.m3/mol and a model river
1 m deep flowing at 1 m/s with a wind velocity of 3 m/s, the half-life of benzene
was 2.7 h at 20°C.
The half-life for the evaporation of benzene from seawater was investigated in a
mesocosm containing planktonic and microbial communities. Half lives for
summer, spring and winter were reported as 3.1, 23 and 13 days respectively.
The half-life for evaporation of benzene from a 1 m thick still water column was
4.8 and 5 h at 25 and 10°C respectively by thermodynamic calculations. The
residence half-time for well-mixed water was 37 min. This half-life of 4.8 h is also
included in the IPCS (1993) and Government of Canada (1993) reports.
An experiment in a wind-wave tank 6 m long, 0.61 m deep and 0.6 m wide with
wind velocities of around 6-13 m/s at a temperature of 20.7°C is described. The
testing period was >50 h so that an approximate 10-fold change of solute
concentration (which was measured by gas chromatography) would occur. The
Benzene 27
mass transfer coefficients of benzene at the water-air interface were 11.4-34 cm/h
dependent on wind velocity. The volatilisation is of first order kinetics. For a wind
speed of 7.09 m/s, a half-life of 5.2 h can be calculated.
While the reliability of these results is unclear, they support that rapid volatilisation
from water will occur.
8.2.3 Terrestrial fate
Adsorption
Documentation on the adsorption of benzene to soil is limited as the exposure of
the terrestrial compartment is likely to be low.
The Government of Canada (1993) report cites Koc values for benzene ranging
from 12-213, indicating the chemical to be moderately to highly mobile in soil.
IUCLID reports a calculated soil absorption coefficient of 71, using equations
developed by Kenaga & Goring and published by the American Society for Testing
and Materials. While this reference has not been obtained, an experimental soil
absorption coefficient value of 83 is reported in IUCLID as well as in IPCS (1993).
IPCS (1993) cites a rounded log Koc range of 1.8-1.9 (Koc = 60-83), indicating fair
mobility in soil, and states that benzene is not expected to adsorb to bottom
sediments based on its Koc, solubility and volatility.
Koc values provided in IUCLID indicate that benzene may exhibit high mobility in
soils and may migrate to groundwater. Several tests are reported and are generally
described as valid, or valid with restrictions. They can be used to provide a guide as
to the adsorptive behaviour of benzene.
One report gave the results of adsorption tests using radioactive labelled test
substance on aquifer material. This test is described as valid with IUCLID noting
that the test procedure was in accordance with generally accepted scientific
standards and described in sufficient detail. The results provided log Koc values
between 2.09 and 3.01 (Koc 123-1023). Experiments were carried out in capped
glass centrifuge tubes on two American groundwater aquifer materials with the
following characteristics:
Material: Sand (%): Silt Clay Organic matter pH:
(%): (%): (%):
A 90 8.0 2.0 4.4 3.8
B 70.4 24.0 5.6 2.2 5.5
Both these materials were acidic, with material A being quite acidic. It cannot be
concluded from the IUCLID summary whether Koc was a function of pH, although
this is not expected to be the case since benzene is a neutral molecule. These results
suggest that benzene is moderately mobile.
A water-soil adsorption coefficient of 18.2 provided in IUCLID was measured in
soil-solution mixtures which were equilibrated for 24 h at 20°C in capped
centrifuge tubes. Losses by volatilisation were avoided by sampling through the
septum of the caps. The substance amounts were corrected by the airspace of the
tubes under consideration of air volume and Henry's Law constant. Soil
characteristics were reported as 9% sand; 68% silt; 21% clay and 1.9% organic
matter. pH was not stated.
Priority Existing Chemical Number 21
28
While IUCLID also provides some calculated results, these are not reported here as
measured values are considered more reliable and the terrestrial compartment is not
expected to be a significant sink for benzene.
Volatilisation
The primary mechanisms responsible for loss of benzene from soil are
volatilisation to the atmosphere and runoff to surface water. Benzene released
below the soil surface may leach to groundwater (Government of Canada, 1993).
The volatility of benzene from soil to air is summarised in two reports in IUCLID.
In one report, the half-lives of volatilisation, without water evaporation, of benzene
uniformly distributed at a rate of 1 kg/ha to 1 and 10 cm in soil with an organic
carbon content of 1.25% were 7.2 and 38.4 days respectively. The second report is
from a model developed to predict the environmental fate of benzene following
leakage of gasoline from an underground storage tank. It estimated that some 67%
of the benzene would volatilise from the soil within 17 months, with 29% leaching
to groundwater and the remainder associating with the soil.
8.2.4 Biodegradation
IPCS (1993) provides the following insight into the biodegradation of benzene:
In surface and ground water benzene is biodegradable by microorganisms
·
under both aerobic and anaerobic conditions with the mechanism of
biodegradation seeming to involve the formation of catechol via cis-1,2-
dihydroxy-1,2-dihydrobenzene followed by ring cleavage.
One study on the aerobic biodegradation of benzene in groundwater utilised a
·
mixed bacterial culture from groundwater and soil bacteria capable of using
gasoline as a sole carbon source. Under closed agitated conditions without
added nutrients the half-life appeared to be <48 h with benzene levels falling
from 480 to 218 µg/L in this time. When ammonium nitrate was added, the
reaction was much faster, with benzene levels decreasing to 35 µg/L in 20 h.
The biodegradation of benzene in ground and river waters appears to follow
·
first-order rate kinetics with reported half-lives of 28 and 16 days respectively.
IUCLID provides results from several biodegradation studies which generally agree
that a significant degree of biodegradation occurs under aerobic conditions. Some
tests classify the substance as readily biodegradable. However, many of the tests
are not ready biodegradation tests and the results do not indicate degradation that
would be fast enough for benzene to be classed as readily biodegradable. While
validity cannot be assumed, they may be used to provide guidance as to the
biodegradability of benzene. As such, for the purposes of this assessment, the
chemical will be considered at least inherently biodegradable.
The majority of tests summarised in IUCLID for anaerobic degradation indicate
degradation is very slow to non-existent. This is supported in the GDCh (1988)
report where it is stated that degradation of benzene has not yet been detected in
anaerobic conditions. However, IPCS (1993) describes a report where samples of
landfill leachate incubated under methogenic conditions in an anaerobic glove box
showed a 72% reduction in benzene concentrations after 40 weeks, although no
significant benzene biodegradation occurred during the first 20 weeks of
incubation. In another study using anaerobic digesting sludge under methano-
trophic conditions, benzene was undegraded after 11 weeks. It is also reported that
Benzene 29
no toxic effects of benzene on the anaerobic digestion of sewage sludges were
observed until levels of 50-200 mg/L had been reached.
8.2.5 Bioaccumulation
Benzene is not expected to bioconcentrate to any significant degree in aquatic or
terrestrial organisms given the reported values for log Po/w of 1.56-2.15 (GDCh,
1988; ICPS, 1993). IPCS (1993) also reports a bioconcentration factor (BCF) for
freshwater algae of 30, for water fleas of 153-225 depending on the concentration
of benzene in their food, and for goldfish of 4.3.
GDCh (1988) reports measured BCF values in Clupea harengus (herring) of 2-6 in
most organs, and 31 in the gall bladder. One study outlined in this document claims
no significant biological accumulation in algae or fish. For fish, the BCF was in the
range of 1-10 after 3 days.
These conclusions are largely supported by data available from USEPA (2000) and
IUCLID. Results are available for several species of fish including Anguilla
japonica (Japanese eel), Leuciscus idus melanotus (golden orfe), Morone saxatilis
(striped bass), Salmo gairdneri (rainbow trout) and Engraulis mordax (Northern
anchovy). BCF values were all under 100, with the exception of the Northern
anchovy. This species provided BCF values of 113-505, with an outlying result of
8450. There is not enough detail to determine whether these factors are for specific
organs or the whole organism, which would impact on the analysis.
Nonetheless, based on the scale provided in Mensink et al. (1995), benzene can be
classed as slightly to moderately concentrating in fish. No data are available on
depuration rates.
Benzene appears to be more concentrating in invertebrates with results generally
indicative of a moderately concentrating chemical. Several species of invertebrates
have test results reported by USEPA (2000). An 8-day static and 9-day flow
through test on Brachionus plicatilis (rotifer) showed BCF values of 100-1000
under static conditions and 10,000 under flow through conditions. The maximum
concentration tested was 900 µg/L, well within the limit of solubility. Three results
are available for Daphnia pulex with BCF values ranging from 153-225.
Several results in IUCLID were also reported by USEPA and have not been
duplicated here. IUCLID provides information on depuration from Daphnia pulex.
Daphnids were exposed to water dosed with 10 µg/L benzene, water containing
algae preloaded by incubation with 50 µg/L benzene, or both dosed water and
preloaded algae. The reported BCF values were 225 for exposure to just dosed
water, 203 for feeding on preloaded algae and 153 after incubation in dosed water
with preloaded algae. Where exposure was through dosed water only, clearance
was 88% after 72 h. Where daphnids were exposed to dosed water and preloaded
algae, 83% clearance was reported when moved to fresh water with unloaded algae,
although the time involved in this depuration was not stated.
Limited data are available for algae, but suggest bioaccumulation will be slight,
with the ECOTOX database (USEPA, 2000) reporting BCF values of 30 for the
green algae Chlorella fusca and Chlorella fusca vacuolata.
Priority Existing Chemical Number 21
30
8.3 Effects on organisms in the environment
Of the three assessments listed at the beginning of this section, only GDCh (1988)
has any detailed discussion of the effects of benzene on organisms in the
environment. As this publication is relatively old, the USEPA's ECOTOX database
was interrogated for more recent results (USEPA, 2000). In addition, IUCLID was
consulted where limited data were reported in the other two sources, but as this is
largely unvalidated, is used for guidance only. Not all available information is
reported here due to its volume. However, the range for each trophic level will be
given with an indication of where the majority of results fall.
8.3.1 Aquatic organisms
Fish
The majority of reported results come from tests performed under static conditions.
GDCh (1988) provides 96-h results for 7 freshwater species including Leuciscus
idus melanotus (golden orfe), Lepomis macrochirus (sun perch), Pimephales
promelas (fathead minnow), Lebistes reticulatus (guppy), Carassius auratus
(goldfish), Gambusia affinis (mosquito fish) and Ictalurus punctatus (channel
catfish), with LC50 values ranging from 15-430 mg/L. Most of these fall in the 10-
100 mg/L range, indicating slight toxicity to fish. This is largely supported by more
recent data from USEPA (2000) where a 48-h LC50 to Mugil curema (white mullet)
of 22 mg/L, 96-h LC50 to fathead minnow of 12.6-24.6 mg/L and 96-h LC50 to
Poecilia reticulata (guppy) of 28.6 are reported. These results are all indicative of
slight toxicity.
Flow through tests provide more sensitive results. All flow through results are for
rainbow trout. Two tests reported in GDCh (1988) give 96-h LC50 values of 5.3 and
9.2 mg/L, while one more recent result gives a 96-h LC50 of 5.9 mg/L (USEPA,
2000). These results are indicative of moderate toxicity.
GDCh (1988) reports a 96-h LC50 of 5.8 mg/L in the saltwater species Morone
saxatilis (striped bass), which is indicative of moderate toxicity. The test conditions
are not known.
As such, benzene can be considered moderately to slightly toxic to fish under acute
exposure.
Chronic and sub-chronic data for fish appear limited with only one study reported
in GDCh (1988). Following 14 days exposure of Lebistes reticulatus (guppy) under
static conditions, a LC50 of 63 mg/L was determined.
The Government of Canada (1993) report highlights an investigation into the
chronic toxicity of benzene to the early life stages of rainbow trout, leopard frog
and the Northeastern salamander. Eggs of each species were exposed continuously
to benzene from within 30 minutes of fertilisation (embryos) through to 4 days
post-hatch (larvae). This resulted in continuous exposures of 27 days for rainbow
trout, 9 days for leopard frog and 9.5 days for Northeastern salamander. The
corresponding LC50 values were 8.3, 3.7 and 5.2 mg/L respectively.
IUCLID provides results of tests in Pimephales promelas (fathead minnow),
Morone saxatilis (striped bass) and Clupea harengus (pacific herring) for 7-day,
28-day and 17-day exposure periods respectively. The striped bass was exposed
under flow through conditions. A no observed effect concentration (NOEC) of
10.2, 3.1 and 0.49-0.88 mg/L was reported for fathead minnow, striped bass and
Benzene 31
pacific herring respectively, although those for pacific herring were the highest
concentrations tested on these fish so no real conclusions can be drawn from the
results.
Overall, these results indicate that benzene is of very low toxicity to fish from
chronic exposure.
GDCh (1988) lists some toxic effects of benzene on developmental stages and
behaviour of fish. Pacific herring demonstrated a decrease of survival time of eggs
after 48-h exposure of sexually mature females at 0.7 mg/L. However, this study
was conducted in a polluted region so other chemicals may have been responsible.
Other tests on pacific herring showed unspecified developmental abnormalities at
31-40 mg/L. Also, 24-h exposure of embryos to sublethal concentrations (up to
1.85 mg/L) under static conditions showed an effect on metabolism. Significantly
less growth of the embryos, altered oxygen consumption and greater food intake in
larvae were reported.
Sublethal effects were reported in the coho salmon at 1.8 mg/L and an increase in
the respiratory rate of chinook salmon was found at 4.4 mg/L. This effect was also
observed at the same concentration in striped bass.
Invertebrates
Invertebrates appear to be the largest group tested. GDCh (1988) provides results
for four freshwater species, of which Daphnia magna, Daphnia pulex and Daphnia
cucullata all had 48-h EC50 values >100 mg/L. One freshwater invertebrate, Aedes
aegypti (mosquito larva) had a 24-h LC50 of 59 mg/L. Of the saltwater species
reported in GDCh (1988), benzene could be considered moderately toxic to four
species: Artemia salina (salt water shrimp), Crango franciscorum (bay shrimp),
Nitroca spinepes and Palaemonetes pugio (grass shrimp), with 24- to 96-h LC50
values ranging from 20-82 mg/L. Two salt water species, Cancer magister
(Dungeness crab) and Crassostrea gigas (oyster), had 96-h LC50 values >100 mg/L.
More recently published data from the ECOTOX database (USEPA, 2000) largely
confirm the moderate to slight toxicity of benzene to aquatic invertebrates outlined
above. Moderate toxicity is reported for Ceriodaphnia dubia (water flea; 24-h EC50
= 18.4 mg/L), Gammarus fossarum (scud; 96-h LC50 = 53 mg/L) and Corixa
punctata (water boatman; 48-h LC50 = 48 mg/L), with LC50 values >100 mg/L
reported for a further three species: Daphnia magna, Lymnaea stagnalis (great
pond snail) and Viviparus bengalensis (snail).
However, one crab species (Scylla serrata) was relatively sensitive to benzene,
with three 96-h LC50 results (mortality as the end point) of 3.7, 6.1 and 7.7 mg/L.
While the majority of results indicate benzene is only moderately to slightly toxic
to aquatic invertebrates, this species shows benzene may be considered toxic to
some aquatic invertebrates.
GDCh (1988) only describes one study where chronic effects were investigated in
Daphnia magna. In a lifetime and partial lifetime test, no toxic effect of benzene
was found at a concentration of 98 mg/L.
Only one chronic test is available in IUCLID where sufficient detail is presented.
This test on a crab species (Cancer magister) indicates slight toxicity to aquatic
invertebrates. Larval stages of the crab were continuously exposed after hatching in
a flowing water laboratory culture system at benzene levels of 0.17-0.18, 1.1-1.2
and 6.5-7.0 mg/L. Benzene had little effect on the duration of the larval stages and
Priority Existing Chemical Number 21
32
no effect on the size of surviving larvae. At the lowest concentration, there was no
effect on survival. At the other two concentrations, benzene led to an accelerated
mortality rate compared to untreated controls. After 10 days of exposure at the
highest concentration, most larvae died. At the middle concentration, most larvae
died before day 20 of exposure. Therefore, the 20-day NOEC was 0.17 mg/L.
Algae
Data covered in GDCh (1988) suggest benzene is only slightly to very slightly
toxic to algae. A 24-h EC50 on the green algae (Chlorella vulgaris) based on cell
division was >>100 mg/L.
Three 72-h EC50 values are reported for sea algae with cell division as the end
point. The green algae (Dunaliella tertiolecta), siliceous algae (Skeletonema
costratum) and yellow-green algae (Cricopshaera carterae) all had EC50 results
>100 mg/L. In a 3-day test in the dinoflagellata Amphidinium carterae, the EC50
with cell division as the end point was reported to be 50 mg/L, indicating slight
toxicity (GDCh, 1988).
More recent data from the ECOTOX database (USEPA, 2000) appear more
indicative of slight toxicity than the earlier studies reported in GDCh (1988). One
72-h EC50 in Selanastrum capricornutum of 29 mg/L and a 24-h EC50 for the
diatom Thalassiosira pseudonana of 40 mg/L are reported.
In summary, benzene can be classed as slightly to very slightly toxic to algae under
acute exposure.
In 8-day tests, >1400 mg/L benzene had no detectable effect on biomass in the
freshwater species Scenedesmus quadricauda and the blue alga Microcystis
aeruginosa (GDCh, 1988). With growth as the end point, the more sensitive
species Selenastrum capricornutum provided an 8-day EC50 of 41 mg/L, although a
14-day EC50 of 292 mg/L is also reported for this species (USEPA, 2000).
Predicted No-Effect Concentration (PNEC) in the aquatic environment
As there are results available for both acute and chronic exposure in three trophic
levels, the lowest NOEC for chronic exposure, in this case to the aquatic
invertebrate crab species Cancer magister, will be used with an assessment factor
of 10. While this result (NOEC = 0.17 mg/L) is based on unvalidated results from
IUCLID, it is considered that there are sufficient data available from published and
peer-reviewed sources for this test to be accepted for use in a worst-case PNEC and
that there is sufficient detail in the IUCLID report to support the results.
Therefore, the PNEC for the aquatic environment is 0.17/10 = 0.017 mg/L, or 17
µg/L.
8.3.2 Terrestrial organisms
A study in Eisenia fetida (earthworm) is reported in the ECOTOX database
(USEPA, 2000), in which an LC50 of 98 µg/cm2 was determined in adult worms
weighing 300-500 mg placed for 48 h on filter paper impregnated with a solution of
benzene in water, acetone and trichloromethane.
According to GDCh (1988), use of benzene as a solvent for plant protection agents
in bioassay tests showed that it is slightly toxic to various insect species. The LD50
for the house fly (Musca domestica) was 0.8 mg per animal. Exposure to benzene
Benzene 33
in the vapour phase exhibited toxic action in the grain weevil (Calandra granaria),
although the concentration is not reported. Benzene acted as a repellent to the
adults of certain species of flies (Diptera).
In plants, air concentrations >50 mg/m3 (>15.5 ppm) have a lethal effect. However,
all plant species investigated recovered from sublethal effects. In water, higher
concentrations of 0.9-1.3 g/L have a growth-inhibiting effect (GDCh, 1988).
It is difficult to translate the earthworm measurement to an application rate likely to
lead to adverse impacts in soil and a PNEC cannot be determined from these data.
8.4 Summary
Benzene is expected to partition predominantly to the atmosphere, with the primary
route of degradation coming from indirect photolysis through reaction with
hydroxyl radicals. Direct photolysis or reactions with oxygen or ozone are not
expected to be major removal processes from the atmosphere. Based on the
accepted global concentration of hydroxyl radicals, the degradation half-life of
benzene from the atmosphere is calculated at 8 days.
Benzene is largely abiotically stable in water, with the major removal process
expected to be volatilisation. The high water solubility and relatively low log Po/w
indicate that benzene will not adsorb significantly to organic matter and sediments.
When released to the terrestrial compartment, benzene may be relatively mobile
and may leach to groundwater if released underground, for example, from leaking
storage tanks. The chemical is unlikely to adsorb readily to soils and may readily
volatilise from soil surfaces.
Benzene may be considered biodegradable under aerobic conditions, although
under anaerobic conditions, biodegradation may be expected to be very slow.
Based on the chemical's low log Pow and experimental results, bioaccumulation is
not expected to any significant degree, and at worst, benzene can be described as
moderately concentrating.
Aquatic organisms exhibit only a low level of sensitivity to benzene, with the
chemical being slightly toxic to fish following acute exposure under static
conditions and moderately toxic under flow through conditions. Chronic exposure
of fish to benzene gave results indicative of slight toxicity. Invertebrates appear to
be the largest group of aquatic organisms tested. For the majority of species tested,
benzene was only slightly to very slightly toxic. However, one crab species was
relatively sensitive, with results in the range of a moderately toxic chemical.
Chronic results show benzene to be slightly toxic to aquatic invertebrates. Benzene
may also be classified as slightly to very slightly toxic to algae. The PNEC for the
aquatic environment is 17 µg/L.
Limited data on the toxicity of benzene to terrestrial organisms show the chemical
to be slightly toxic to various insect species and the earthworm. In plants, high
concentrations in air have a lethal effect, although all plants investigated recovered
from sublethal effects.
Priority Existing Chemical Number 21
34
9. Kinetics and Metabolism
The toxicokinetics and metabolism of benzene have been extensively investigated
in several animal species and, to a lesser extent, in humans. Studies relevant to the
toxicokinetics of benzene have been reviewed and summarised in this section. The
metabolites and their modes of action are further discussed in Section 12. A
number of reviews of benzene toxicokinetics and metabolism are available,
including IPCS (1993) and ATSDR (1997).
9.1 Absorption
9.1.1 Animal studies
Inhalation
Schrenk et al. (1941) found the absorption of benzene vapour by dogs after
inhalation exposure to be rapid. Inhalation of the vapour (800 ppm) over 4-7 h
resulted in the concentration of benzene in arterial blood approaching equilibrium
conditions by 30 minutes. Although considerable inter-animal variation was noted,
a linear relationship was demonstrated between the concentration of benzene in air
over the range from 200-1300 ppm and the equilibrium concentration in blood. In
another study, the absorbed dose after inhalation (nose-only) of [14C]-benzene
(approximately 10-1000 ppm) for 6 h by rats (F344) and mice (B6C3F1) was found
to be non-linear. The percentage of benzene absorbed decreased from 33% to 15%
in rats and from 50% to 10% in mice as the exposure concentration increased from
10 to 1000 ppm. Due to apparent physiological differences in respiration between
the two species, mice inhaled approximately twice the amount of benzene
compared to rats (Sabourin et al, 1987). Similarly, Eutermoser et al. (1986) found
that the absorption rate of benzene vapour (300 ppm) by male rats (Sprague-
Dawley) decreased with increasing duration of exposure. When determined after 1,
3 and 6 h of continuous exposure and compared to baseline values, benzene
absorption decreased to 33, 22 and 9% respectively. Male mice (Swiss) when
exposed to benzene (310 ppm) for 1, 3 and 6 h of continuous exposure and
compared to baseline values gave values of 65, 76 and 81% respectively. Thus after
the first hour, the rate of benzene uptake by rats decreased significantly compared
to mice.
Dermal
Dermal absorption of liquid benzene was investigated by Maibach & Anjo (1981)
using intact and abraded skin of rhesus monkeys. Under conditions where
evaporative losses were allowed, the application of a single dose of benzene to
intact skin resulted in absorption of 0.17% of the dose while the application of
multiple doses (11 exposures with an interval of 15 min) resulted in 0.85% of the
dose being absorbed. In contrast, abraded skin resulted in 0.91% of the applied
dose being absorbed. Similar results were obtained by Franz (1984) who found that
after a single dermal application of benzene, 0.14% and 0.09% was absorbed by
rhesus monkeys and miniature pigs respectively. It was concluded from the in vitro
studies that the major factor influencing percutaneous absorption of benzene was its
contact time with the skin. Susten et al. (1985) reported similar findings with
Benzene 35
hairless mice (HRS/J) using a skin chamber where less than 1% of the applied dose
was absorbed.
Adsorption of benzene onto soil matrices (sandy or clay soil) was found to modify
the dermal absorption of [14C]-benzene when applied topically to male rats
(Sprague-Dawley) over a 72-h period using a glass skin chamber. While the peak
plasma level of radioactivity after exposure to benzene adsorbed onto sandy soil
was comparable to that obtained for pure benzene, a statistically significant lower
plasma level was obtained for benzene adsorbed onto clay, however, neither soil
type altered the time to reach peak plasma levels which was 12 h (Skowronski et al,
1988).
Dermal absorption of benzene vapour has also been addressed. Dermal absorption
(whole-body) of benzene vapour over 2, 4 or 6 h was investigated by the use of
male nude mice attached to respirators. The dermal absorption rates for exposures
of 200, 1000 and 3000 ppm were 4.11, 24.2 and 75.5 nmol/cm2/h (0.3, 1.9 and 5.9
µg/cm2/h) respectively, demonstrating a linear relationship between the two
parameters. Dermal absorption was also found to be linear with respect to exposure
time. The dermal absorption coefficient for the mouse was determined to be 0.619
cm/h (Tsuruta, 1989). McDougal et al. (1990) exposed male rats (F344; whole-
body) to benzene vapour (40,000 ppm) for periods up to 4 h. The rats were shaved
of all fur prior to exposure and provided with latex face masks attached to a fresh
air supply during exposure. Benzene blood levels at 0.5 h were 8 µg/mL and rose to
11 µg/mL at 4 h indicating that benzene is rapidly absorbed by the dermal route.
The dermal absorption rate was determined to be 0.0191 mg/cm2/h and the dermal
absorption coefficient 0.152 cm/h.
Oral
Following the administration by gavage of [14C]-benzene (340-500 mg/kg) to
rabbits, approximately 80% of the ingested radiolabel was recovered in exhaled
breath and urine indicating substantial gastrointestinal absorption at these dose
levels (Parke & Wiliams, 1953). Similar results were obtained by the
administration of lower doses of [14C]-benzene (0.5-150 mg/kg) to rats (F344 and
Sprague-Dawley) and mice (B6C3F1) where it was determined that >97% of the
dose was absorbed (Sabourin et al, 1987).
9.1.2 Human studies
Inhalation
In a study of 23 subjects exposed to benzene vapour (47-110 ppm) over 2-3 h,
maximal absorption (70-80% of dose) occurred within 5 min of initial exposure.
Subsequent absorption declined rapidly and reached a plateau at 15 min.
Absorption remained constant for the remainder of the exposure duration at
approximately 50% (range: 20-60%) of the exposure dose (Srbova, et al, 1950).
Comparable results have been obtained in a number of other studies. Nomiyama &
Nomiyama (1974a) exposed 6 volunteers (3 males and 3 females) to benzene
vapour (52 to 62 ppm) for 4 h and showed that after an initially high absorption rate
(50-60%), the rate decreased to reach a plateau of approximately 30-40% after 3 h
of exposure. The mean retention of benzene, after allowances for respiratory
excretion, was determined to be 30.2% for a 3-h exposure. Similarly, Snyder et al.
(1981) demonstrated that during continuous exposure to benzene vapour
approximately 50% of the dose is absorbed by the lungs. Pekari et al. (1992)
Priority Existing Chemical Number 21
36
exposed 3 non-smoking volunteers to benzene vapour (1.6-9.4 ppm) for 4 h. The
absorbed dose was estimated to be 52% and 48% for the low and high dose
exposure respectively based on the average difference in concentration between the
inhaled and exhaled air.
Further evidence for the absorption of benzene by inhalation is provided by studies
of cigarette smokers. Analysis of cigarette smoke has shown the presence of
substantial amounts of benzene, with the yield within the range of 0.4-104
µg/cigarette (see Section 16.1). Analysis of breath samples from 198 smokers and
322 non-smokers showed significantly higher (p <0.001) benzene concentrations in
the breath of smokers (16 µg/m3) compared to non-smokers (2.5 µg/m3). Benzene
breath levels were significantly correlated (p <0.01) with the number of cigarettes
smoked per day (Wallace & Pelizzari, 1986; Wallace et al, 1987). Pekari et al.
(1992) found 6 non-smokers to have venous blood benzene levels of <1-2 nM
compared to 3 smokers (1 pack/day) with 4-13 nM in the morning and 5-8 nM in
the afternoon. Cessation of smoking for a period (duration not stated) resulted in a
reduction of blood benzene levels to <2 nM. In a similar study, it was found that
the mean venous blood benzene levels of 14 smokers were significantly higher (7.0
nM; range 3.7-12.1 nM) compared to 13 non-smokers (2.8 nM; range 1.4-5.8 nM);
however, the number of cigarettes consumed were not stated (Hajimiragha et al,
1989). With the exception of cigarette smoking, there were no other known
activities undertaken by the subjects that may have resulted in benzene exposure
prior to or during either study.
Dermal
A number of studies indicate that benzene is absorbed via the dermal route in
humans. A study of 2 men exposed to benzene (approximately 0.06 g/cm2 applied
to the forearm, 35-43 cm2, under occluded conditions for 1.25-2 h) determined the
dermal absorption rate to be 0.4 mg/cm2/h based on urinary excretion of phenol
(Hanke et al, 1961). Approximately 0.05% of the applied dose of [14C]-benzene
(0.0026 mg/cm2) was absorbed when applied to the forearm skin of 4 volunteers.
Absorption was determined by urinary excretion of radiolabel which indicated that
absorption was rapid. Evaporative losses during the absorption period were not
accounted for (Franz, 1984).
The absorption of benzene due to dermal exposure to petrol has been studied in car
mechanics having direct skin contact with petrol for 30-150 min during work on car
fuel systems, with the concentration of benzene in the breathing zone ranging from
0.2 ppm (detection limit) to 3.7 ppm averaged over the duration of the task. Blood
benzene levels determined 2-9 h after exposure ranged from 3-16 nM. Based on
expected benzene blood levels derived from the airborne concentrations, it was
estimated that dermal absorption accounted for up to 80% of the total absorbed
dose of benzene. The mechanics did not wear protective gloves (Laitinen et al,
1994). However, the estimation assumed that the mechanics were exposed to non-
detectable benzene air concentrations during the remainder of the working day and
would therefore have overestimated the extent of skin absorption if this were not
the case.
In an in vitro study, benzene (pure benzene, air saturated with benzene vapour or a
saturated aqueous solution of benzene) was shown to diffuse across hydrated
stratum corneum prepared from human skin. Absorption, initially preceded by a lag
phase (range: <1-1.5 h), was linear over the duration of the experiment (4 h). The
rates of benzene absorption due to pure benzene and air saturated with benzene
Benzene 37
vapour were 2.1 and 1.0 µL/cm2/h (1.8 and 0.88 mg/cm2/h) respectively. It was
further demonstrated that the barrier characteristics of human skin alter in response
to the presence of other solvents (Blank & McAuliffe, 1985). Lodén (1986)
determined the amount of benzene absorbed by excised human skin to be 0.17
mg/cm2 after 0.5 h and 0.93 mg/cm2 at steady state (13.5 h). The total absorption of
benzene over 13.5 h in skin and receptor fluid was 1.92 mg/cm2 and the resorption
rate (that is, the amount of substance migrating to the receptor fluid below the skin)
was determined to be 99 µg/cm2/h.
Oral
No studies were identified addressing the absorption of benzene by the oral route in
humans. Cases of accidental or intentional ingestion indicate that benzene is readily
absorbed by the gastrointestinal tract, with a dose of 125 mg/kg proving fatal (see
Section 11.1).
9.2 Distribution
9.2.1 Animal studies
Inhalation
Schrenk et al. (1941) observed that in dogs continuously exposed to the vapour,
benzene preferentially partitions to the organs and tissues with a higher fat content,
although considerable inter-animal variation was noted. The establishment of an
equilibrium between most tissues (except fat) and blood levels appeared to be rapid
(15.5 h). When exposed to various concentrations of benzene vapour (850-1320
ppm) for periods ranging from 0.65-5 days, benzene levels were highest in bone
marrow (57.6-64.1 mg/100 g tissue) followed by peritoneal fat (40.3-61.4 mg/100 g
tissue) and subcutaneous fat (39.9-48.6 mg/100 g tissue). All other tissues or
organs had substantially (generally 20-fold) lower levels of benzene. A comparable
distribution pattern was observed when dogs were exposed to 800 ppm benzene
vapour for 8 h/day for 38-272 days. Rickert et al. (1979) studied the distribution
and residence times of benzene and three major metabolites, phenol, hydroquinone
and catechol, in male rats (F344) exposed to benzene vapour (500 ppm) for up to 8
h. The steady-state benzene concentrations at 6 h were determined for the following
tissues: fat (164.4 µg/g), bone marrow (37.0 µg/g), kidney (25.3 µg/g), lung (15.1
µg/g), liver (9.9 µg/g), brain (6.5 µg/g) and spleen (4.9 µg/g), while blood
contained 11.5 µg/mL. The half-times for tissues to reach steady-state levels for
benzene were essentially the same for all tissues (0.9-2.0 h) as were the elimination
times (0.4-0.8 h), with the exception of fat which was 1.6 h. The concentrations of
phenol in blood and bone marrow were maximal within 2 h after cessation of
exposure and declined rapidly thereafter. Hydroquinone and catechol
concentrations were sustained for 9 h after exposure with higher concentrations
found in bone marrow.
Ghantous & Danielsson (1986) demonstrated the transplacental distribution of
benzene and its metabolites in mice following inhalation exposure to [14C]-
benzene. Benzene was detected in the placenta and the foetus immediately
following and for up to 1 h after exposure, as were benzene metabolites. The
metabolites did not reach the same tissue concentrations as in maternal tissues and
no metabolites were retained in the placenta or the foetus.
Priority Existing Chemical Number 21
38
Dermal
Susten et al. (1985) examined the distribution of radiolabel after dermal application
of undiluted [14C]-benzene and a 0.5% (v/v) solution in rubber solvent using a skin
chamber attached to male hairless mice (HRS/J) for 4 h. Approximately 5% and
8% of the benzene in the pure sample and rubber solvent respectively remained
associated with the site of application while approximately 23% and 22%
respectively was associated with the carcass. Skowronski et al. (1988) examined
the tissue distribution of radiolabel in male rats (Sprague-Dawley) 48 h following
the topical application of benzene (300 µL) using a glass skin chamber. The highest
levels of radiolabel (expressed as % of initial dose per g of tissue) were found in
the kidneys (0.026%), liver (0.013%) and treated skin (that is, below the site of
application; 0.011%). Untreated skin gave a value of 0.002%. Subcutaneous fat
from below the area of application gave 0.008% while subcutaneous fat from a
different site gave 0.005% as did bone marrow. All other tissues and organs
examined (including the brain) accounted for less than 0.04% of the initial dose.
Oral
Analysis of rabbit tissues and organs (1 animal) 2 days after dosing by gavage with
[14C]-benzene (500 mg/kg) showed the highest level of radioactivity to occur in
muscle (1.6%), fat (1.5%), liver (0.07%), stomach (0.05%), testes (0.02%) and
kidneys (0.015%). No radioactivity was detected in the brain, spinal cord or blood
(Parke & Wiliams, 1953). Low et al. (1989) found that the distribution of radiolabel
in female rats (Sprague-Dawley) varied with the dose of [14C]-benzene
administered. At 1 h after a single dose of 0.15 or 1.5 mg/kg by gavage, radiolabel
was highest in the liver and kidneys (0.198-2.043 µg/g tissue), intermediate in
blood (0.086-0.769 µg/mL), and lowest in the Zymbal gland, nasal cavity tissue,
oral cavity tissue, mammary gland and bone marrow (0.034-0.547 µg/g tissue). In
contrast, at 15 mg/kg, the amount of radiolabel found in the mammary gland and
bone marrow had substantially increased in comparison to other tissues. At the
highest dose, bone marrow and adipose tissue had the highest concentrations of
benzene.
9.2.2 Human studies
Studies of the distribution of benzene in humans are generally limited to a number
of fatal cases of accidental or deliberate benzene exposure. Autopsy data from such
cases indicate that benzene preferentially partitions into lipid-rich tissues.
Analysis of tissue and fluid samples from a youth who died after sniffing benzene
(reagent grade) showed the following order for tissue benzene content: brain, 39
mg/kg; abdominal fat, 22.3 mg/kg; blood, 0.02 mg/mL; kidneys, 19 mg/kg; liver,
16 mg/kg; bile, 0.011 mg/mL; stomach, 10 mg/kg and urine, 0.0006 mg/mL
(Winek & Collom, 1971). Similar findings were demonstrated at autopsy of 3 cases
of acute industrial benzene poisoning indicating that benzene preferentially
distributes to lipid-rich tissues such as body fat (range: 68->120 mg/kg) and brain
tissue (range: 58-63 mg/kg) with lesser amounts in blood (range: 30-129 mg/mL),
liver (range: 15-38 mg/kg), lungs (positive findings) and bile (range: trace to 45
mg/mL) (Avis & Hutton, 1993). In a similar industrial accident involving acute
fatal benzene poisoning analysis of tissue and fluid samples revealed the following
benzene concentrations: blood, 0.0317 mg/mL; brain, 178.7 mg/kg; lungs, 22.2
mg/kg; heart, 182.6 mg/kg; liver, 378.6 mg/kg; kidneys, 75.2 mg/kg and urine,
0.0023 mg/ml (Barbera et al, 1998). In the above case reports the individuals are
Benzene 39
believed to have inhaled benzene vapour for some time before death occurred. In
one case in which an individual died suddenly during an industrial accident
involving benzene, precluding extensive inhalation of the vapour, autopsy findings
revealed the following benzene concentrations: blood, 0.0038 mg/mL; brain, 13.8
mg/kg; liver, 2.6 mg/kg (Tauber, 1970).
Limited data indicate that developing foetuses and infants may be exposed to
benzene as a result of maternal exposure. Benzene can cross the placenta and
concentrations in umbilical cord blood have been shown to be equal to or greater
than in maternal blood (Dowty et al, 1976). Due to the richly perfused nature of
breast tissue and the high fat content of human milk (approximately 4%), benzene
is expected to partition from blood into human milk from which it can transfer to
nursing infants (Fisher et al, 1997). Qualitative analysis of 12 human milk samples
revealed the presence of benzene in 8 of them (Pellizzari et al, 1982).
9.3 Metabolism
The metabolism of benzene has been extensively investigated in several species of
animals including humans. Benzene toxicity has been attributed to the formation of
reactive metabolites that appear to exert their toxic effect in combination, with no
one metabolite accounting for all of the observed effects. The metabolism of
benzene has been reviewed by Ross (1996) and Snyder & Hedli (1996).
9.3.1 General metabolic pathways
Urinalysis of several species exposed to benzene has demonstrated qualitative
similarities in the spectrum of metabolites produced, indicating that the metabolism
of benzene follows similar pathways between species. Urinary benzene metabolites
identified from rabbits, rats, mice, monkeys and humans include conjugates of
phenol, hydroquinone, catechol and 1,2,4-trihydroxybenzene while phenyl-
mercapturic acid and trans,trans-muconic acid have also been identified. The
conjugates are principally glucuronides and sulfates (Parke & Williams, 1953;
Rothman et al, 1998; Sabourin et al, 1988, 1992). Analysis of rat and human blood
samples has further revealed the presence of benzene oxide and its S-
phenylcysteine adducts following benzene exposure (Bechtold et al, 1992a, 1992b;
Lovern et al, 1997). The general metabolic pathways for benzene metabolism are
provided in Figure 9.1. The initial step in the formation of toxic metabolites is the
conversion of benzene to the benzene oxide/oxepin which can be further
metabolised to phenolic compounds or cleaved to give trans,trans-muconaldehyde.
Detoxification pathways primarily involve conversion of benzene oxide to pre-
phenylmercapturic acid and phenylmercapturic acid while the phenolic compounds
form glucuronide and sulfate conjugates.
Priority Existing Chemical Number 21
40
Figure 9.1. The metabolism of benzene, with question marks indicating
suspected pathways for which definitive evidence is lacking (after Sabourin
et al. (1988) and Schlosser et al. (1993))
O
Benzene oxide
oxepin OH
HO
trans, trans-
Benzene O
O Muconic acid
?
Pre-phenylmercapturic acid O
OH ?
OH H
H
O
O trans, trans-
OH
S-N-Acetyl-Cys
Muconaldehyde
Benzene
Benzene
dihydrodiol
oxide
OH
OH
OH
S-N-Acetyl-Cys
Phenylmercapturic acid Phenol Catechol
OH
OH
HO OH
HO
1,2,4-Trihydroxybenzene
Hydroquinone
The primary site for benzene metabolism is the liver. It has been observed that
animals that have undergone partial hepatectomy metabolise less benzene and
exhibit reduced benzene-mediated toxicity compared to animals with intact livers
(Sammet et al, 1979). The initial biotransformation of benzene involves oxidation
by the action of a cytochrome-P450 (CYP) to produce the benzene oxide/oxepin
intermediate (Jerina et al, 1968). Studies of liver microsomal preparations from rats
and rabbits, using inhibitor and immunochemical techniques, have identified the
cytochrome as CYP2E1 (Johansson & Ingelman-Sundberg, 1988; Koop et al, 1989;
Nakajima et al, 1989). Similar studies with human liver microsomal preparations
have shown CYP2E1 to be the major cytochrome involved in the metabolism of
benzene by humans (Guengerich et al, 1991). Valentine et al. (1996) confirmed the
role of CYP2E1 in the in vivo metabolism of benzene using transgenic CYP2E1
knockout mice (cyp2e1-/-). Analysis of urine samples after exposure to [14C]-
benzene by nose-only inhalation showed reduced levels of urinary metabolites
compared to wild-type mice (cyp2e1+/+). The study further demonstrated that, while
oxidative metabolism of benzene occurs primarily through CYP2E1, other
cytochromes are involved.
Studies of rat liver microsomes have shown there to be high affinity (Km = 20 µM)
and low affinity (Km = 0.3 mM) binding sites for benzene (Johansson & Ingelman-
Sundberg, 1988). Nakajima et al. (1989), using monoclonal antibodies, identified
two distinct rat enzymes, a high affinity and a low affinity binding type involved in
benzene oxidation. Subsequent studies of rat microsomal P450 isozymes, CYP2E1,
CYP2C11/6, CYP1A1/2 and CYP2B1/2, by Nakajima et al. (1992) using
monoclonal antibodies showed that all four isozymes are involved in the initial
oxidation of benzene. However, while CYP2E1 has been characterised as a high
affinity enzyme with respect to benzene metabolism, CYP2B1/2 exhibits low
affinity but high capacity (Gut et al, 1996; Nakajima et al, 1989) and CYP2C11/6
Benzene 41
and CYP1A1/2 exhibit low affinity and low efficiency towards benzene (Nakajima
et al., 1992).
Several studies have demonstrated the inducible nature of CYP2E1 and subsequent
enhancement of benzene metabolism by phenobarbital, acetone or ethanol
treatment of rats (Johansson & Ingelman-Sundberg, 1988; Koop et al., 1989;
Nakajima et al., 1989). In addition, it has been demonstrated that benzene is able to
stimulate its own metabolism by inducing CYP2E1 activity (Arinç et al., 1991; Gut
et al., 1993). However, it has also been demonstrated in mice that repeated oral
exposure to benzene can diminish CYP2E1 activity (Daiker et al., 1996). One
postulated mechanism for reduced cytochrome activity, demonstrated in vitro,
requires inactivation of the cytochrome by quinones formed by oxidation of
hydroquinone, catechol and 1,2,4-trihydroxybenzene (Soucek et al., 1994).
The initial oxidation product of benzene, benzene oxide, has been estimated to have
a half-life, in vitro, of approximately 8 min in blood (Lindstrom et al., 1997). Thus
the oxide has sufficient stability to allow it to participate in a variety of reactions.
Minor reactions of benzene oxide include alkylation with DNA and RNA (Mueller
et al, 1987) and proteins (Bechtold et al, 1992a, 1992b) while epoxide hydrolase
converts it to benzene dihydrodiol (1,2-dihydroxycyclohexadiene).
The presence of benzene oxide in blood has been detected by the presence of S-
phenylcysteine adducts of haemoglobin and albumin (Bechtold et al, 1992a;
1992b). Haemoglobin adducts were detected in the blood of rats (F344) and mice
(B6C3F1) after inhalation exposure (600 ppm, 6 h/day, 5 days/week for 2 weeks) or
gavage (rats only; 0, 100, 1000 or 10,000 µmol/kg). Albumin adducts were also
detected in the plasma of rats exposed to benzene vapour (Bechtold & Henderson,
1993). While the haemoglobin adduct has been found to be relatively stable in the
rat (F344) with decay rates consistent with the life-span of erythrocytes
(approximately 60 days), albumin adducts were found to have a half-life of 0.4
days compared to unmodified albumin (half-life approximately 3 days) (Troester et
al, 2000). Lindstrom et al. (1998) estimated the half-life of benzene oxide in blood,
under in vitro conditions, to be approximately 6.6 min in mice (B6C3F1), 7.9 min
in rats (F344) and 7.2 min in humans. When benzene oxide (0-184 µM) was
incubated with mouse, rat or human blood for 3 h it was observed that haemoglobin
adduct formation was proportional to the oxide concentration. The order of
reactivity for the oxide with haemoglobin was rat >> mouse > human. Negligible
haemoglobin adduct formation was observed with human blood. All three species
formed albumin adducts with the order being rat human > mouse.
9.3.2 Formation of phenolic metabolites
Studies with microsomal preparations, which preclude conjugation (detoxification)
pathways, indicate that the major pathway for the further metabolism of benzene
oxide involves the spontaneous rearrangement to phenol (Jerina & Daly, 1974;
Jerina et al, 1968). It has been demonstrated, using liver microsomes and
reconstituted enzyme systems, that phenol can also arise by the direct oxidation of
benzene by hydroxyl radicals derived from the reduction of molecular oxygen by
cytochrome P450 activity (Johannson & Ingelman-Sundberg, 1983). However,
Gorsky & Coon (1985) observed that when benzene is present at concentrations
approaching the Km of CYP2E1 for benzene, hydroxyl radicals do not contribute
significantly to benzene oxidation. The further oxidation of phenol by cytochrome
P450 results in hydroquinone (Koop et al, 1989; Valentine et al, 1996) and catechol
while 1,2,4-trihydroxybenzene arises from the P450-mediated oxidation of either
Priority Existing Chemical Number 21
42
hydroquinone or catechol (Schlosser et al., 1993). In addition, catechol can be
produced from benzene dihydrodiol by the action of dihydrodiol dehydrogenase
(Bolcsak & Nerland, 1983).
The hydroquinone species derived from benzene, that is, hydroquinone, catechol
and 1,2,4-trihydroxybenzene, readily undergo autoxidation to their respective
semiquinone and quinone forms and the presence of peroxidases facilitate the
oxidation process (Schlosser et al., 1989; Smith et al., 1989). Quinones are
chemically reactive and capable of forming adducts with macromolecules. Further
discussion of the secondary metabolism of benzene-derived phenol and
hydroquinone species, along with their biological effects, is presented in Section
12.
The detoxification of the phenolic benzene metabolites occurs primarily by
conjugation to glutathione (GSH), glucuronide or sulfate (Parke & Williams, 1953).
Conjugation of benzene oxide with GSH by glutathione-S-transferase (GST) results
in the formation of pre-phenylmercapturic acid and, by dehydrogenation,
phenylmercapturic acid, both of which have been identified as urinary metabolites.
The major metabolites, phenol, hydroquinone, catechol and 1,2,4-
trihydroxybenzene, all form glucuronide and sulfate conjugates (Parke & Williams,
1953; Sabourin et al., 1988).
9.3.3 Formation of trans,trans-muconaldehyde
Cleavage of the oxidised aromatic ring results in the formation of trans,trans-
muconaldehyde which is subsequently converted to trans,trans-muconic acid prior
to excretion. A number of in vivo studies of several animal species, including
humans, have shown trans,trans-muconic acid to be an end-stage product of
benzene metabolism (Parke & Williams, 1953; Rothman et al., 1998; Sabourin et
al., 1988). While the opening of the benzene ring and subsequent formation of
muconic acid have been shown to occur in isolated perfused rat livers, as has the
conversion of trans,trans-muconaldehyde to trans,trans-muconic acid (Grotz et al.,
1994), the precise mechanism of ring opening remains elusive. It has been
proposed that benzene oxide, while in the oxepin state, undergoes secondary
oxidation by cytochrome P450 to produce trans,trans-muconaldehyde (Davies &
Whitham, 1977) and small quantities of trans,trans-muconaldehyde have been
found to be produced by mouse liver microsomal preparations on incubation with
benzene (Latriano et al, 1986). However, hydroxyl radicals have also been
implicated in the formation of trans,trans-muconaldehyde. Incubation of benzene
with Fenton's reagent, which produces reactive oxygen species, results in the
formation of cis,trans-muconaldehyde which, through a series of rearrangements,
yields the trans,trans-isomer (Zhang et al., 1995). An alternative proposed
mechanism for aldehyde formation requires cleavage of benzene dihydrodiol
(Latriano et al., 1986), however, the aldehyde was not produced when benzene
dihydrodiol was incubated with Fenton's reagent (Zhang et al., 1995). The
subsequent conversion of trans,trans-muconaldehyde to trans,trans-muconic acid
involves several steps requiring the action of an aldehyde dehydrogenase (Kirley et
al, 1989; Zhang et al, 1993). Conjugation of trans,trans-muconaldehyde with GSH
via hepatic GST has been demonstrated as a detoxification pathway for this
metabolite (Goon et al., 1993a, 1993b).
Benzene 43
9.4 Elimination and excretion
9.4.1 Animal data
Inhalation
Benzene was detected in the urine of dogs exposed to benzene vapour (850-1320
ppm) at levels ranging from 29.3-48.3 mg/100 g (Schrenk et al, 1941). Sabourin et
al. (1989) identified the urinary metabolites of benzene metabolism in rats and mice
following inhalation exposure to benzene vapour (nose-only) for 6 h. The results
are presented in Table 9.1.
Table 9.1: Major urinary metabolites after inhalation of benzene, expressed as
a percentage of total urinary metabolites from 24-h samples (adapted from
Sabourin et al. (1989))
Dose Phenol Hydroquinone Catechol Pre-phenyl- Muconic
Species (ppm) conjugates conjugates conjugates mercapturic acid acid
Rat (F344) 5 57 5.7 ND* 9.5 19
600 74 2.0 ND 17 4.0
5 37 33 ND 6.0 23
Mouse
(B6C3F1) 600 67 11 ND 15 5.0
* ND = not detected.
Dermal
Franz (1984) observed that peak excretion of radioactivity in the urine of rhesus
monkeys after the application of 0.5 mL of [14C]-benzene occurred during the first
2 h and decreased rapidly thereafter but remained detectable for up to 30 h.
Skowronski et al. (1988) found that after the topical application of 300 µL of [14C]-
benzene to male rats (Sprague-Dawley) by means of a glass skin chamber, the
major excretory route for radiolabel was in the urine, with substantially lesser
amounts in faeces and expired air. Excretion in the urine was greatest during the
12- to 24-h interval after application, accounting for 58.8% of the initial dose with
68.4% recovered in 24 h and 86.2% after 48 h. In contrast, 0.2% of the initial dose
was recovered over 48 h in the faeces. Expired air accounted for 12.0% in the first
24 h with the following 24 h accounting for only a further 0.8%.
Oral
Analysis of urinary benzene metabolites from several species following
administration of [3H]-benzene by gavage has shown similar profiles of
metabolites. As indicated in Table 9.2, the principal urinary metabolites for the
species shown are conjugates (glucuronides and sulfates) of phenol and, to a lesser
extent, hydroquinone. The mouse is quantitatively different from the other species
with a substantially higher production of hydroquinone conjugates and trans,trans-
muconic acid. In addition to species differences, the table shows the effect of
changes in dose levels on urinary metabolites for rats and mice. Increasing the dose
of benzene leads to an increase in the excretion of phenol conjugates and a decrease
in hydroquinone conjugates by the mouse but no substantial change in the rat. In
contrast, trans,trans-muconic acid excretion is diminished in both the rat and the
mouse at higher doses.
Priority Existing Chemical Number 21
44
Table 9.2: Major urinary metabolites after oral administration of benzene,
expressed as a percentage of total urinary metabolites from 24-h samples
(adapted from Parke & Williams (1953), and Sabourin et al. (1989, 1992))
Dose Phenol Hydroquinone Catechol Pre-phenyl- Muconic
Species (mg/kg) conjugates conjugates conjugates mercapturic acid acid
Rat (F344) 1 70 4.0 ND* 11 13
10 71 2.8 ND 15 10
200 75 2.0 ND 18 5.0
1 30 47 ND 2.7 20
Mouse
(B6C3F1) 10 38 39 1.0 4.5 16
200 63 16 ND 11 9.0
Rabbit 340 24 4.8 2.2 No data 1.3
* ND = not detected.
Within 2 days of administering [14C]-benzene (0.34-0.5 g/kg) to rabbits by gavage,
approximately 45% of the dose was detected in expired air (43% as unchanged
benzene and 1.5% as carbon dioxide) and approximately 35% appeared in the
urine. Urinary radiolabel was predominantly in the form of conjugated phenols,
with phenol comprising approximately 23% of the administered dose and with
hydroquinone, catechol and 1,2,4-benzenetriol making up 4.8%, 2.2% and 0.3%
respectively (as conjugates). Approximately 1.3% of the dose was recovered as
trans,trans-muconic acid and a further 0.5% as phenylmercapturic acid. No
diphenyl or its derivatives were detected in the urine. The residual radioactivity
(5% to 10%) was associated with the tissues and faeces (Parke & Williams, 1953).
The elimination of benzene by the metabolic route appears to be saturable. Oral
doses of [14C]-benzene 15 mg/kg resulted in the excretion in the urine over 48 h of
>89% of the administered radioactivity by rats (F344 or Sprague-Dawley). Doses
50 mg/kg bw resulted in a dose-dependent reduction in urinary excretion and a
corresponding dose-dependent increase in exhaled 14C, predominantly as the parent
molecule. At all doses, residual 14C in the carcass amounted to less than 8%.
Excretion in the faeces did not exceed 11% of the administered dose up to the
maximum dose of 300 mg/kg. Mice (B6C3F1) demonstrated similar elimination
characteristics to rats (Sabourin et al, 1987).
Other routes
Analysis of urine from male cynomolgus monkeys administered [14C]-benzene (5,
50 and 500 mg/kg) by intraperitoneal injection revealed that urinary excretion of
radiolabel diminished with increasing dose. At 5 mg/kg an average of 56% of the
administered dose was recovered in the urine compared to 13% at 500 mg/kg over
a 95-h period. In contrast, recovery of radiolabel from the urine of chimpanzees
administered a dose of benzene (1 mg/kg) by intravenous injection was complete
after 24 h with >90% of the radiolabel collected within the first 8 h. As shown in
Table 9.3, phenyl sulfate was found to be the major metabolite (45-74% of total
urinary metabolites) for all doses. Lesser amounts of hydroquinone glucuronide,
muconic acid, phenyl glucuronide, hydroquinone sulfate and catechol sulfate were
also present. No unconjugated metabolites were detected. The amount of excreted
hydroquinone sulfate and muconic acid decreased and phenyl glucuronide and
catechol glucuronide increased as the benzene dose increased. A similar urinary
profile of metabolites was obtained with female chimpanzees administered an
intravenous dose of [14C]-benzene (1 mg/kg), although the formation of catechol
conjugates was not detected (Sabourin et al, 1992).
Benzene 45
Table 9.3: Major urinary metabolites after intraperitoneal administration of
benzene, expressed as a percentage of total urinary metabolites from 24-h
samples (adapted from Sabourin et al. (1992))
Dose Phenol Hydroquinone Catechol Pre-phenyl- Muconic
Species (mg/kg) conjugates conjugates conjugates mercapturic acid acid
5 61 27 8.0 No data 4.4
Cynomolgus
monkey 50 73 15 6.0 No data 3.1
500 78 8.9 9.9 No data 1.3
Chimpanzee 1 75 8.0 ND* 0.5 5.5
* ND = not detected.
9.4.2 Human data
Inhalation
The elimination of benzene across the lungs of 10 subjects was studied. Subjects
inhaled benzene (47-84 ppm) for 2-3 h after which breath samples were taken over
a further 5-7 h. The results showed 16.4-41.6% of the absorbed benzene to be
exhaled with the greatest rate occurring during the first hour. Excretion in the urine
accounted for a maximum of 0.2% of the absorbed dose (Srbova et al, 1950). It
appears that urinary benzene metabolites were not measured by the protocol
employed. Comparable results were produced after a 4-h exposure to benzene
vapour (52-62 ppm) where 6 volunteers (3 males and 3 females) were shown to
exhale 16.3% (men) and 17.2% (females) of the inhaled benzene (Nomiyama &
Nomiyama, 1974a). The ratio of respiratory elimination of non-metabolised
benzene to retained benzene was determined to be 114.8% for males (considered by
the authors to be unreliable) and 39.8% for females (Nomiyama & Nomiyama,
1974b). Using 4 volunteers (male non-smokers) exposed to benzene vapour (mean
daily exposure 26.2-42.2 ppm) for 5 consecutive daily 6-h periods, Berlin et al.
(1980) showed the clearance of benzene across the lungs to be biphasic with a half-
time of 2.6 h for the rapid phase and 24 h for the slow phase. At higher benzene
concentrations (99 ppm for 1 h), Sherwood (1988) identified one individual with an
initial rapid phase and 2-3 slower phases while urinary excretion displayed a
biphasic pattern.
Ghittori et al. (1993) found a linear correlation between benzene in the breathing
zone and unmetabolised benzene in the urine of workers. Subsequently, Ghittori et
al. (1995) identified a linear relationship between benzene in the breathing zone of
workers and urinary levels of trans,trans-muconic acid and phenylmercapturic
acid.
Dermal and oral
Peak excretion of radioactivity in the urine of 4 human volunteers after the
application of 0.4 ml of [14C]-benzene to the ventral forearm occurred rapidly
within the first 2 h and decreased rapidly thereafter but remained detectable for up
to 30 h (Franz, 1984).
No studies were identified that address the elimination of benzene or its metabolites
from humans after exposure by the oral route.
Priority Existing Chemical Number 21
46
9.5 Comparative kinetics and metabolism
As discussed above, CYP2E1 is a high affinity, low capacity enzyme.
Consequently, the pathway for the hepatic metabolism of benzene becomes
saturated at relatively low doses. Henderson et al. (1989) found a single oral dose
of benzene of 50 mg/kg or greater, when administered to rats or mice, resulted in
saturation of the metabolic pathway with consequent loss of unmetabolised
benzene by exhalation. Rats and mice administered an oral dose of 150 mg/kg
exhaled approximately 50% and 85% respectively as unmetabolised benzene. This
loss of benzene by exhalation becomes a limiting factor in the maximal tissue
concentrations of metabolites that can be achieved following oral dosing. However,
higher tissue concentrations of benzene metabolites can be achieved by the
inhalation route. It has been observed that mice accumulate substantially more
benzene metabolites than rats during inhalation exposure. This appears to be due to
physiologic and metabolic differences between the two species. Mice have a higher
respiratory minute volume per kg body weight compared to rats allowing for the
blood benzene level to achieve equilibrium faster than in the rat (Sabourin et al,
1989, 1990). Mice also have a higher metabolic rate, based on increased oxygen
consumption (approximately 1.8 times greater than the rat), resulting in faster
removal of benzene from the circulation. This allows for higher levels of
metabolites to accumulate within body tissues compared to the rat. Doses of
benzene that lead to metabolic saturation also produce changes in the metabolic
profile of benzene metabolites (Sabourin et al, 1989; Daiker et al, 1996).
9.5.1 Oral studies
The content of water-soluble benzene metabolites in bone marrow has been
examined following oral administration of benzene to male rats and mice. Table 9.4
shows that phenol conjugates accounted for the major proportion of metabolites in
the rat and remained relatively constant over the dose range as did the mercapturic
acid derivatives. The trans,trans-muconic acid content diminished with increasing
doses of benzene and hydroquinone conjugates remained at relatively low levels. In
contrast, the mouse produced comparable bone marrow levels of phenol and
hydroquinone conjugates at the lowest dose with the amount of hydroquinone
conjugates decreasing as the benzene dose increased. Both phenyl mercapturic acid
and trans,trans-muconic acid increased with the dose of benzene (Sabourin et al,
1989).
Table 9.4: Major bone marrow metabolites after oral administration of
benzene, expressed as a percentage of total water-soluble metabolites from
pooled samples (adapted from Sabourin et al. (1989))
Pre-phenyl- Phenyl-
Hydroquinone mercapturic mercapturic Muconic
Dose Phenol
acid
Species (mg/kg) conjugates conjugates acid acid
Rat (F344) 1 75 ND* 15 ND 10
10 74 3.0 15 4.0 4.0
200 84 2.3 13 ND 0.2
1 50 50 ND ND ND
Mouse
(B6C3F1) 10 52 35 ND 13 ND
200 56 9.0 ND 27 8.0
* ND = not detected.
Benzene 47
9.5.2 Inhalation studies
Animal data
The profile of metabolites produced by male rats (F344) and mice (B6C3F1)
exposed (nose-only) to benzene vapour for 6 h at 5 ppm or 600 ppm is presented in
Table 9.1. Phenol conjugates account for the major proportion of metabolites
produced at either concentration by both species. At 5 ppm, hydroquinone
conjugates and trans,trans-muconic acid are present in higher amounts than at 600
ppm while pre-phenylmercapturic acid increases with the administered dose
(Sabourin et al, 1989).
Tissue and blood levels of non-conjugated benzene metabolites were determined in
male rats (F344) and mice (B6C3F1) after inhalation of benzene vapour (50 ppm)
for 6 h. While phenol, hydroquinone and catechol could not be detected in the liver,
lung or blood of the rat, detectable levels of phenol and hydroquinone were found
in the mouse with catechol detected only in the liver. The data for male rats and
mice are presented in Table 9.5.
Table 9.5: Major non-conjugated benzene metabolites (nmol/g tissue) in rat
and mouse tissues (adapted from Sabourin et al. (1988))
F344 rats B6C3F1 mice
Metabolite Liver Lung Blood Liver Lung Blood
Phenol ND* ND ND 0.3 0.6 1.3
Hydroquinone ND ND ND 2.1 1.2 4.3
Catechol ND ND ND 0.3 ND ND
* ND = not detected.
In contrast, conjugated derivatives of phenol, hydroquinone and catechol were
detected in substantially greater amounts in the tissues and blood of both species
(Table 9.6).
Table 9.6: Major conjugated benzene metabolites (nmol/g tissue) in rat and
mouse tissues (adapted from Sabourin et al. (1988))
F344 rats B6C3F1 mice
Metabolite Liver Lung Blood Liver Lung Blood
Phenylglucoronide ND* ND ND 6.3 1.2 ND
Cathecholglucoronide 1.0 ND ND 0.8 0.5 ND
Hydroquinoneglucoronide 0.4 ND ND 26 15 12
Phenylsulfate 1.5 15 20 28 36 36
Hydroquinone monosulfate ND ND ND 2.8 0.6 ND
Pre-phenylmercapturic acid 6.9 0.9 ND 44 2.1 2.3
Phenylmercapturic acid ND ND ND ND ND ND
Muconic acid 8.4 1.9 0.7 228 18 1.0
* ND = not detected.
As shown in the table, the level of trans,trans-muconic acid in the mouse liver was
very much greater than observed in lung tissue or blood for either species (Sabourin
et al, 1988). Henderson et al. (1989) observed that the mouse metabolised more of
an inhaled dose of benzene than the rat under comparable conditions and that a
greater proportion was converted to the putative toxic metabolites. It was found that
Priority Existing Chemical Number 21
48
detoxification (conjugation) pathways were low-affinity, high-capacity whereas
toxic metabolite formation appeared to be high-affinity, low capacity.
A short-term benzene inhalation study (exposure for 6 h/day for 6 days) with male
Swiss mice showed that at 199 ppm or less, the major metabolite in blood was
phenylsulfate while above 199 ppm a dose-dependent increase in phenyl-
glucuronide occurred. At all benzene concentrations, the blood phenol level
increased in a dose-dependent manner (Wells & Nerland, 1991).
Human data
Studies of humans are generally limited to analysis of urine or blood samples of
workers occupationally exposed to benzene.
Bechtold and Henderson (1993) conducted analyses on the urine and blood of non-
smoking female workers exposed to benzene vapour. Five women exposed to
approximately 4.4 ppm for 8 h showed the presence of elevated urinary levels of
trans,trans-muconic acid (6.2 µg/mg creatinine) compared to 8 females with no
known exposure (0.27 µg/mg creatinine). Blood samples from 10 women exposed
to benzene vapour (0-23.1 ppm) showed a linear relationship between benzene
exposure levels and albumin-S-phenylcysteine adducts, however, no haemoglobin-
S-phenylcysteine adducts were detected.
9.5.3 Dermal studies
Comparative studies of the metabolism of benzene after dermal absorption were not
identified.
9.5.4 In vitro studies
The metabolism of low levels of benzene by microsomes prepared from 10 human
liver samples was investigated. When [14C]-benzene (3.4 µM) was incubated with
microsomal preparations, the major metabolites were phenol and hydroquinone
accounting for up to 48% of the recovered radiolabel while minor metabolites were
catechol and 1,2,4-trihydroxybenzene which accounted for <2% and 0.2%
respectively. A further metabolite, tentatively identified as 2,2'-biphenol,
accounted for approximately 4% of radiolabel. The CYP2E1 activities of the
individual liver samples were found to vary 13-fold as determined by a standard
hydroxylation assay with activities ranging between 0.253 to 3.266 nmol/mg/min.
When benzene was used as the substrate, a 6-fold difference between liver samples
was noted that ranged from 10% to 59%. Comparison of individual liver samples
over a 16-min incubation period showed that phenol was the major metabolite
formed with the exception of two samples where hydroquinone predominated.
These latter two samples had higher CYP2E1 activities and the sample with the
highest activity produced equal quantities of phenol and hydroquinone. The rate of
benzene metabolism by each of the 10 liver samples correlated with their CYP2E1
activity (Seaton et al, 1994). In a subsequent report by Seaton et al. (1995)
addressing the in vitro sulfonation of phenol and glucuronidation of hydroquinone
by human liver cytosolic and microsomal preparations from 10 donors, there was a
3-fold difference in the rates of conjugation for each reaction.
Benzene 49
9.6 Summary
Benzene is readily absorbed by the inhalation, oral and dermal routes in all animal
species tested. In humans, the absorption of benzene by the inhalation route is
maximal within minutes of exposure and subsequently declines to a constant level.
Dermal absorption is generally low compared to inhalation due to volatilisation,
with less than 1% of an applied dose being absorbed unless skin exposure is
prolonged. The variation in benzene absorption between individuals following
inhalation is high. Partitioning of benzene is expected to occur into lipid-rich
tissues due to the lipophilic nature of benzene. Several studies have confirmed that
benzene accumulates in the adipose tissue, bone marrow and brain of animals and
humans.
The metabolism of benzene is qualitatively similar between various animal species,
including humans, and proceeds predominantly by hepatic CYP2E1-mediated
oxidation of the aromatic ring to yield benzene oxide/oxepin. Subsequent pathways
for metabolism of the oxide/oxepin include spontaneous rearrangement to phenol
or ring cleavage to give trans,trans-muconaldehyde. Phenol can be further oxidized
to polyphenols (hydroquinone, catechol and 1,2,4-trihydroxybenzene).
Detoxification pathways involve conjugation of benzene oxide or trans,trans-
muconaldehyde with GSH while the phenolic metabolites are conjugated to either
glucuronate or sulfate. The metabolism of benzene is rapid with water-soluble
metabolites appearing in the urine within 2 h of exposure. The major urinary
metabolites from several species are conjugates of phenol followed by lesser and
variable amounts of hydroquinone conjugates and of pre-phenylmercapturic acid
and trans,trans-muconic acid. Conjugates of catechol have been detected in small
amounts in the urine of mice, rabbits and primates.
Due to the limited capacity of hepatic CYP2E1 to metabolise benzene, a substantial
proportion of absorbed benzene is eliminated unchanged in exhaled air, with the
remainder being eliminated via the urine, principally as metabolites. Urinary
excretion appears to be biphasic with a fast phase followed by a prolonged phase,
suggesting the slow removal of benzene from adipose tissue. Due to the readily
saturable nature of benzene metabolism, exposure at higher doses results in greater
elimination of unmetabolised benzene via exhalation.
While comparative studies of urinary benzene metabolites have shown common
pathways for benzene metabolism to exist between various species, physiological
as well as metabolic differences contribute to some of the observed differences.
The easily saturated nature of benzene metabolic pathways and greater respiratory
minute volume of the mouse allow the mouse to expire more of an oral dose of
benzene compared to the rat. Similarly, respiratory differences and the greater
metabolic rate of the mouse allow tissue levels of benzene metabolites to reach
higher levels compared to the rat.
Priority Existing Chemical Number 21
50
10. Effects on Laboratory
Mammals and Other Test
Systems
The aim of this section is to describe the toxic effects and corresponding effect
levels of benzene in animals. In the case of end points studied extensively in
humans (mainly haematological and genetic toxicity), the assessment is based on
recent reviews by ATSDR (1997) and USEPA (USEPA 1998a, 1998c). For other
adverse effects, individual animal studies have been reviewed for this assessment.
These include all carcinogenicity studies as well as investigations of the toxic
effects of benzene on the central nervous system (CNS), immune function,
reproductive organs and foetus.
Most of the available studies do not comply with Good Laboratory Practices (GLP)
or international standards such as the OECD Test Guidelines. In consequence, all
available publications with a relevant end point have been included in the review
irrespective of their compliance with formal quality criteria. However, studies
providing insufficient scientific detail to permit a critical appraisal of their findings
are clearly identified as such. Unless otherwise indicated, only effects that were
statistically different (p <0.05) from controls have been considered.
10.1 Acute toxicity
The acute toxicity of benzene in experimental animals is summarised in Table 10.1,
which includes the highest and the lowest values reported in the published
literature. Mortality is due to cardio-respiratory arrest from severe CNS depression
and/or cardiac arrhythmia (Nahum & Hoff, 1934).
Table 10.1: Acute toxicity of benzene
Route Species Measure* Results Sex Reference
Inhalation Mouse LC50(7 h) 9980 ppm Not specified Svirbely et al. (1943)
Rat LC50(4 h) 13,700 ppm Females Drew & Fouts (1974)
16,000 ppm Males Smyth et al. (1962)
ALD(4 h)
45,000 ppm Both sexes Carpenter et al. (1944)
Rabbit LC100(30 min)
Mouse LD50
Oral 4700 mg/kg Not specified RTECS (2000)
6500 mg/kg Males Spanò et al. (1989)
810 mg/kg Males Cornish & Ryan (1965)
Rat LD50
5600 mg/kg Males Wolf et al. (1956)
9900 mg/kg Males Smyth et al. (1962)
LD50 >8200 mg/kg Roudabush et al. (1965)
Dermal Male guinea pigs
Guinea
Male and female
pig, rabbit
rabbits
SC Mouse ALD 3500 mg/kg Males Watanabe & Yoshida (1970)
* ALD = approximate lethal dose; LC50 = median lethal concentration; LC100 = concentration leading to
100% mortality; LD50 = median lethal dose; SC = subcutaneous.
Benzene 51
10.2 Irritation and corrosivity
Several rabbit tests for skin and eye irritation have been reported. From 10-20 daily
applications of undiluted benzene to the skin caused redness, oedema, skin peeling
and blistering (Wolf et al, 1956). The chemical was also reported to cause skin
irritation in a test according to OECD Test Guideline No. 404, however, further
details were not provided (Jacobs, 1992, as cited in OECD, 2000). One or two
drops of undiluted benzene applied to the eye produced moderate irritation of the
conjunctiva and very slight, transient corneal injury (Wolf et al, 1956). Smyth et al.
(1962) reported similar skin and eye lesions rated as grade 3 on a 10-point scale.
Rats exposed to benzene vapours for 6 h/day, 5 days/week for 10 weeks exhibited
lacrimation during the first 3 weeks of exposure to levels 10 ppm (Shell, 1980, as
cited in ATSDR, 1997).
10.3 Sensitisation
There are no studies of skin or respiratory sensitisation to benzene in animals.
10.4 Repeated dose toxicity (other than carcinogenicity)
10.4.1 Short-term exposure
The toxic effects of benzene have been investigated in numerous short-term studies
in mice and rats. In these studies, benzene was administered orally in vegetable oil
or drinking water for 2 days to 24 weeks, or by whole-body exposure to vapours,
usually for 6 h/day, 5 days/week. The dose levels tested ranged from 1-600
mg/kg/day by mouth and from 0.44-6600 ppm by inhalation. Repeated dose dermal
studies could not be identified.
Neurotoxicity
Evans et al. (1981) exposed male CD-1 and C57BL mice to inhalation of 0, 300 or
900 ppm benzene for 6 h/day. After the 5th exposure, the mice were observed and
scored for 7 behavioural categories at 30 and 75 min post-exposure. In both strains,
there were increases in the frequency of eating and grooming, and a decrease in
sleeping and resting. These stimulatory effects were more pronounced at 75 than at
30 min post-exposure and in the 300 than in the 900 ppm exposure groups,
indicating an association with brain concentrations below a certain level.
Immediately following single and repeated 6-h exposures of male mice to 100, 300,
1000 or 3000 ppm benzene, there were increased milk licking at 100 ppm and a
reduction in hind limb grip strength at 1000 ppm, but no effects on locomotor
activity (Dempster et al, 1984). In the absence of motor disturbances, hind limb
grip strength is a test for unconditional reflexes. Milk licking, however, may be
influenced by hunger and mucosal membrane irritation.
In groups of 10 adult male mice exposed to 0, 0.78, 3.13 or 12.52 ppm benzene for
2 h/day, 6 days/week for 30 days, tests for behaviour (time taken to run to a safety
area in a Y-maze following an electric shock) and forelimb grip strength showed
stimulation at 0.78 ppm and depression at 12.52 ppm, but no effect on locomotor
activity (Li et al, 1992). Compared to unexposed controls, the average change in
the frequency of rapid shock responders was +30% at 0.78 ppm and 24% at 12.5
ppm. For grip strength, it was +77% and 11% respectively. As the concentration
Priority Existing Chemical Number 21
52
of benzene in the air was not checked after the first three days of the experiment
and there were extraordinary changes in bone marrow morphology on day 30,
actual benzene exposure may have been higher than reported (USEPA, 1998c). On
the other hand, the changes in behaviour were observed already on the first 1-2
days of exposure when air level monitoring did take place.
Tegeris & Balster (1994) evaluated the acute behavioural effects in mice of a 20-
min inhalation exposure to 2000, 4000 and 8000 ppm benzene and five derivatives
(toluene, ethyl benzene, propyl benzene, m-xylene and cumene). All six chemicals
produced a nearly identical profile of CNS depressant effects that paralleled those
of the anaesthetic drug pentobarbital, except that they were short-lived, with
recovery beginning within minutes of cessation of exposure.
In mice, administration for 4 weeks of 8-180 mg/kg/day benzene in drinking water
had no behavioural effects, but induced a dose-related increase in the level of
noradrenaline, dopamine, serotonin and their metabolites in a number of brain
regions (Hsieh et al, 1988b). There was also a dose-dependent stimulation of
hypothalamic-pituitary-adrenocortical activity (Hsieh et al, 1991). Changes in brain
noradrenaline, dopamine, serotonin and/or their metabolites were also found in rats
2 h after a single oral dose of 950 mg/kg benzene or following inhalation of 1500
ppm benzene, 6 h per day for 3 days (Andersson et al, 1983; Kanada et al, 1994).
The most consistent finding in these studies was an increase in noradrenaline and
dopamine levels in the hypothalamus and other subcortical brain regions. In a rat
inhalation study, benzene also induced noradrenaline release from post-ganglionic
sympathetic nerves in the ovaries and uterus (Ungváry & Donáth, 1984).
A 30-day drinking water study found a reduction in brain weight in mice at 350 but
not at 195 mg/kg/day (Shell 1992, as cited in ATSDR, 1997). No studies were
identified that specifically looked for benzene-induced morphological changes in
nervous organs or tissues.
Immunotoxicity
When administered to mice by inhalation or in drinking water, benzene suppressed
a number of lymphocyte (LC) functions. These included T- and B-LC response to
mitogens; interleukin-2 production in T-helper LC; the activity of cytotoxic,
alloreactive and suppressive T-LC; B-LC antibody production; and T-LC and
macrophage resistance to intracellular infection with Listeria monocytogenes (Fan,
1992, as cited in USEPA, 1998c; Hsieh et al, 1988a; Irons et al, 1983; Rosenthal &
Snyder, 1985, 1987; Rozen et al, 1984; Rozen & Snyder, 1985; Stoner et al, 1981,
as cited in IPCS, 1993; White et al, 1984, as cited in USEPA, 1998c). Based on the
above effects, the lowest observed adverse effect level (LOAEL) was 10 ppm by
inhalation (Rozen et al, 1984) and 12 mg/kg/day by the oral route (White et al,
1984, as cited in USEPA, 1998c). A no observed adverse effect level (NOAEL)
was not achieved, although Hsieh et al. (1988a) found a bimodal response with a
reduction in LC proliferation at 40 mg/kg/day and an increase at 8 mg/kg/day, the
lowest dose tested. However, Daiker et al. (2000) recently found no changes in
spleen LC cellularity, subtype profile or function in mice exposed to inhalation of
0.44 ppm benzene for 7 h/day, 5 days/week for 6 weeks.
In these studies in mice, immunosuppression generally occurred at exposure levels
which were also associated with reduced absolute lymphocyte counts (ALC).
However, in one 30-day study, mitogen-stimulated LC proliferation was decreased
at 12 mg/kg/day (the lowest dose tested) in the absence of any other blood or bone
marrow toxicity (White et al, 1982, as cited in USEPA, 1998c). There is no
Benzene 53
evidence of specificity with respect to antigen or immune response type. Overall,
these findings indicate that benzene-induced immunosuppression is the outcome of
a general impairment of the ability of LC to respond to antigenic stimuli by rapid
clonal expansion, with little, if any, interference with antigen recognition.
In wild cotton rats given three consecutive intraperitoneal injections of benzene at a
dosage of 0, 100, 300, 600 or 1000 mg/kg/day and a battery of tests for cellular and
humoral immune functions on days 1-9 after the last treatment, there was no
evidence of immunosuppression in any of the treatment groups (McMurry et al,
1991).
In a subacute inhalation study in male Sprague-Dawley rats exposed to 0, 30, 200
or 400 ppm benzene for 6 h/day, 5 days/week for 2-4 weeks, Robinson et al. (1997)
determined a NOAEL/LOAEL of 200/400 ppm based on spleen weight, cellularity,
total T-LC, T-helper LC, antigen-stimulated and unstimulated B-LC content, and
thymus weight. As such, rats appear to be less sensitive than mice to the
immunotoxic effects of benzene.
Effects on blood and blood forming organs
ATSDR (1997) and USEPA (1998c) have reviewed a large number of published
and unpublished study reports that address the effects of short-term exposure to
benzene on the blood and blood forming organs of mice and rats. Based on these
reviews, the overall findings can be summarised as follows:
In peripheral blood, there was a decrease in the quantity of some or all of the
·
formed elements, including red blood cells (RBC), white blood cells (WBC),
LC and blood platelets (Plt). In some studies, there was also a reduction in
haemoglobin (Hb) levels and the average RBC size (mean corpuscular volume
(MCV)) was either increased or decreased.
There was bone marrow hypoplasia, with decreased numbers of multipotential
·
stem cells and cells that differentiate into RBC, WBC and macrophages, but an
increase in immature RBC such a micronucleated polychromatic and
normochromatic RBC.
In the spleen, there was a decrease in the number of all LC types, but an
·
increase in haematopoiesis in general3.
There was a decrease in the weight of the thymus, which is the main site of T-
·
LC proliferation.
The order of susceptibility to these effects was male mice > female mice > rats. In
terms of target organ, it was spleen peripheral blood bone marrow > thymus. In
mice, the most sensitive indicators of benzene toxicity were spleen weight, bone
marrow cellularity, and WBC and RBC counts, one or more of which were affected
at concentrations around 10 ppm and above for inhalation and at 8 mg/kg/day and
above for oral exposure. In rats, the most sensitive end points were ALC and WBC
counts, which were affected at 100 ppm by inhalation and 25 mg/kg/day by oral
administration.
3
Haematopoiesis is the sum of processes involved in the production and development of the blood
cells. The haematopoietic processes are usually confined to the bone marrow, but may also take place
in the spleen and liver, for example, in foetuses and newborn animals, or when there is a substantial
increase in the demand for blood cells.
Priority Existing Chemical Number 21
54
Two 2-16 week studies in the mouse showed that all haematological abnormalities
returned to normal or near-normal within 4-25 weeks post-exposure (Cronkite et al,
1985; Snyder et al, 1988).
Other effects
There are no consistent reports of respiratory, cardiovascular, gastro-intestinal,
hepatic or renal effects of short-term exposure to benzene by any route (ATSDR,
1997). Effects on reproductive organs are reviewed in Section 10.5. Mortality was
generally low and only a few studies reported decreases in body weight (BW) gain.
Other experimental animals
There is limited evidence that airborne exposure to benzene for 3-4 weeks induces
leukopoenia in guinea pigs and lymphocytopoenia, leukopoenia and impaired
cellular immunity in pigs at levels 88-100 ppm (Dow, 1982, as cited in ATSDR,
1997; Wolf et al, 1956).
10.4.2 Long-term exposure
Blood and blood forming organs
ATSDR (1997) and USEPA (1998c) have reviewed more than a dozen published
reports which describe the results of nine separate studies of the long-term effects
of benzene exposure on blood and blood forming organs. In these studies, mice or
rats were exposed to benzene for 26 weeks by oral administration in vegetable oil
or by whole-body inhalation for 5-6 h/day, 4-5 days/week, at dose levels ranging
from 1-500 mg/kg/day by mouth and from 88-300 ppm by inhalation.
The most consistent long-term effect on the blood was a reduction in RBC, WBC
and LC counts. In some studies, the number of neutrophilic granulocytes and
reticulocytes (young RBC) was increased. The bone marrow and the spleen showed
hypoplasia in some studies and an increase in haematopoietic tissue in others.
Haematological effects were recorded at the lowest dose level examined in all long-
term tests, except for one poorly reported rat study in which the NOAEL was 1
mg/kg/day by mouth (Wolf et al, 1956). This study also recorded a lower LOAEL
than any of the other available studies, namely 88 ppm by inhalation based on
WBC count and spleen weight and 10 mg/kg/day by oral administration based on
WBC count. In more adequately reported studies, the LOAEL was 100 ppm by
inhalation and 25 mg/kg/day by mouth in both mice and rats (ATSDR, 1997;
USEPA, 1998c).
Other effects
There are no consistent reports of non-neoplastic cardiovascular, liver or kidney
abnormalities from long-term exposure to benzene by any route (ATSDR, 1997).
There was chronic irritation of the forestomach epithelium in male rats and mice
and hyperplasia of the Harderian gland4 and pulmonary alveolar epithelium in male
and female mice in 2-year, but not in 17-week oral gavage studies (NTP, 19865).
Lesions occurred at 200 mg/kg/day in rats and at dose levels 25 mg/kg/day in
4
A tear gland in the median angle of the eye which is rudimentary in humans.
5
The major findings in the studies conducted by the National Toxicology Program (NTP) have been
published by Huff et al. (1989).
Benzene 55
mice. Reproductive effects are described below. The median survival time and BW
gain were generally reduced in a dose-dependent manner.
Other experimental animals
There is limited evidence that airborne exposure to benzene for 35-38 weeks
induces leukopoenia and increased spleen weight in guinea pigs and leukopoenia in
rabbits at dose levels 80-88 ppm (Wolf et al, 1956).
10.5 Reproductive toxicity
10.5.1 Effects on fertility and lactation
Data on fertility and lactation are available from three repeated-dose toxicity tests,
three one-generation fertility studies and a limited number of other studies.
Repeated-dose toxicity and one-generation fertility studies
In a 13-week inhalation study, Ward et al. (1985) exposed groups of 150 mice and
50 rats per sex to inhalation of 0, 1, 10, 30 or 300 ppm benzene for 6 h/day, 5
days/week. There was clear evidence of haematological toxicity at the highest dose
level in both species and sexes, but no consistent exposure-related trends in
mortality, clinical observations or mean BW data. The testes and ovaries from 20
mice/sex/group and from 10 rats/sex exposed to either 0 or 300 ppm benzene were
examined microscopically. In mice from the highest exposure group (300 ppm),
there were 4 animals with cystic ovaries, 7 with bilateral testicular atrophy or
degeneration, 6 with decreases in the number of spermatozoa in the epididymal
ducts and 9 with an increase in abnormal sperm forms. Similar lesions of doubtful
biological significance were seen in both sexes at lower dose levels. No
abnormalities were found in the gonads of rats exposed to 300 ppm benzene.
A study in mice administered benzene at 25, 50 or 100 mg/kg/day by gavage for 2
years found the following number of animals with epithelial hyperplasia or
follicular atrophy of the ovaries (NTP, 1986):
Ovarian lesions: 0 mg/kg/day 25 mg/kg/day 50 mg/kg/day 100 mg/kg/day
No. of mice examined 47 44 49 48
Epithelial hyperplasia 12 39 31 29
Senile atrophy 15 35 32 22
The statistical significance of these findings is not reported. However, when
analysed for this assessment, the increase in the incidence of epithelial hyperplasia
was significant at all dose levels, whereas the incidence of senile atrophy was
significantly elevated at 25 and 50, but not at 100 mg/kg/day (p <0.05; test for
exact confidence limits). Testes were not examined microscopically, as they had no
grossly visible lesions. A parallel study in rats found no macroscopic abnormalities
in the gonads, even at the highest dose level tested (100 mg/kg/day in females, 200
mg/kg/day in males) (NTP, 1986).
In an early inhalation study, Wolf et al. (1956) observed sedation, growth
depression, mortality and a moderate increase in testis weight in male rats exposed
to 6600 ppm benzene for 13 weeks. The testes were normal in rats exposed to 88 or
2200 ppm for 30 weeks. At these dose levels there were mild lesions of the blood
and lymphatic system, but no mortality. In guinea pigs and rabbits exposed to 80-
88 ppm benzene for 35-38 weeks, there was no mortality, mild haematological
Priority Existing Chemical Number 21
56
changes and a slight increase in testis weight and in mild degenerative lesions of
the seminiferous tubules. Further details were not reported and it is unclear whether
these findings were statistically significant.
In female mice, a single intraperitoneal injection of benzene at the maximum
tolerated dose of 1250 mg/kg did not reduce the number of offspring and litters
produced in a reproductive capacity test involving the observation of 35 treated and
untreated breeding pairs for 347 days (Bishop et al, 1997).
In a one-generation fertility study, groups of 26 female rats were exposed to 0, 1,
10, 30 or 300 ppm for 6 h/day, 5 days/week for 10 weeks prior to mating and then
daily on days 0-20 of gestation and days 5-20 of lactation (Kuna et al, 1992). In the
dams, there was no exposure-related effect on BW gain, clinical observations or
necropsy findings and no fertility-related effects. When the neonates were
examined at weaning on day 21 postpartum, there was a dose-related, 6-9%
increase in relative kidney weight in female offspring of dams exposed at 10 ppm
and above. Female offspring of dams exposed to 300 ppm benzene also had a 10%
BW reduction and a 14% reduction in absolute liver weight.
In another fertility study, female rats were kept in inhalation chambers where they
were exposed for 24 h/day to 0, 0.3, 1.6, 6.3, 15, 18, 20 or 200 ppm benzene
(Gofmekler, 1968). Exposure began 10-15 days prior to mating, when males were
introduced into the chambers for 6-10 days, and continued throughout the entire
pregnancy period until spontaneous delivery. There were no pregnancies at the
highest dose level. In dams exposed to 0.3-20 ppm benzene, the average litter size
was 7.5 compared to 8.4 in the controls, but there was no exposure-related effect on
the birth weight of the pups.
Other studies
In early experimental studies, benzene caused degenerative changes in the testes
and severely hypoplastic ovaries, degenerated ovarian follicles and chromosomal
damage and mitotic interruption in the ova when administered by subcutaneous
injection or inhalation to male and female mice and female rabbits (Hett & Mark,
1938; Vara & Kinnunen, 1946). The dose levels used in mice were not given, but
were high enough to induce marked leukopoenia. Rabbits were administered 1000
mg/kg/day for 10 days.
In a study in adult mice, testicular germ cell suspensions were examined for DNA
content by flow cytometry at 1, 2, 3, 4 and 10 weeks after a single sublethal dose of
1-7 mL (880-6160 mg) benzene/kg administered by oral gavage (Spanò et al,
1989). These doses had no effect on body or testis weight, but resulted in a dose-
dependent reduction in the relative cell count in the primary spermatocyte and
spermatid fractions. The primary spermatocyte fraction was most affected at 2
weeks, the round spermatid fraction at 3 weeks and the elongated spermatid
fraction at 4 weeks post-treatment, as one would expect from a cytotoxic insult
resulting in a transient reduction in the number of differentiating spermatogonia.
Conclusions
Overall, the above studies indicate that benzene exposure may cause degenerative
changes in the gonads of mice, whereas there is insufficient evidence of similar
effects in other species. There was also epithelial hyperplasia in the ovaries of mice
in the NTP (1986) 2-year oral bioassay. However, this is likely to represent a
preneoplastic lesion as ovarian tumours occurred with a significant positive trend in
Benzene 57
this study and epithelial hyperplasia was found in other organs with neoplastic
lesions, namely the Harderian gland and the lungs.
Compound-related testicular atrophy or degeneration was observed in male mice
exposed to 300 ppm benzene by inhalation. Ovarian atrophy was observed in mice
at 25 mg/kg/day by mouth and cystic ovaries at 300 ppm by inhalation. In both
sexes, these lesions occurred at dose levels that were associated with
haematological effects, but not with mortality or other signs of generalised toxicity.
The available data on reproductive capacity are inconclusive.
The changes in body, liver and relative kidney weights observed by Kuna et al.
(1992) in 21-day old female neonates of rats exposed to inhalation of benzene
during pregnancy and lactation are modest, but nonetheless indicative of
developmental toxicity. Because of the study design it cannot be determined
whether these effects were lactational or the result of exposure in utero.
10.5.2 Developmental toxicity
Standard tests
Developmental toxicity tests have been conducted in mice, rats and rabbits exposed
to benzene by inhalation, mouth or subcutaneous injection during the gestation
period (Table 10.2). All foetuses were examined for external defects and in all but
two studies (Exxon Chemical Company, 1986, as cited in USEPA, 1998c;
Watanabe & Yoshida, 1970) for visceral and skeletal abnormalities as well.
Overall, there were no major structural abnormalities in the foetuses, except in one
study in the mouse in which a single SC injection of a maternally toxic dose of
2600 mg/kg benzene on GD 13 was associated with cleft palate and jaw
malformations (Watanabe & Yoshida, 1970). However, several inhalation and oral
studies conducted in mice or rats found evidence of other foetal effects at dose
levels where no toxic effects were recorded in the dams. These include a small (4-
6%), but statistically significant reduction in foetal BW (Coate et al, 1984; Murray
et al, 1979; Seidenberg et al, 1986) and a significant increase in the frequency of
minor skeletal abnormalities (Green et al, 1978; Murray et al, 1979). Moreover, in
two studies on which little experimental detail is available, there was an increase in
resorptions in rats (Litton Bionetics, 1977, as cited in USEPA, 1998c) and a
decrease in foetal BW in mice (Nawrot & Staples, 1979), in both cases in the
absence of any signs of maternal toxicity.
In rabbits, continuous inhalation of 310 ppm benzene was associated with
abortions, an increase in resorptions or foetal deaths, a decrease in foetal BW and
an increased incidence of minor abnormalities in the presence of maternal toxicity
(reduced BW gain), whereas 155 ppm had neither foetal nor maternal effects
(Ungváry & Tátrai, 1985).
Priority Existing Chemical Number 21
58
Table 10.2: Summary of developmental toxicity tests*
Species Study design Daily dose Foetal effects Maternal effects Reference
Benzene
None Decreased Hgb
Mouse SC injection on GD 8-9 or GD 1760 mg/kg Matsumoto et al.
12-13 (1975)
Decreased BW (3%) Decreased Hgb
8-11 pregnancies per group 3520 mg/kg
Decreased placenta weight (5%) in GD 12- Decreased WBC count
13 group
Delayed ossification in GD 12-13 group
Inhalation, 7 h/day, GD 6-15 500 ppm Decreased BW (6%) None Murray et al.
26-30 pregnancies/group Unspecified increase in `minor skeletal (1979)
variants'
Oral gavage 3 times daily, 800 mg/kg Decreased BW None Nawrot &
GD 6-15 Staples (1979)
1300 mg/kg Increase in resorptions Increase in mortality
No. of pregnancies not given
Decreased BW
2600 mg/kg Increase in resorptions Increase in mortality
Decreased BW
59
2600 mg/kg Increase in late resorptions Increase in mortality
Oral gavage 3 times Nawrot &
Decreased BW
daily,GD 12-15 Staples (1979)
No. of pregnancies not given
155 ppm No information
`Weight retardation' (25 vs. 7% of foetuses)
Inhalation, 24 h/day, GD 6- Ungváry & Tátrai
Delayed ossification (10 vs. 5% of foetuses)
15 (1985)
15 exposed pregnancies
115 control pregnancies 310 ppm No information
`Weight retardation' (27 vs. 7% of foetuses)
Delayed ossification (11 vs. 5% of foetuses)
1300 mg/kg None
Oral gavage, GD 8-12 Decreased BW (4%) Seidenberg et al.
28 pregnancies/group (1986)
2600 mg/kg Decrease in WBC count in all
Single SC injection on GD Watanabe &
Increased incidence of cleft palate and jaw
dams
11, 12, 13, 14 or 15 Yoshida (1970)
malformations in offspring of dams injected
No difference in fall in WBC
15 pregnancies per group on GD 13 compared to foetuses of dams
count or in BW gain between
No controls injected on GD 11-12 or 14-15
dams with or without malformed
External foetal examination
foetuses
only
Table 10.2: Continued
Species Study design Daily dose Foetal effects Maternal effects Reference
Rat Inhalation, 6 h/day, GD 6-15 10 ppm Litton Bionetics
Increase in resorptions None
(1977), as cited
26-31 pregnancies/group
in EPA (1998c)
40 ppm Increase in resorptions None
Green et al.
None
100 ppm Missing sternebrae (9/18 vs. 1/16 litters)
Inhalation, 7 h/day, GD 6-15
(1978)
14-18 pregnancies/group
None
300 ppm Delayed ossification of sternebrae (10 vs.
2% of female foetuses)
Decreased BW (10%) Decreased BW gain
2200 ppm
Decreased crown-rump length (5%) Lethargy
Delayed ossification of sternebrae (11 vs.
1% of female foetuses)
Missing sternebrae (11/15 vs. 2/14 litters)
Decreased BW (12%) Hudák &
Decreased BW gain (57%)
313 ppm
Inhalation, 24 h/day, GD 9-
Delayed ossification (11 vs. 0% of foetuses) Ungváry (1978)
14
Fused sternebrae and extra ribs (9 vs. 1% of
19 exposed pregnancies
60
foetuses)
28 controls
Decreased BW (5%) Tátrai et al.
Inhalation, 24 h/day, GD 7- Decreased BW gain (27%)
50 ppm
(1980)
14 Decreased placenta weight (9%)
17-20 pregnancies/
Resorbed or dead foetuses (42 vs. 6%) Mortality (3/20 vs. 0/48)
150 ppm
exposure group
Decreased BW (28%) Decreased BW gain (45%)
46 control pregnancies
Skeletal abnormalities (57 vs. 5% of Increased relative liver weight (9%)
foetuses) Decreased placenta weight (7%)
Resorbed or dead foetuses (32 vs. 6%) Mortality (1/22 vs. 0/48)
500 ppm
Decreased BW (20%) Decreased BW gain (55%)
Skeletal abnormalities (66 vs. 5% of Increased relative liver weight (14%)
foetuses) Decreased placenta weight (16%)
Resorbed or dead foetuses (29 vs. 6%) Mortality (3/22 vs. 0/48)
1000 ppm
Decreased BW (22%) Decreased BW gain (41%)
Priority Existing Chemical Number 21
Skeletal abnormalities (55 vs. 5% of Increased relative liver weight (10%)
foetuses) Decreased placenta weight (20%)
Table 10.2: Continued
Species Study design Daily dose Foetal effects Maternal effects Reference
Rat Inhalation, 7 h/day, GD 6-15 10 ppm Kuna & Kapp
None Increased BW gain (33%) on GD 15-
Benzene
14-15 pregnancies/ (1981)
20
exposure group
Decreased BW gain (34%) on GD 5-
50 ppm Decreased BW (14%)
11 control pregnancies
15
Increase in foetuses with skeletal and/or
visceral variations (18 vs. 3%)
Decreased BW gain (37%) on
500 ppm Decreased BW (18%)
GD 5-15
Decreased crown-rump length (7%)
Increased BW gain (40%) on GD 15-
Increase in foetuses with skeletal and/or
20
visceral variations (21 vs. 3%)
Coate et al.
None
Inhalation, 7 h/day, GD 6-15 1 ppm None
(1984)
32-38 pregnancies/group
None
2 control groups 10 ppm None
None
40 ppm None
None
100 ppm Decreased BW (6%)
61
Exxon Chemical
None
Oral gavage, GD 6-15 50 mg/kg None
Company
20-22 pregnancies/group
(1986), as cited
Decreased feed consumption
250 mg/kg
External foetal examination None
in EPA (1998c)
only
Decreased BW gain
500 mg/kg Decreased BW
Decreased feed consumption
Decreased BW gain
1000 mg/kg Decreased BW
Decreased feed consumption
Alopecia
500 ppm
Rabbit Murray et al.
None
Inhalation, 7 h/day, GD 6-18 Increased feed and water
(1979)
18-19 pregnancies/group consumption
Ungváry & Tátrai
Inhalation, 24 h/day, GD 7-20 155 ppm None None
(1985)
11-15 exposed pregnancies/
group 310 ppm Abortions (6/15 vs. 0/60 dams) Decreased BW gain (62%; not
Resorbed or dead foetuses (16 vs. 5%) corrected for the effect of
60 control pregnancies
Decreased BW (17%) abortions)
`Minor anomalies' (86 vs. 34% of foetuses) Increased relative liver weight (17%)
* BW = body weight; GD = gestation day; Hgb = haemoglobin; SC = subcutaneous; WBC = white blood cell.
Where available, information on the incidence or magnitude of effects compared to non-exposed controls is shown in brackets.
Other studies
According to Ungváry (1985), continuous inhalation of benzene (125 ppm),
benzene plus toluene, or benzene plus xylenes had foetotoxic effects in rats, but
only in the presence of maternal toxicity. Exposure to 830 ppm benzene over 48 h
on GD 10-13 increased the severity of maternal toxicity and incidence of
malformations induced by a single oral dose of 250-500 mg/kg acetyl salicylic acid
administered at the end of the exposure period.
Keller & Snyder (1986, 1988) investigated the effects of low level maternal
exposure to benzene on the blood and blood forming organs of mouse foetuses,
neonates and young adults. Groups of 5-10 dams were exposed to inhalation of 0,
5, 10, or 20 ppm benzene for 6 h/day on GD 6-15. Tests on the progeny comprised
RBC, WBC, differential blood cell count, and blood cell morphology; Hb and
haematocrit (Hct); quantification of colony forming units of erythrocyte (CFU-E)
and granulocyte/macrophage (CFU-GM) progenitor cells; and microscopic
examination of blood forming tissue in the liver, bone marrow and spleen. The
number of progeny examined included 2/sex/litter/dose on GD 16, 2/sex/litter/dose
on day 2 after birth, and 1/sex/ litter/dose at 6 weeks after birth.
In 16-day old foetuses exposed to 20 ppm benzene in utero, liver CFU-E was
depressed in both sexes. In peripheral blood from the 2-day old neonates, there was
a dose-related, marked decrease in the number of nucleated RBC. At 20 ppm, there
was also an increase in CFU-E (males only), CFU-GM, non-dividing and dividing
granulocytes in hematopoietic liver tissue, and in non-dividing granulocytes in
peripheral blood. In 6-week old young adult progeny, bone marrow CFU-E was
depressed and spleen CFU-E increased in males exposed to 10 but not to 20 ppm in
utero. In the 20 ppm group, there was a decrease in early nucleated RBC in the
bone marrow and an increase in blast cells in the spleen. There were no effects on
BW or BW gain, feed consumption and clinical signs in the dams, or on BW and
structural abnormalities in the foetuses at any dose level.
In a subsequent study on the interaction between alcohol and inhaled benzene in
mice, CFU-E was significantly depressed in the liver of 16-day old male (but not
female) foetuses exposed in utero to 10 ppm benzene for 6 h/day on GD 6-15
(Corti & Snyder, 1996). This study comprised a total of 9 exposed and 12 control
litters.
The embryotoxicity of benzene and its major metabolites has also been investigated
in vitro in GD 10-12 rat conceptuses. There were no toxic effects at concentrations
of 0.4-0.8 mM (32-64 mg/L) benzene (Brown-Woodman et al, 1994; Chapman et
al, 1994). Phenol was not toxic at 1.6 mM, but caused 100% lethality at 0.2 mM in
the presence of several CP450-dependent bioactivating systems. In the absence of
metabolic activation, catechol, hydroquinone, and quionone each produced 100%
lethality at 0.1 mM and the combination of phenol and hydroquinone showed a
greater than additive effect (Chapman et al, 1994).
Conclusions
In several studies in pregnant animals exposed to benzene by inhalation or
ingestion, there was a small, but statistically significant decrease in foetal BW and
an increase in the incidence of minor skeletal abnormalities at dose levels at which
there was no evidence of maternal toxicity. Major structural abnormalities and
abortions only occurred at dose levels that also caused marked toxicity in the dams.
As such, benzene can be characterised as foetotoxic, but not teratogenic. Based on
Priority Existing Chemical Number 21
62
adequately reported rat studies that found foetal effects in the absence of any signs
of maternal toxicity, the inhalation NOAEL for foetal growth disturbances is 40
ppm (Coate et al, 1984), with a LOAEL of 100 ppm (Coate et al, 1984; Green et al,
1978). Reliable oral effect levels cannot be determined from the available data.
In a small number of pregnant mice, inhalation of 10-20 ppm benzene resulted in
specific adverse effects on multipotential haematopoietic stem cells (colony
forming units) and other blood cells in the liver, bone marrow or spleen of the
offspring (Corti & Snyder, 1996; Keller & Snyder, 1986, 1988). These effects
occurred in the absence of any other signs of developmental toxicity and at levels
similar to those that are known to be toxic to the blood and blood forming organs of
adult mice (Section 10.4).
In vitro studies indicate that some benzene metabolites, including catechol,
hydroquinone and quinone (but not phenol) are substantially more toxic to rat
embryos than benzene itself.
10.6 Genotoxicity
The toxic effects of benzene on human genetic material have been investigated in
numerous in vivo studies which are addressed in Section 11.6 below. As such, the
assessment of studies conducted in animals and various in vitro systems is limited
to an overview of the most significant findings. Unless otherwise indicated, the
information presented is summarised from ATSDR (1997), IARC (1987), IPCS
(1993) and USEPA (1998a).
In vitro tests
Tests with benzene itself have predominantly produced negative results in
conventional in vitro gene mutation assays in bacteria and mammalian cell systems,
with and without metabolic activation. In vitro assays for chromosome aberrations
have also generally been negative, unless special precautions were taken to prevent
the evaporation of benzene from the test system (Randall et al, 1993). Likewise,
conventional in vitro tests for DNA breaks, unscheduled DNA synthesis and DNA
synthesis inhibition have produced inconsistent results. However, sister chromatid
exchanges (SCE), micronuclei (MN) and unscheduled DNA synthesis have been
induced in vitro by metabolites such as catechol, hydroquinone and/or quinone, and
DNA adducts with phenol, hydroquinone and quinone have been detected in a
number of in vitro systems. Benzene itself has recently been shown to induce
morphological transformation, gene mutations through base substitutions, and
aneuploidy in Syrian hamster embryo cells, but is less potent than its metabolites
(Tsutsui et al, 1997). In the alkaline single cell gel electrophoresis (Comet) assay,
pronounced, contact time-dependent DNA damage has been detected in non-
cycling (G0) human LC after treatment not only with catechol, hydroquinone,
quinone, 1,2,4-trihydroxybenzene or muconic acid, but also with benzene itself
(Anderson et al, 1995).
Tests in Drosophila
Benzene was consistently negative in the sex-linked recessive lethal test in
Drosophila melanogaster, which is a specific, but insensitive test for the potential
of chemicals to cause heritable gene mutations and chromosome aberrations.
However, benzene has been shown to induce a statistically significant number of
Benzene 63
so-called delayed lethal mutations, which may be the result of heritable mutations
in one rather than in both DNA strands of the X chromosome (Kale & Kale, 1995).
In vivo tests in rodents
There is ample evidence that benzene is genotoxic in a broad spectrum of in vivo
tests in rodents, in which the chemical was administered by inhalation, oral gavage
or parenteral injection. These include tests for SCE and MN induction in peripheral
blood cells, bone marrow cells, foetal liver cells, lung fibroblasts (Ranaldi et al,
1998), and Zymbal gland cells (Angelsanto et al, 1996); gene mutations in LC, lung
and spleen cells; chromosome aberrations in LC, bone marrow cells, spleen cells,
and spermatogonia; and DNA adducts in nucleated blood and bone marrow cells.
Furthermore, many of these effects have been shown to be mitigated by inhibitors
of benzene metabolism and reproduced by benzene metabolites such as
hydroquinone and 1,2,4-trihydroxybenzene.
In an in vivo chromosome aberration study in male mice, the sensitivity and dose
response to a single oral dose of benzene was found to differ markedly between
bone marrow cells and differentiating spermatogonia, as illustrated in Figure 10.1
(Ciranni et al, 1991).
Figure 10.1: Chromatid aberrations excluding gaps in mouse bone marrow
cells (broken lines) and spermatogonia (solid lines) at 6-48 h after oral
treatment with 880 mg/kg benzene (left) and at 24 h after oral treatment with
88, 220, 440 or 880 mg/kg benzene (right) (Ciranni et al, 1991)
25 25
Per cent aberrant cells
20 20
15 15
10 10
5 5
0 0
0 200 400 600 800 1000
0 6 12 18 24 30 36 42 48
Dose (mg/kg)
T i m e (h )
Sensitivity to SCE and MN induction was 2- to 3-fold higher in male than in
female mice and male sensitivity to MN induction was markedly decreased by
castration and restored by testosterone treatment (Luke et al, 1988, as cited in
USEPA, 1998c). Immature mice showed no gender difference in sensitivity to MN
induction (Siou & Conan, 1980, as cited in USEPA, 1998c).
10.7 Carcinogenicity
Table 10.3 highlights the principal findings in the 23 carcinogenicity tests that have
been reported in the open literature. They include oral gavage studies in B6C3F1,
RF/J and Swiss mice and F344, Sprague-Dawley and Wistar rats and inhalation
studies in AKR, C57BL, CBA, CD-1 and HRS mice and Sprague-Dawley rats.
Priority Existing Chemical Number 21
64
Table 10.3: Principal findings in inhalation (I) and oral gavage (O)
carcinogenicity studies in mice and rats
Strain I/O Protocol Principal findings* Reference
Mice
AKR No increase in tumour incidence
I Snyder et al.
0, 100 ppm for 6 h/day, 5
(1980)
days/week for life (72 weeks)
I No increase in tumour incidence
0, 300 ppm for 6 h/day, 5 Snyder et al.
days/week for life (28 weeks) (1978)
O
B6C3F1 NTP (1986)
0, 25, 50, 100 mg/kg/day for 5 Harderian gland tumours, lung
days/week, 103 weeks tumours, lymphoma, mammary
gland carcinoma, ovarian granulosa
cell and mixed benign tumours,
preputial gland carcinoma, Zymbal
gland carcinoma
C57BL I 0, 300 ppm 6 h/day, 5 Lymphoma, ovarian tumours, Cronkite et al.
days/week for 16 weeks, with Zymbal gland carcinoma (no (1985)
life-long observation (about statistical analysis)
110 weeks)
Lymphoma
I Snyder et al.
0, 300 ppm for 6 h/day, 5
(1980)
days/week for life (70 weeks)
I Zymbal gland carcinoma
0, 300 ppm 6 h/day, 5 Snyder et al.
days/week, 1 out of every 3 (1988)
weeks for life (118 weeks)
I No increase in tumour incidence
0, 1200 ppm 6 h/day, 5 Snyder et al.
days/week for 10 weeks, with (1988)
life-long observation (about
146 weeks)
CBA§ I 0, 100 ppm for 6 h/day, 5 Unspecified, non-hepatic, non- Cronkite et al.
days/week for 16 weeks, with haematopoietic tumours (1989)
life-long observation (about
135 weeks)
I 0, 300 ppm for 6 h/day, 5 Non-hepatic, non- haematopoietic Cronkite et al.
days/week for 16 weeks, with tumours (including Harderian gland (1989)
life-long observation (about carcinoma, lung adenocarcinoma,
115 weeks) mammary gland carcinoma,
neoplasms resembling acute
myeloblastic and chronic
granulocytic leukaemia, Zymbal
gland carcinoma)
I 0, 300 ppm for 6 h/day, 5 Lung adenoma, lymphoma, Farris et al.
days/week for 16 weeks, with preputial gland carcinoma (1993)
life-long observation (78
weeks)
CD-1 I 0, 300 ppm for 6 h/day, 5 Sporadic cases of suspected Goldstein et al.
days/week for life (not further myeloid leukaemia (p = 0.147) (1982)
specified)
I Lung adenoma
0, 300 ppm for 6 h/day, 5 Snyder et al.
days/week 1 out of every 3 (1988)
weeks for life (about 60
weeks)
I 0, 1200 ppm for 6 h/day, 5 Lung adenoma, Zymbal gland Snyder et al.
days/week for 10 weeks, with carcinoma (1988)
life-long observation (about
130 weeks)
HRS I 0, 400 ppm for 6 h/day, 5 No leukaemia or lymphoma in either Stoner et al.
days/week for 26 weeks (1980), as cited
hr/hr (leukaemia-prone) or hr/-
in Cronkite et al.
(leukaemia-resistant) strains
(1985)
RF/J O 0, 500 mg/kg/day for 5 Lymphatic neoplasms, lung Maltoni et al.
days/week, 52 weeks tumours, mammary gland (1989)
carcinoma (no statistical analysis)
Swiss O 0, 500 mg/kg/day for 5 Lung adenomas, mammary gland Maltoni et al.
days/week, 78 weeks carcinoma, Zymbal gland carcinoma (1989)
(no statistical analysis)
Benzene 65
Table 10.3: Continued
Strain I/O Protocol Principal findings Reference
Rat
F344 O NTP (1986)
0, 50, 100, 200 mg/kg/day in Oral cavity tumours, skin tumours,
males and 0, 25, 50, 100 Zymbal gland carcinoma
mg/kg/day in females for 5
days/week, 103 weeks
No increase in tumour incidence
I Snyder et al.
0, 100 ppm for 6 h/day, 5
Sprague-
(1984)
days/week for life (123
Dawley
weeks)
I No increase in tumour incidence
0, 300 ppm for 6 h/day, 5 Snyder et al.
(1978)
days/week for life (99
weeks)
I 0, 200-300 ppm for 4-7 Oral cavity carcinoma, Zymbal Maltoni et al.
h/day, 5 days/week, 104 gland carcinoma (no statistical (1989)
week analysis)
O 0, 50, 250 mg/kg/day, 5 Mammary gland tumours (lowest Maltoni et al.
days/week, 52 weeks dose level only), Zymbal gland (1989)
carcinoma in females (no statistical
analysis)
O 0, 500 mg/kg/day, 5 days/ Forestomach carcinomas, liver Maltoni et al.
week, 104 weeks angiosarcomas, nasal and oral (1989)
cavity carcinomas, skin carcinomas,
Zymbal gland carcinoma (no
statistical analysis)
Wistar O 0, 500 mg/kg/day, 5 Nasal and oral cavity carcinoma, Maltoni et al.
days/week, 104 weeks Zymbal gland carcinoma (no (1989)
statistical analysis)
* Positive findings were statistically significant (p <0.05) unless otherwise indicated.
Carries a virus causing spontaneous lymphoma in 90% of animals by 52 weeks of age.
Carries a virus yielding a high incidence of lymphoma from exposure to radiation, immunosuppression
and certain carcinogens.
§
Highly susceptible to radiation-induced thymic lymphoma.
There was a frequent association between benzene exposure and the occurrence of
solid tumours in epithelia of the mouth, nasal cavities, lung alveoli, Harderian,
Zymbal, preputial and mammary glands, and the ovary.
With regard to the blood and lymphatic system, the incidence of lymphoma was
elevated in several studies conducted in B6C3F1, C57BL, CBA and RF/J mice.
There was also a statistically significant increase in the incidence of lesions
resembling acute myeloblastic and chronic granulocytic leukaemia in a study in
CBA mice exposed to 300 ppm benzene for 16 weeks (Cronkite et al, 1989). In
addition, 3/40 CD-1 mice exposed to 300 ppm and 1/40 Sprague-Dawley rats
exposed to 100 ppm benzene developed suspected myeloid leukaemia after 27-38
weeks of exposure (Goldstein et al, 1982; Snyder et al, 1984). However, the
increase in lymphoma incidence was limited to strains where this is a common
spontaneous tumour type and the lesions resembling leukaemia may not have been
malignant but rather an intense proliferation of myeloid cells caused by infections
or necrotic processes in benzene-induced tumours in other organs (Farris et al,
1993). Furthermore, early findings of lymphoma or leukaemia-like lesions in a
given strain have not been consistently reproduced in later studies (Farris et al,
1993; Snyder et al, 1988).
Some of the target organs in rodents such as the forestomach, Harderian, Zymbal
and preputial glands have no anatomical equivalent in humans. Moreover, human
exposure to benzene is not associated with tumours of the mouth, nasal cavities or
lung alveoli (see Section 11). However, as there is limited evidence of an elevated
risk of malignant melanoma and breast cancer in humans exposed to benzene-
containing products, skin and mammary tumours are further analysed below.
Priority Existing Chemical Number 21
66
Skin tumours
The incidence of skin tumours was increased in F344 and Sprague-Dawley rats in
2-year oral gavage studies (NTP, 1986; Maltoni et al, 1989). By contrast, no skin
tumours developed in groups of 10 mice after oral, subcutaneous or topical
application of 800 mg/kg benzene followed 4 weeks later by topical application of
the tumour promoter 12-o-tetradecanoylphorbol-13-acetate 3 times a week for 20
weeks (Bull et al, 1986). Furthermore, although benzene was once widely used as a
solvent in tests for skin cancer induction in mice resulting in large numbers of
controls being topically exposed to benzene alone, there has been no indication that
it induced skin tumours in these models (IARC, 1982a).
In F344 rats, skin tumours were found on the face, back, flank, and other locations.
Microscopically, they represented a spectrum from pure squamous cell papillomas
or carcinomas to mixed tumours containing basal cell, sebaceous gland or hair
follicle elements. By incidental tumour tests, the incidence was elevated in male
rats at 200 mg/kg/day (12/50 vs. 1/50 in controls; p <0.01), but not at 50 mg/kg/day
(7/50) or 100 mg/kg/day (5/50), or in female rats treated with 25, 50 or 100
mg/kg/day. Based on mortality and BW data, high dose male rats were probably
exposed to benzene levels that exceeded the maximal tolerated dose (NTP, 1986).
In Sprague-Dawley rats, skin carcinomas (not further specified) occurred in 9/40
male animals administered 500 mg/kg/day by oral gavage for 2 years. The
incidence was zero in male controls and treated females (Maltoni et al, 1989). The
authors did not comment on the statistical significance of these results, however,
when analysed for this assessment, the difference in incidence between exposed
and control males was statistically significant (p <0.05; test for exact confidence
limits). Compared to their controls, male rats had an increased survival rate but a
reduction in BW that ranged from 6-18% during the course of the study (Maltoni et
al, 1983).
Mammary gland tumours
Mammary tumours have been found in B6C3F1, CBA, RFJ and Swiss mice and
Sprague-Dawley rats (Cronkite, 1986; NTP, 1986; Maltoni et al, 1989).
In a 2-year oral bioassay in B6C3F1 mice, benzene induced a significantly elevated
incidence of carcinomas and carcinosarcomas in mid- and high-dose females, with
a trend for dose-dependence (Table 10.4). The carcinomas often showed extensive
squamous cell metaplasia, whereas the carcinosarcomas contained a prominent
spindle-cell component resembling malignant fibroblasts. The historical incidence
of mammary gland carcinoma in this strain is approximately 1% (NTP, 1986).
Table 10.4: Mammary gland lesions in female B6C3F1 mice in a 2-year oral
carcinogenicity study (NTP, 1986)
Lesions Controls 25 mg/kg/day 50 mg/kg/day 100 mg/kg/day
Hyperplasia 2/49 (4%) 4/45 (9%) 2/50 (4%) 1/49 (2%)
Carcinoma 0/49 (0%) 2/45 (4%) 5/50 (10%)* 10/49 (20%)
Carcinosarcoma 0/49 (0%) 0/45 (0%) 1/50 (2%) 4/49 (8%)*
* p <0.05 (incidental tumour tests).
p <0.01 (incidental tumour tests).
Among male and female CBA mice exposed to 100 ppm benzene for 6 h/day, 5
days/week for 16 weeks, 20% had developed mammary gland tumours at follow-up
Benzene 67
102 weeks after the last exposure (Cronkite, 1986). Details on tumour incidence in
concurrent or historical controls were not given and the histopathology of the
tumours was not described, although a later publication refers to them as
adenocarcinomas (Cronkite et al, 1989).
In female RF/J mice administered 500 mg/kg/day by oral gavage for 52 weeks, the
incidence of mammary carcinomas was 22.5%, compared to 2.5% in controls. In
female Swiss mice receiving the same treatment for 78 weeks, the incidence was
47.5% compared to 5.0% in controls (Maltoni et al, 1989). The statistical
significance of these findings is not discussed in the paper. When analysed for this
assessment, the incidence in exposed females was significantly different from
controls (p <0.05; test for exact confidence limits) in Swiss but not in RF/J mice.
In female Sprague-Dawley rats given benzene by oral gavage for 1 year, the
incidence of total/malignant mammary tumours was 53.3/13.3% in controls,
73.3/13.3% in animals treated with 50 mg/kg/day, and 45.7/20.0% in animals
treated with 250 mg/kg/day. In female rats given 500 mg/kg/day for 2 years, the
incidence was 32.5/17.5% compared to 42.0/14.0% in controls (Maltoni et al,
1989). The tumours comprised fibroadenomas, adenocarcinomas and carcino-
sarcomas similar to the spontaneous mammary gland tumours commonly found in
ageing female Sprague-Dawley rats (Maltoni et al, 1983). The investigators did not
report on the statistical significance of their findings. When analysed for this
assessment, there was no difference between any of the groups in the incidence of
either total or malignant mammary tumours (p>0.05; test for exact confidence
limits).
Conclusions
The available carcinogenicity studies provide clear evidence of a causal
relationship between benzene exposure and malignant neoplasms in mice and rats.
The tissues most commonly involved are various glandular or non-glandular
epithelia of the oral cavity, nasal cavity, lungs and skin (Table 10.3). The incidence
of lymphoma was increased in several studies, but only in mice where this is a
common spontaneously occurring tumour type. In one study in mice, there was a
significant increase in bone marrow lesions described as resembling myeloblastic
or granulocytic leukaemia (Cronkite et al, 1989), but this may have been the result
of an intense inflammatory response (Farris et al, 1993). As such, a proven
reproducible animal model for benzene-induced leukaemia is not available.
The lowest exposure levels associated with an increase in tumour incidence in
rodents was 100 ppm by inhalation for 16 weeks in CBA mice and 25 mg/kg/day in
a 2-year oral gavage test in B6C3F1 mice and F344 rats (Cronkite, 1989; NTP,
1986). However, there was no increase in tumour incidence in two out of three
inhalation tests in rats exposed to 100-300 ppm benzene for 99-123 weeks (Maltoni
et al, 1989; Snyder et al, 1978, 1984).
There was an increased incidence of epithelial skin tumours in male rats in two 2-
year oral bioassays. In both studies, however, the increase only occurred at the
highest dose level tested (200 and 500 mg/kg/day respectively), which may have
exceeded the maximum tolerated dose. Mammary gland carcinomas were increased
in female mice at 50 and 100 mg/kg/day in a 2-year and at 500 mg/kg/day in a 78-
week test.
Priority Existing Chemical Number 21
68
10.8 Summary and conclusions
Taken together, the tests summarised above clearly demonstrate that benzene is not
highly acutely toxic to experimental animals, whereas it is a potent, multi-organ
toxicant by repeated administration. The target organs include the CNS, skin, eyes,
immune system, blood and blood forming organs, gonads and developing foetus.
Benzene is also toxic to genetic material and induces a variety of solid tumours,
including mammary cancer in female mice.
The only consistently reported acute systemic effects are CNS depression and
cardio-respiratory arrest. In rats, the median lethal dose is 810-9900 mg/kg by
mouth and 13,700 ppm by 4-h inhalation.
Topically, benzene appears to be irritating to the skin and eyes.
Of the available repeated dose oral studies, only the US National Toxicology
Program's 2-year bioassays in mice and rats have been conducted and reported in
full compliance with GLP and other internationally recognised quality standards
(NTP, 1986). In these studies, benzene administered by oral gavage induced
leukopoenia and lymphocytopoenia and an increase in the incidence of malignant
tumours at the lowest dose level tested, namely 25 mg/kg/day in male and female
mice and female rats and 50 mg/kg/day in male rats. In other oral studies of a lesser
quality, benzene produced leukopoenia in mice and rats and signs of
immunosuppression in mice at dose levels from 8-12 mg/kg/day.
With regard to repeated exposure by inhalation, which is the predominant route in
humans, the studies available for assessment were either poorly reported or
inadequate for the determination of dose-response relationships for other reasons,
such as an insufficient number of animals or range of exposure levels. Nonetheless,
the weight of evidence indicates that the following approximate effect levels are
likely to apply:
In mice but not in rats, subtle signs of neurobehavioural stimulation may be
·
detectable at vapour concentrations around 1 ppm, whereas gross CNS
impairment only occurs at and above 1000 ppm. ;
There are functional disturbances of the immune system at and above 10 ppm
·
in mice, but no such effects in rats below 400 ppm. The NOAELs were
determined to be 0.44 and 200 ppm for mice and rats respectively;
Abnormal blood counts and morphological abnormalities in blood forming
·
organs are found at and above 10 ppm in mice (including mouse foetuses
exposed in utero) and at and above 100 ppm in rats. As effects were observed
at all concentrations tested, a NOAEL could not be determined;
There are degenerative changes in the gonads at 300 ppm in mice, but not in
·
rats. The NOAEL was determined to be 30 ppm for mice ;
Benzene is foetotoxic, but not teratogenic in rats and mice exposed during
·
pregnancy at levels in the 100-500 ppm range, with an inhalation NOAEL for
foetotoxicity of 40 ppm in rats; and
The incidence of solid tumours is increased in mice exposed to 100-300 ppm
·
benzene for 16 weeks, but not consistently in mice exposed to 1200 ppm for 10
weeks or in rats exposed to 100-300 ppm for 99-123 weeks.
The relevance of these findings for human risk characterisation will be examined in
Section 13, in the context of the interspecies variations in benzene metabolism
Benzene 69
addressed in Section 9, the human health effects reviewed in Section 11, and the
molecular mechanisms of action discussed in Section 12.
Priority Existing Chemical Number 21
70
11. Human Health Effects
The literature on human health effects of benzene is extensive and contains data on
hundreds of thousands of people. This section summarises and reviews studies that
are relevant to the characterisation of the toxic effects of benzene and the
corresponding effect levels. Because of the nature of the available studies, the
review is predominantly based on findings in people who were exposed to benzene
at work or held jobs with the potential for exposure to the chemical.
The findings reported below must be interpreted with caution, as they rely on
inherently uncertain information about the exposure of individuals or populations
to benzene, which was either inferred or, at best, estimated from limited monitoring
data. Furthermore, in the vast majority of cases there was co-exposure to other
chemicals. These may be hazardous in their own right or inhibit the metabolism of
benzene to toxic metabolites, thus resulting in either an over- or underestimation of
the toxic potential of benzene. For example, the aromatic organic solvent toluene
may interfere with the metabolism of benzene as well as cause brain atrophy and
developmental toxicity (IPCS, 1985; Wilkins-Haug, 1997); some of the polycyclic
aromatic hydrocarbons (PAHs) that occur in petroleum, coal gas, coal tar and
vehicle exhaust are genotoxic and cause anaemia, immunosuppression and non-
melanoma skin cancer (IPCS, 1998); and 1,3-butadiene found in vehicle exhaust is
genotoxic and may increase the risk of blood and lymphatic cancers (IARC, 1999).
Moreover, many studies are not controlled for confounding by smoking, although
tobacco smoke contains benzene (see Section 16.1) and several studies have found
an association between active smoking and leukaemia and reproductive effects
such as semen quality and pregnancy outcome (Brownson et al, 1993; Vine, 1996;
Werler, 1997).
Unless otherwise mentioned, all results were statistically significant in comparison
with unexposed controls (p <0.05). In occupational studies, chronic inhalation
exposures refer to 8-h TWA (TWA8) concentrations. Technical terms used to
describe epidemiological study designs and statistics have the meaning given in
Last (1995).
11.1 Acute toxicity
Cases of acute intoxication have occurred because of workplace accidents and in
persons sniffing benzene-containing products for recreational purposes (Avis &
Huton, 1993; Barbera et al, 1998; Tauber, 1970; Winek & Collum, 1971). The
approximate lethal dose is 20,000 ppm by inhalation for 5-10 min, or 125 mg/kg by
ingestion, whereas exposure to 25 ppm for 8 h is reported to be without clinical
effects (Gerarde, 1960; Thienes & Haley, 1972, as cited in IPCS, 1993). No
adverse effects were reported in three kinetic studies in healthy volunteers exposed
to benzene levels of 26-42 ppm for 6 h, 52-62 ppm for 4 h or 47-110 ppm for 2-3 h
(Berlin et al, 1980; Nomiyama & Nomiyama, 1974a; Srbova et al, 1950). Clinical
signs at higher exposure levels include generalised symptoms such as dizziness,
headache and vertigo at levels of 250-3000 ppm, leading to drowsiness, tremor,
delirium and loss of consciousness at 700-3000 ppm (ATSDR, 1997; USEPA,
1998c). Unless fatal, the CNS symptoms are reversible following cessation of
exposure. Autopsy findings are typical of cardio-respiratory arrest.
Benzene 71
11.2 Irritation
Aspiration of liquid benzene has been observed to cause immediate pulmonary
oedema and bleeding at the site of contact (Gerarde, 1960). Benzene vapours have
been reported to cause eye and mucous membrane irritation in workers exposed at
33-59 ppm and irritation of the skin, nose, mouth and throat at levels 60 ppm
(Midzenski et al, 1992; Yin et al, 1987a). Acute tracheitis, laryngitis, bronchitis and
massive haemorrhage of the lungs were observed in a youth who died from an
overdose of intentionally inhaled benzene (Winek & Collum, 1971). Second degree
burns to the face, trunk and limbs were reported in chemical cargo ship crew
accidentally exposed to fumes at a concentration resulting in death within minutes
(Avis & Hutton, 1993).
11.3 Sensitisation
There are no reports of skin or respiratory sensitisation to benzene in humans.
11.4 Repeated dose toxicity (other than carcinogenicity)
11.4.1 Neurological effects
Yin et al. (1987a) found a dose-dependent increase in the prevalence of dizziness
and headache in a survey of female Chinese workers in the footwear and printing
industries. This study included 87 unexposed controls and two groups exposed to
benzene at levels ranging from 1-40 ppm (40 cases) or 41-210 ppm (47 cases). In
the two groups combined, benzene levels averaged 59 ppm. Both groups were co-
exposed to low levels of toluene (16 ppm).
Peripheral neuropathy was reported in a small number of Turkish workers with
benzene-induced aplastic anaemia or preleukaemia (Baslo & Aksoy, 1982). At a
benzene-manufacturing petrochemical plant in Estonia, frequent headaches at the
end of the shift, tiredness, sleep disturbances and memory loss occurred in 61% of
workers exposed to levels in the 2-16 ppm range for several years (Kahn &
Muzyka, 1973). In a survey of deck crew on nine Norwegian petroleum product
tankers, headache, dizziness or nausea were reported by 5/11 workers exposed to
>0.3 ppm benzene whereas there were no CNS complaints in 10 workers exposed
to 0.3 ppm (Moen et al, 1995). Psychological examinations in 28 men exposed to
a mixture of benzene (0.56-1.8 ppm), toluene (2.1-9.8 ppm) and xylenes (0.43-12
ppm) indicated diminished function of some cortical centres and impaired motor
reaction time (Sikora & Langauer-Lewowicka, 1998). Varelas et al. (1999) used
computed tomography imaging to visualise abnormal calcifications and cortical
atrophy in the brains of 122 petrol station workers, taxi and bus drivers in central
Athens. The subjects had been in their present employment for a minimum of 3 and
an average of 16-17 years. Whereas blood lead levels were unremarkable in all
three groups, there was mild to moderate cortical atrophy in 19/37 petrol station
workers, 14/44 taxi drivers and 14/41 bus drivers. The prevalence in petrol station
workers was higher than in taxi and bus drivers and unrelated to smoking or
alcohol habits. None of these studies included an unexposed control group.
11.4.2 Effects on the immune system
There was a decrease in circulating IgA and IgG immunoglobulins, accompanied
by an increase in IgM and an elevated occurrence of leukocyte auto-antibodies, in
Priority Existing Chemical Number 21
72
painters co-exposed to benzene, toluene and xylenes at air levels ranging from 3-
57, 21-71 and 27-680 ppm respectively (Lange et al, 1973a, 1973b). In workers co-
exposed to benzene, toluene and xylenes at air levels that averaged from 1-35, 2-32
and 4-28 ppm respectively over an 11-year period, total LC and T-LC counts were
slightly lower in workers exposed for 55-122 months than in an unexposed control
group, whereas there were no differences in LC function as determined by LC
transformation and tuberculin tests (Moszczynsky & Lisiewicz, 1984).
11.4.3 Cardiovascular effects
Kotseva & Popov (1998) conducted a routine cardiological examination of a
sample of male and female petrochemical workers aged 20-60 years. It included
118 workers concomitantly exposed to 20 ppm benzene and low levels of toluene
and petrol as well as 154 workers concomitantly exposed to <3 ppm benzene, 32
ppm xylenes and low levels of toluene and petrol. Compared to unexposed controls
matched for age, sex, salt intake, smoking and body mass index, the prevalence of
arterial hypertension and minor electrocardiographic abnormalities was
approximately twice as high in the exposed groups.
11.4.4 Haematological effects
Benzene has been known to be toxic to the blood for more than a hundred years
and in the past was sometimes given orally to leukaemia patients to reduce WBC
count (ATSDR, 1997; Landrigan, 1996).
Occupational exposure
Table 11.1 summarises a number of surveys of non-cancerous blood disorders in
workers exposed to airborne benzene. Overall, these studies point to a strong
association between recent or current exposure to airborne benzene and the
occurrence of decreased ALC, WBC, RBC and Plt counts, Hb and haematocrit
(Hct), and an increase in MCV. Such cases are sometimes described as `benzene
poisoning' (BP). Depending on the pattern and magnitude of these changes and the
histological findings in a bone marrow biopsy, they may be clinically diagnosed as
lymphocytopoenia, leukopoenia, anaemia, thrombocytopoenia, pancytopoenia,
agranulocytosis, myelofibrosis, or aplastic anaemia. They may be accompanied by
clinical signs such as paleness, increased susceptibility to infections, and a
tendency to bruising and bleeding. BP is generally reversible upon cessation of
exposure, except aplastic anaemia which may be fatal or progress to acute myeloid
leukaemia (AML) (Aksoy, 1989).
Among the studies summarised in Table 11.1, three surveys comprising a total of
795 workers found no adverse haematological effects from long-term benzene
exposure at levels averaging 0.55, 0.81 and 0.53 ppm respectively (Collins et al,
1997; Khuder et al, 1999; Tsai et al, 1983). These studies have limitations with
respect to blood analysis methodology, exposure assessment and/or control for
confounders such as smoking and co-exposure to other chemicals. Nevertheless,
taken together they indicate that the NOAEL for bone marrow toxicity is likely to
be >0.5 ppm.
Benzene 73
Table 11.1: Summary of haematological effects in workers exposed to airborne benzene
Industry Condition(s) observed Comments Reference
Country Benzene exposure (TWA8)*
Chemical USA Range = 0.01-1.40 ppm for an Collins et al.
In 200 exposed compared to 268 non-exposed workers there was no
average of 7.3 years (1991)
consistent benzene-related effect on haematology surveillance
indicators
USA Mean (range) = 0.55 (0.01-88) ppm, Collins et al.
In 387 exposed compared to 553 non-exposed workers there was no
(1997)
with <5% of workers exposed to difference in the prevalence of decreased ALC, RBC, WBC or Plt
levels >2 ppm counts, decreased Hb levels, or increased MCV values
USA Fishbeck et al.
Mean >24 ppm for an average 10/10 workers had increased MCV values and 9/10 also had
(1978)
of 9.6 years decreased Hb levels
USA 2/282 exposed workers died from Townsend et al.
From <2 to about 30 ppm for 1-20 Marginally lower RBC count and total bilirubin in 282 exposed
(1978)
leukaemia during the study
years workers compared to an equal number of matched controls
period (1967-74)
Coke oven USA Hancock et al.
0.1-31.4 ppm No differences in WBC, RBC or Hb values between groups of 17-37
by-products (1984)
workers with no, low (<2 ppm-years), intermediate (2-20 ppm-years)
or high (>20 ppm-years) exposure
Footwear Turkey Aksoy et al.
Exposed to adhesives. No
15-210 ppm for 3 months Increased incidence of reduced WBC and/or Plt or CBC counts in
manufacturing (1971)
correlation with duration of
217 workers compared to 100 controls matched for sex, age and
to 17 years
74
exposure
general living conditions
Croatia Bogadi-Sare et
Benzene contaminated glues,
Median (range) = 5.9 (1.9-14.8) ppm Decreased mean Hb concentration and percentage of B-LC and
al. (1997, 2000)
cleaners and paints; co-exposed
increased MCV and band neutrophils in 49 exposed females
(area monitoring)
to 11-50 ppm toluene
compared to 27 unexposed controls
Miscellaneous Italy Vai et al. (1989)
There was a highly significant
>20 ppm Of 301 workers referred to an occupational health clinic with
uses of benzene- correlation between severity of
suspected benzene intoxication, 153 had transient and 39
based solvents bone marrow disease and current
progressive bone marrow abnormalities; 11 died from aplastic
or recent exposure
anaemia and 21 developed cancers of the blood and lymphatic
system
China Rothman et al.
No correlation between any
Median (range) current personal In 44 exposed workers compared to an equal number of controls
(1996a, 1996b)
exposure = 31 (1.6-328.5) ppm, matched for sex, age, cigarette and alcohol consumption, WBC, ALC, haematological parameter and
with an average duration of Plt, RBC and Htc levels were reduced and MCV values increased. In cumulative exposure
exposure of 6.3 years 11 workers exposed to a median (range) level
of 7.6 (1-20) ppm, only ALC was decreased
China Xia et al. (1995)
Mean (range) = 5.8 (0.7-139) ppm 26% of 326 exposed workers had leukopoenia (WBC count <4.5 x
(assessment method not specified) 109/L) compared to 8.9% of 236 non-exposed workers
Priority Existing Chemical Number 21
China Yin et al. (1987a)
Co-exposed to 6-7 ppm toluene
Mean (maximum) = 59.2 (210) in Decrease in ALC in 83 exposed women compared to 85
women and 47.9 (210) ppm in unexposed controls, but no differences between 61
men, for an average of 5 years exposed men and 44 unexposed controls
Table 11.1: Continued
Condition(s) observed Comments Reference
Industry Country Benzene exposure (TWA8)
Canada Khuder et al. (1999)
Petroleum Mean (range) = 0.81 (0.14-2.08) ppm In 105 exposed workers, levels of WBC, RBC, Hb, MCV and Plt were
Benzene
for an average of 10 years
refining generally in the low normal range. MCV and Plt values were
negatively correlated with duration of employment, but not with
individual benzene exposure
USA Median = 0.53 ppm Tsai et al. (1983)
All haematological parameters generally within normal limits in 303
workers followed from 1959-1980
UK Yardley-Jones et al.
In 66 exposed compared to 33 non-exposed workers, All absolute MCV values were
10 ppm
(1988)
within the normal clinical range
there was no difference in various unspecified
haematology and serum biochemistry values, except
for a small increase in MCV values in exposed workers
USA 11-1060 ppm for 3-5 years Greenburg et al.
Printing 130/332 exposed workers showed signs of intoxication, including No cases of relapse after
(1939)
anaemia, increased MCV, reduced Plt counts and/or reduced benzene use was discontinued
WBC counts
USA Median exposure estimated at Cody et al. (1993)
Rubber In 161 workers hired between 1946-49, there was a 10% decline Pliofilm cohort
30-54 ppm
manufacturing in WBC counts over the first 4 months of employment, but no (Section 11.6.1)
consistent changes in RBC levels
75
USA Mean estimated at 75 ppm during Kipen et al. (1988,
In a longitudinal study of 459 workers, WBC, RBC and Hb levels Pliofilm cohort
1940-48 and at 15-20 ppm during 1989)
decreased with total exposure between 1940-48, but showed no (Section 11.6.1)
1949-78 persistent trends over the ensuing 25 years
Range estimated at <5-34 ppm
USA Ward et al. (1996)
Haematological screening data for 657 workers exposed between Pliofilm cohort
1939 to 1975 showed a relationship (Section 11.6.1)
between benzene exposure and the risk of a low WBC or RBC
count which was stronger for WBC, with no evidence
for a threshold exposure level
USA Mean and range estimated at 100 Wilson (1942)
Following complaints of malaise, nausea, vomiting, and bleeding, Outbreak coincided with large
and 50-500 ppm respectively blood counts were done on 1104 workers. ALC was abnormally war orders for synthetic rubber
low in 83 of them and 25 had severely reduced WBC, RBC and Plt
counts. Of these, 9 were hospitalised, where aplastic anaemia
was diagnosed by bone marrow biopsy; 3 died.
USA 60-600 ppm Midzenski et al.
Ship repair No relationship between blood
9/15 workers exposed over several days from the
(1992)
changes and duration of
degassing of shipboard tanks developed abnormal WBC, ALC,
exposure
Hb, Plt and/or MCV values within 4 months. At 12 months, 7/15
still had one or more abnormal values.
Turkey 0-110 ppm (area monitoring) Aksoy et al. (1987)
Used thinners and solvents
Tyre cord Decreased WBC count in 9, decreased Plt count in 4 and
containing up to 6-8% benzene
manufacturing decreased WBC, RBC and Plt count in 1 of 231 exposed workers
* TWA8 = 8-h time-weighted average.
CBC = complete blood cell count; for other abbreviations, see text.
In 44 Chinese workers exposed to a median benzene concentration of 31 ppm, with
a range from 1.6-328.5 ppm, Rothman et al. (1996a, 1996b) found a decrease in
WBC, RBC and Htc, an increase in MCV and an inverse correlation between ALC
and benzene exposure. In a subgroup of these workers with a median exposure
level of 7.6 ppm (range: 1-20 ppm), the lowest exposure group examined, the only
haematological finding was a 16% decrease in ALC (1.6 x 109/L compared to 1.9 x
109/L in controls; p = 0.03). This study is small, but had a well-matched control
group, minimal exposure to other chemicals (toluene and xylenes) and a dose-
response relationship was established between ALC and benzene exposure as
measured by repeated personal monitoring as well as with benzene metabolites in
the urine.
Three other studies reported haematological effects in workers whose exposure was
stated to range from 1.9-14.8 (median: 5.9), <5-34 and 0.7-139 (mean: 5.8) ppm
respectively (Bogardi-are et al, 1997; Ward et al, 1996; Xia et al, 1995). However,
these studies assessed exposure by means of area monitoring, a job-exposure
matrix or unspecified methods and are therefore less suitable for dose-response
characterisation.
Repeated exposure at higher levels was usually associated with clear signs of BP in
some workers, with little or no relationship with cumulative exposure (Aksoy et al,
1971; Midzenski et al, 1992; Rothman et al, 1996a, 1996b; Vai et al, 1989).
Dosemeci et al. (1997) evaluated the statistical relationship between a clinical
diagnosis of BP and benzene exposure in a subgroup of 412 cases drawn from a
large Chinese cohort study (Hayes et al, 1997), which is described in detail in
Section 11.6.1. The cumulative incidence of BP (defined as (1) a WBC count <4 x
109/L or a WBC count <4.5 x 109/L and a Plt count <80 x 109/L over several
months, (2) occupational benzene exposure for 6 months and (3) exclusion of
other causes of abnormal blood counts) rose sharply with increasing estimated
intensity of benzene exposure over a period of 18 months prior to diagnosis, as
shown by the following relative risks (RRs):
Exposure <5 ppm 5-19 ppm 20-39 ppm 40 ppm
RR (95% CI)6 1.0 (reference level) 2.2 (1.7-2.9) 4.7 (3.4-6.5) 7.2 (5.3-9.8)
The clear trend with the level of exposure is noteworthy, even if the absolute
exposure levels may have been underestimated, as discussed in Section 11.6.1
below.
The risk of BP developing into cancer was assessed in a subgroup of 11,177
benzene-exposed workers from the same Chinese cohort, 103 of whom had BP as
defined above (Rothman et al, 1997). At follow-up 4-14 years after diagnosis, three
of the cases had developed acute non-lymphocytic leukaemia (ANLL), non-
Hodgkin's lymphoma (NHL) and myelodysplastic syndrome (MDS) respectively,
compared to 6 cases of cancer of the blood and lymphatic system (including 2 with
ANLL) and 1 case of MDS among the 11,074 workers without a diagnosis of BP
(Table 11.2)7. The corresponding RRs indicate that a diagnosis of BP is associated
6
Throughout this section, ranges in brackets immediately following a relative risk (RR), odds ratio
(OR), standardised mortality rate (SMR) or standardised incidence rate (SIR) represent the 95%
confidence interval (CI) of the statistic.
7
ANLL comprises all acute leukaemias other than acute lymphocytic leukaemia and can usually be
equated to AML. MDS is a term that encompasses a variety of preleukaemic disorders.
Priority Existing Chemical Number 21
76
with a 42-fold increase in the risk of pre-cancer or cancer of the blood and
lymphatic system and with a 71-fold increase in the risk for ANLL/MDS. The RRs
changed little upon adjustment for cumulative exposure, indicating that the elevated
cancer risk was not due to a higher cumulative exposure in the 103 BP cases.
Table 11.2: Benzene poisoning and subsequent risk of blood and lymphatic
system cancer and related disorders (Rothman et al, 1997)
Without
benzene With benzene
Parameter* poisoning poisoning
Person-years of follow-up 122,620 848
RR (95% CI) of all pre-cancer or cancer of the blood and
lymphatic system 1.0 42.3 (10.7-167.0)
RR (95% CI) of ANLL/MDS 1.0 70.6 (11.4-439.3)
RR (95% CI) of all pre-cancer or cancer of the blood and
lymphatic system, adjusted for cumulative benzene 1.0 47.4 (11.7-191.9)
exposure
RR (95% CI) of ANLL/MDS, adjusted for cumulative
benzene exposure 1.0 61.3 (9.8-384.3)
* ANLL = acute non-lymphocytic leukaemia; CI = confidence interval; MDS = myelodysplastic syndrome;
RR = relative risk.
Similarly, Vai et al. (1989) reported 28 cases of fatal blood cancer among 304
workers in Northern Italy who were hospitalised 15-35 years earlier with suspected
BP, representing a 13.3-fold increase over the incidence in the general population
in the region.
Public exposure
In the 1980s, the US Federal Department of Health and Human Services created a
National Exposure Registry to assess the health consequences to the general
population from long-term, low-level exposure to specific substances in the
environment. A Benzene Subregistry was established in 1991 based on a
population health survey in a community in Texas, USA, where tap water from the
public water system was known to have contained 66 µg/L benzene since 1
January 1979 (Burg & Gist, 1998). The survey included 1,143 persons who had
used contaminated water as the sole source of drinking, bathing and cooking for at
least 30 consecutive days. These persons were administered a questionnaire and
follow-up telephone interviews were conducted one and two years later. The
questions asked were similar to those used in the National Health Interview Survey
(NHIS) conducted every year in USA, except that the benzene questionnaire
contained a qualifier relating to professional rather than self diagnosis of ailments,
so as to minimise reporting bias. Findings were compared with concurrent NHIS
data subsets matched for demographic variables and current and ever smoking
rates. The initial response rate was 97%. There was a loss of 9% for each follow-up
from the previous data collection.
The outcome of anaemia and related blood disorders within the last 12 months was
reported in excess at all three data collections, with 40 observed vs. 14.1 expected
cases at baseline, 32 observed vs. 11.6 expected cases at one year (p <0.01), and 28
observed vs. 11.9 expected cases at two years. There was no difference in the
reporting of cancer.
Benzene 77
Conclusions
Several occupational surveys show that chronic exposure to benzene may lead to
bone marrow depression, with manifestations that range from small reductions in
blood count parameters to aplastic anaemia. The available data indicate that both
the incidence and severity of this effect is dose-related. In a small, but reliable
study, the only haematological effect in workers with a median (range) exposure of
7.6 (1-20) ppm (TWA8) was a modest reduction in ALC. As this was the lowest
exposure group examined, 7.6 ppm (TWA8) is currently considered the best
estimate for a LOAEL which may be close to the point of departure for the onset of
haematological effects (USEPA, 1998c). An appropriate NOAEL has not been
determined, but studies with various limitations indicate that it is likely to be >0.5
ppm (TWA8).
The only available epidemiological study in the general population found an excess
occurrence of anaemia and related disorders in a community whose tap water
contained 66 µg/L benzene.
There is some evidence that bone marrow depression is associated with a
substantially increased risk for ANLL/MDS.
11.4.5 Reproductive effects
Effects on fertility
Vara & Kinnunen (1946) reported a variety of gynaecological disorders in 12
female rubber workers who were exposed to unspecified levels of benzene on a
daily basis. All 12 women had menstruation disorders, with sparse bleeding being
the most common complaint. Although most of the women practised regular
unprotected intercourse, only two of them had conceived since they started working
and both pregnancies ended in spontaneous abortions (SAb) by the first trimester.
Five had ovarian hypoplasia. Other common findings included excessive bruising,
tiredness, dizziness, headaches and abnormal haematological findings, particularly
low WBC and Plt counts.
Menstruation abnormalities have also been reported in surveys of female workers
exposed to mixed aromatic hydrocarbons including benzene or to benzene,
petroleum and chlorinated hydrocarbons in Poland and Russia in the 1960s
(Michon, 1965; Mukhametova & Vozovaya, 1972; both as cited in ATSDR, 1997)
and in female workers at a large petrochemical company in China (Thurston et al,
2000). Huang (1991) reported menstruation disorders in 49% of 223 Chinese
leather footwear workers co-exposed to an average of 29 (range: 1-132) ppm
benzene and 19 (range: 1-136) ppm toluene compared to a prevalence of 16% in
unexposed controls (p <0.01). There is no information on the smoking habits of the
study population.
Benzene exposure as a risk factor for fecundability (time to pregnancy) was
assessed in a Norwegian case-control study in 558 female dental surgeons and 450
high school teachers with at least one child (Dahl et al, 1999). Forty percent of the
dentists reported daily exposure to a now discontinued disinfectant containing
0.25% v/v benzene. There was no difference in fecundability between dental
Priority Existing Chemical Number 21
78
surgeons exposed to benzene and the controls. Potential confounders were
considered, but the level of benzene exposure resulting from the disinfectant was
not assessed.
In males, De Celis et al. (2000) studied the sexual functioning and semen profile of
48 Mexican rubbers workers exposed to a mixture of benzene (10-15 ppm), ethyl
benzene (~50 ppm), toluene (~50 ppm) and xylenes (~12 ppm) for 2 years. Mean
sperm count and the mean percentage of motile and normal sperm forms were
reduced by 78, 62 and 24% respectively, compared to a group of 42 age-matched
controls. There was no correlation between smoking or alcohol intake and
alterations in the semen profile. Longer abstinence intervals may have contributed
to the reduced sperm concentration and motility in exposed workers as they also
had an increased prevalence of reduced libido.
Effects on pregnancy outcome
Huang (1991) investigated pregnancy outcome in 106 Chinese leather footwear
workers co-exposed to an average of 29 ppm (range: 1-132) ppm benzene and 19
(range: 1-136) ppm toluene and 209 unexposed controls. Exposure to benzene and
toluene was associated with an elevated incidence of SAb (5.8 vs. 2.4%; RR = 2.4;
p <0.01), whereas there was no difference in the incidence of preterm delivery or
stillbirth. There is no information on the smoking habits of the study population.
Lindbohm et al. (1991) used Finnish census data, hospital records and industry-
wide air monitoring results collected in 1975-82 to study the outcome of 11,570
pregnancies with potential paternal exposure to hazardous chemicals compared
with a control group of 87,616 unexposed pregnancies. The RR for SAb was
elevated for paternal exposure to solvents used in petroleum refineries, but did not
differ significantly from unity when analysed separately for exposure to benzene.
Although not quantified, benzene exposure levels were estimated to be low.
In a case-control study of female workers in the Finnish pharmaceutical industry
which included 44 cases of SAb and 130 matched controls, Taskinen et al. (1986)
found a non-significant association between abortion risk and benzene exposure
(OR = 2.4 (0.5-12.0)).
The effects of parental occupational exposures on foetal development were
investigated in an exploratory case-control study based on probability samples of
live births and foetal deaths obtained by the US National Natality and Fetal
Mortality survey conducted in 1980 among married women (Savitz et al, 1989).
The samples included case groups of stillbirths (2096 mothers, 3170 fathers),
preterm deliveries at <37 weeks of pregnancy (363 mothers, 552 fathers) and
small-for-gestational age (SGA) infants (218 mothers, 371 fathers). Control
pregnancies were drawn from the same survey. Occupational exposures within the
last 12 months were defined by industry of employment and relative levels of
exposure to individual agents estimated on the basis of a job-exposure linkage
system. In computing the OR, adjustments were made for known confounding
factors for each pregnancy outcome, such as child's race, receipt of prenatal care,
mother's age, number of previous miscarriages, previous induced abortions and
maternal smoking and alcohol consumption.
Overall, this study found a significantly elevated SGA risk in the offspring of
fathers exposed to benzene at work (OR = 1.5 (1.1-2.3)), with a strong dose-
response gradient. Benzene-exposed fathers of SGA infants included a large
percentage of engine mechanics and repairers, welders and flame cutters. Maternal
Benzene 79
benzene exposure showed a marginally significant association with stillbirths (OR
= 1.3 (1.0-1.8)), which was supported by the demonstration of a dose-response
gradient. In these mothers, benzene exposure was attributed mainly to work in the
textile industry, barbering and cosmetology, with smaller contributions from the
chemical, pharmaceutical and paint industries.
Stücker et al. (1994) evaluated the risk of SAb before 28 weeks among the spouses
of 1077 male workers at two organic chemical factories in France, on the basis of
exposures estimated by plant occupational physicians and questionnaires
administered to the men and their wives. Medical records of the women were not
examined. There was a total of 1739 pregnancies, of which 171 (9.8%) ended in
SAb. The abortion rate was 8.8% in the wives of unexposed workers. Workers
were divided into low (<5 ppm) and high (5 ppm) exposure categories depending
on their estimated past exposure to benzene. After adjustment for maternal tobacco
consumption, age and pregnancy order, the risk of SAb did not differ from unity in
either of the two exposure groups. Similar results were obtained in analyses of first
pregnancies only, and when pregnancy outcome was examined against a more
detailed exposure graduation.
A Finnish case-control study of 206 cases of SAb in laboratory workers and 329
individually matched controls identified 11 cases of benzene exposure in the SAb
group compared to 25 among the controls and concluded that benzene exposure
was not a significant risk factor (Taskinen et al, 1994).
In a Chinese study, the overall risk of SAb in 3070 non-smoking, primiparous
women employed at a large petrochemical complex and married to male workers at
the same facility was 8.8% in chemical compared to 2.2% in non-chemical workers
(Xu et al, 1998a). Benzene, toluene, xylenes and styrene exposure levels in 38
breathing zone samples collected throughout the complex averaged 0.86, 0.40, 0.50
and 0.03 ppm respectively. In analyses for exposure to specific chemicals during
the first trimester of pregnancy, the estimated ORs of SAb were significantly
elevated for benzene (2.5 (1.7-3.7)) and petrol (1.8 (1.1-2.9)).
Chen et al. (2000) conducted a prospective study of pregnant workers at a Chinese
petrochemical plant producing benzene, toluene, xylenes, styrene and phenol.
Compared with 459 mothers not exposed to organic solvents, there was a small
reduction in birth weight (58 g; 95% CI = 115 to 2 g) among 366 mothers
exposed to 0.02-0.2 ppm benzene with or without other exposures.
Conclusions
There are several reports of menstruation disturbances in female workers and one
of reduced semen quality in male workers exposed to benzene.
The available studies of pregnancy outcome have produced mixed results with
regard to the risk for SAb. One study found an elevated SGA risk for fathers with
occupational exposure to benzene. In another study, there was a marginally
significant reduction in birth weight in infants whose mothers had been exposed to
low levels of benzene at work.
However, all of the available studies have one or more limitations, such as multiple
exposures, inadequate adjustment for other confounders and/or inadequately
quantified exposure to benzene as well as other chemicals. Therefore, there is at
present no convincing evidence from human studies that benzene may have adverse
effects on reproduction.
Priority Existing Chemical Number 21
80
11.4.6 Other health effects
Chronic tiredness and headache and large, spreading bruises on the arms and legs
have been described in a number of workers exposed to benzene air levels in the
order of 100-200 ppm (Helmer, 1944).
Yin et al. (1987a) conducted a survey of the prevalence of symptoms of
intoxication in Chinese factory workers exposed to high levels of benzene or
benzene and toluene for up to 40 years. There was a slight decrease in ALC in both
groups. Sore throat and episodes of nose bleeding were common in all exposed
workers and their frequency was related to benzene exposure levels.
In an uncontrolled case report, Davidoff et al. (1998) described a group of workers
who began complaining about petrol odour and symptoms of nausea, headache,
throat and eye irritation, and cough while tunnelling underneath a former service
station site. An air sample from the tunnel contained 60 ppm benzene. Eight out of
30 randomly selected workers subsequently investigated in detail reported the post-
incident onset of chemical hypersensitivities and other characteristics which,
according to the authors, fitted conservative criteria for the diagnosis of multiple
chemical sensitivities syndrome.
11.5 Genotoxic effects
Several occupational studies conducted over the past 30 years point to a link
between a number of unstable or stable, numerical or structural chromosome
aberrations and benzene exposure (ATSDR, 1997; IPCS, 1993). In most cases,
these studies were conducted in workers exposed to benzene levels >10 ppm.
However, Tompa et al. (1994) analysed whole blood metaphase spreads from
workers employed in an environment where improved working conditions over a 3-
year period reduced average peak exposures from 21 ppm in 1990 to 8.4 ppm in
1991 and 5.7 ppm in 1992. As shown in Figure 11.1, the reduction in benzene
levels was paralleled by a decrease in the frequency of chromosome aberrations,
but not in SCEs. These findings provide evidence of a direct relationship between
benzene exposure and the extent of chromosome damage, but do not establish a
threshold level for the effect.
Figure 11.1: Changes in the frequency of SCEs and chromosome aberrations
(excluding gaps) in workers exposed to progressively reduced benzene
levels (Tompa et al, 1994)
10
SCEs/cell
8
Frequency
C hro m o so m e
6
ab erra tio ns (%)
4
2
0
25 20 15 10 5 0
B enz en e concentration (ppm)
Benzene 81
Table 11.3 summarises a number of recent occupational studies which used
personal air monitoring to measure benzene exposure and modern cytogenetic
techniques such as polymerase chain reaction methods, 32P-postlabelling,
fluorescence in situ hybridization and alkaline single cell gel electrophoresis to
determine the genotoxic effects in various cell samples.
Table 11.3: Genotoxic effects in workers exposed to airborne benzene
Study population (number)
Effects and effect levels (TWA8)* Reference
Exposed Controls
Petrol station Matched for Excess of overall DNA damage and highly damaged Andreoli et
attendants (12) sex, age and cells in freshly isolated non-cycling peripheral blood al. (1997)
smoking LC in subjects exposed to a mean air level of 0.11
habits (12) ppm (range: 0.03-3.0 ppm) (Lagorio et al, 1997)
Petrol station Matched for No evidence of numerical aberrations involving Carere et al.
attendants (12) sex, age and chromosomes 7, 11, 18 or X in peripheral blood LC (1998)
smoking of subjects exposed to an average air level of 0.1
habits (12) ppm
Increase in kinetochore-positive MN in T-LC, but no Holz et al.
Styrene plant Matched for
changes in DNA adducts in MC or in DNA single (1995)
workers (25) sex and age
strand breaks, SCE or total MN in LC at average
(25)
exposure levels corresponding to 0.24 ppm benzene
and 0.31 ppm styrene (as well as toluene, xylenes
and ethylbenzene)
Coke gas plant Matched for No increases in the frequency of MN, MN harbouring Surrallés et
workers (56) age (28) whole chromosomes or acentric chromosomal al. (1997)
fragments or chromosome 9 numerical abnormalities
in LC and buccal cells at exposure levels from 0.5-
1.2 ppm
Coke gas plant Unmatched Small but statistically significant increase in Marcon et
workers (12) inhabitants of centromeric breakage of chromosomes 1 and 9 in al. (1999)
and oven neighbouring interphase LC in benzene workers exposed to a
operators (5) rural village geometric average of 1.3 ppm benzene, but not in
(8) coking oven workers exposed to a geometric
average of 1.0 ppm
Workers using Matched for In heterozygous individuals, the frequency of NN but Rothman et
benzene-based sex, age, not Nø GPA mutants was doubled in peripheral RBC al. (1995,
solvents (24) smoking, and strongly correlated with lifetime cumulative 1996b)
drinking and benzene exposure, at a mean exposure level of 72
obesity (23) ppm (range: 2-301 ppm)
Workers using Matched for There was a dose-related increase in hyperdiploidy Smith et al.
benzene-based sex and age at chromosomes 8 and 21 and in hypodiploidy at (1998)
solvents (43) (44) chromosome 8 in LC of workers with a median
exposure level of 31 ppm (range not specified).
There was also a 15-fold increase in t(8;21) (27
versus 2% LC) and a doubling of t(8;?) and t(21;?) in
LC at exposures >31 ppm. All increases were
related to current but not to cumulative exposure
Increased frequency of hyperdiploidy at chromo- Zhang et al.
some 9, mainly trisomy, in LC at exposure levels (1996)
>31 ppm, which correlated with ALC decreases
Increased frequency of monosomy at chromosomes Zhang et al.
5 and 7, in trisomy and tetrasomy at chromosomes (1998)
1, 5 and 7, and a dose-dependent, up to 3.5-fold
increase in long arm deletions of chromosomes 5
and 7 in whole blood metaphase spreads, at a
median exposure level of 31 ppm (range: 2-329
ppm)
* ALC = absolute lymphocyte count RBC = red blood cells
GPA = glycophorin A SCE = sister chromatid exchange
LC = lymphocytes t(a;b) = translocations between chromosomes a and b
TWA8 = 8-h time-weighted average
MC = monocytes
MN = micronuclei ? = unidentified chromosome.
The studies of Andreoli et al. (1997) and Carere et al. (1998) of petrol station
attendants exposed to benzene concentrations that averaged around 0.1 ppm are
Priority Existing Chemical Number 21
82
difficult to interpret as there is no information on the nature and extent of co-
exposure to other chemicals in petrol or vehicle exhaust fumes, which would
include 1,3-butadiene and a number of genotoxic PAHs (IARC, 1999; IPCS, 1998).
Furthermore, Carere et al. (1998) investigated only one of the six chromosomes in
which aberrations have been found at high levels of benzene exposure.
Holz et al. (1995) reported kinetochore-positive (that is, whole chromosome) MN
in workers with an average exposure of 0.24 ppm benzene and 0.31 styrene.
However, styrene alone is known to cause chromosome damage in human
lymphocytes at low concentrations (IARC, 1994).
In coal gas by-product workers, Surrallés et al. (1997) found no chromosome
aberrations at benzene levels 1.2 ppm, whereas Marcon et al. (1999) found a small
increase in centromeric breakages in chromosomes 1 and 9 at 1.3 ppm, but not in
coke oven workers exposed to a slighter lower level averaging 1.0 ppm. However,
coke oven and coal gas by-product workers are co-exposed to numerous PAHs,
many of which have a variety of genotoxic effects at low concentrations (IPCS,
1998).
The main findings at exposure levels 31 ppm benzene were aneuploidy, long-arm
deletions and translocations involving chromosomes 1, 5, 7, 8, 9 and 21 and gene
duplication in nucleated RBC stem cells at the glycophorin A locus on
chromosome 4 (Rothman et al, 1995, 1996b; Smith et al, 1998; Zhang et al, 1996,
1998). The subjects of these studies were co-exposed to toluene and xylenes, which
may inhibit the metabolism of benzene, but have not been shown to cause
chromosome lesions that resemble the above (IPCS, 1997; McGregor, 1994).
As such, whereas studies using modern cytogenetic techniques have shown a clear
association between extensive chromosome damage and exposure to high benzene
levels, they have not contributed to the definition of a threshold level for genotoxic
effects in humans.
11.6 Carcinogenicity
11.6.1 Cohort studies
Cohort studies with poorly characterised benzene exposure levels
Table 11.4 summarises a number of occupational cohort studies with a combined
study population approaching 450,000 workers holding jobs with the potential for
exposure to benzene, mainly in the petroleum industry. They include the ongoing,
prospective Health Watch (1998) cohort study, which covers about 95% of the
Australian petroleum industry's 18,000 employees in refineries, natural gas plants,
distribution terminals and production sites. They also include two meta-analyses
based on a large number of petroleum industry cohorts (Raabe & Wong, 1995;
Wong & Raabe, 1996, 2000). The most important limitation of these studies and
meta-analyses is their lack of adequate data on benzene exposure levels.
Benzene 83
Table 11.4: Summary of cohort studies in workers exposed to poorly characterised benzene levels
Exposed population Controls Ratio (95% CI) Comments Reference
Health outcome*
Chemical industry
Any death rate
National population Decouflé et al.
No difference
259 workers ever employed at a US benzene In 194 workers employed for 12 months,
(1983)
alkylation plant from 1947-60 and followed up SMR for CBLS = 3.77 (1.09-10.24)
for 17-30 years
All-cause mortality
Architects from same Olin & Ahlbom
RR = 1.14 (0.91-1.37)
822 chemists graduated from university in There were 10 cases of CBLS among the
All cancer mortality
school (1980)
RR = 2.54 (p <0.05)
Stockholm, Sweden, between 1930-50 and chemists compared to 0 among the
All CBLS
followed up till the end of 1974 architects; nine were organic chemists
RR = (p = 0.02)
Coke oven and coal gas by-product workers
Regional population Hurley et al.
Benzene breathing zone levels were
Leukaemia:
2708 men employed by British Steel
(1991)
SMR = 0.41 (0.05-1.47) reported to average 1.3 ppm in by-product
Corporation at 14 coke works in the UK in All workers
SMR = 0.98 (0.02-5.57) and 0.3 ppm in coke oven workers in the
1967 and followed up for 20 years By-product workers
SMR = 0.35 (0.01-1.92) 1980s
Coke oven workers
Regional population
3812 men employed by National Smokeless Leukaemia:
SMR = 0.42 (0.09-1.23)
Fuels Ltd at 13 coke works in the UK in 1967 All workers
SMR = 0.76 (0.02-4.29)
and followed up for 20 years By-product workers
84
SMR = 0.34 (0.00-1.86)
Coke oven workers
SMR = 0.58 (0.01-3.28)
Maintenance workers
National population Swaen et al.
SMR >1.00 (p< 0.05) Among 222 benzene plant workers, death
All-cause mortality, all
5659 coke oven workers employed for 6
(1991)
rates were similar to the expected figures
cancer, liver cancer, and
months from 1945-69 at a Dutch coke plant and
respiratory disease
followed up for 15-40 years
Footwear manufacturing
Paci et al.
National population Workers in some departments were
Male workers (n = 1008):
2013 men and women ever employed at an
SMR <1.00 (p <0.05) exposed to glues containing >70% benzene (1989)
GI disease and accidents
Italian shoe manufacturing plant from 1939-64
SMR = 15.66 (5.47-32.64)
Blood disease
and followed up for 20-45 years
All non-cancer blood disease cases were
SMR = 1.40 (1.09-1.81)
All cancer
aplastic anaemia
SMR = 2.40 (1.37-3.78)
Stomach cancer
SMR = 4.00 (1.46-8.70)
Leukaemia No relationship between leukaemia risk and
duration of exposure
Female workers (n = 1005):
No information on job categories, which
No difference
Any cause of death
likely may have explained negative findings
in female workers
Priority Existing Chemical Number 21
Table 11.4: Continued
Exposed population Controls Ratio (95% CI) Comments Reference
Health outcome*
Benzene
Highway maintenance workers
All-cause mortality and
Regional white Bender et al.
4849 men employed for at least 1 year SMR <1.00 (p <0.05) In workers with 30-39 years of
all cancer
population (1989)
between 1945-84 as highway maintenance employment, the SMR for leukaemia
workers by the Department of Transportation was 4.25 (1.71-8.76)
All CBLS
in Minnesota, USA SMR = 0.95 (0.66-1.33)
Leukaemia SMR = 1.07 (0.62-1.71) No observed deaths from melanoma
compared to 2.9 expected
Petrol and petroleum distribution
Male workers (n = 16,524): Lynge et al.
National population No information on employment status
18,969 men and women employed as petrol
All malignant neoplasms (1997)
of gainfully SIR = 1.1 (1.0-1.1) before or after the census date and
station attendants on the day of the 1970
Cancer of the nose
employed SIR = 3.1 (1.5-5.7) hence no adjustment for person-years
censuses in Denmark, Norway, Sweden and
Lung cancer SIR = 1.3 (1.1-1.4) at risk
Finland and followed up for deaths and
Non-Hodgkin's lymphoma SIR = 1.1 (0.8-1.5)
incident cancer cases for 15-20 years
Hodgkin's disease SIR = 1.0 (0.5-1.8)
Multiple myeloma SIR = 0.6 (0.3-1.1)
Leukaemia SIR = 0.9 (0.6-1.3)
Female workers (n = 2445):
85
All malignant neoplasms SIR = 1.0 (0.8-1.1)
Cancer of the nose SIR = 8.0 (1.0-28.9)
Non-Hodgkin's lymphoma SIR = 0.6 (0.0-2.0)
Hodgkin's disease SIR = IC
Multiple myeloma SIR = IC
Leukaemia SIR = 0.7 (0.1-2.4)
All-cause mortality, all
Regional Lagorio et al.
SMR <1.00 (p <0.05) Benzene exposure levels were reported
2665 petrol station workers in the Latium
cancer, and CV disease
population (1994a)
to be in the order of 0.2 ppm
(greater Rome) region in Italy in 1980 and
followed up for 10 years
All CBLS SMR = 0.40 (0.07-1.26)
All-cause mortality,
Regional Rushton
SMR <1.00 (p <0.05)
23,306 workers employed for 1 continuous
respiratory, liver and
populations (1993b)
year between 1950-75 at UK oil distribution
kidney disease, all cancer
centres and followed up for 15-40 years
and cancer of the
oesophagus, lung and
pleura
Leukaemia SMR = 1.08 (0.83-1.40)
Table 11.4: Continued
Controls Ratio (95% CI) Comments Reference
Exposed population Health outcome*
Petrol and petroleum distribution: Continued
All-cause mortality,
National population Wong et al.
SMR <1.00 (p <0.01)
18,135 US workers employed for 1 year
all cancer and circulatory, (1993)
at land-based petrol terminals or on marine
respiratory and liver disease
petrol tankers from 1946-1985 and followed
up for 5-55 years
Leukaemia SMR = 0.89 (0.59-1.29) Land-based workers
SMR = 0.70 (0.42-1.09) Marine workers
Lymphoma SMR = 0.75 (0.58-0.97) Land-based workers
SMR = 0.61 (0.43-0.83) Marine workers
Petroleum production, refining and distribution
All-cause mortality, all CV Consonni et al.
SMR <1.00 (p <0.05) Limited monitoring data indicate that a
1583 workers ever employed at an Italian refinery National population
disease, stroke, respiratory (1999)
substantial fraction of the workforce had
from 1949-82 and followed up for
disease, liver, and GI been exposed to benzene levels >1 ppm
10-42 years
disease
86
CBLS (all workers) SMR = 1.79 (1.00-2.95)
CBLS (employment >15 SMR = 2.71 (1.09-5.59)
years) Leukaemia SMR = 3.77 (1.01-9.65)
(employment >15 years)
CBLS (employment pre- SMR = 2.82 (1.13-5.81)
1961)
Lymphoma (employment SMR = 4.02 (1.08-10.28)
pre-1961)
All-cause mortality,
National population Health Watch
SMR <1.0 (p <0.05) Elevated incidence of leukaemia mainly
15,732 male workers employed for 5 years
ischaemic heart disease, (1998)
accounted for by refinery and terminal
in the Australian petroleum industry from
stroke, and respiratory, liver workers; no definite relationship with length
1981-96 and followed up for 5-15 years
and GI disease of employment
Bladder cancer SIR = 1.4 (1.0-1.9) Estimated long-term benzene exposure
Multiple myeloma SIR = 1.9 (1.0-3.3) levels were 5 ppm in all cases; estimated
Leukaemia SIR = 2.0 (1.3-2.9) cumulative exposures ranged from 0.005-
Lymphocytic leukaemia SIR = 2.0 (1.0-3.5) 50.9 ppm-years (Glass et al, 1998)
Myeloid leukaemia SIR = 2.2 (1.2-3.6)
Melanoma SIR = 1.6 (1.3-1.9) No excess mortality from melanoma (SMR
= 0.7 (0.4-1.4)
Priority Existing Chemical Number 21
Table 11.4: Continued
Controls Ratio (95% CI) Comments Reference
Exposed population Health outcome*
Benzene
Petroleum production, refining and distribution: Continued
National population Järvholm et al.
4319 Swedish refinery operators and All male workers (n = 4128):
(1997)
All-cause mortality, CV disease, SMR <1.00 (p <0.05)
distribution workers employed for 1 year
and lung cancer
between 1958-91 and followed up for
5-35 years
Refinery operators (n = 1339):
Leukaemia SIR = 3.6 (1.5-7.0)
Distribution workers (n = 1391):
All cancer and lung cancer SIR <1.00 (p <0.05)
Leukaemia SIR = IC (0-2.0)
Lewis et al.
National population There was an increase in multiple
Male workers (n = 26,322):
34,560 workers employed 1 year in refinery,
(2000)
myeloma (SMR = 1.94 (1.11-3.15)) in
All-cause mortality, endocrine, SMR <1.00 (p <0.05)
petrochemical, distribution, marketing,
marketing and distribution workers
circulatory, respiratory and GI
production, drilling and pipeline locations
disease, all cancer
throughout Canada from 1964-83 and
Leukaemia SMR = 0.89 (0.67-1.16)
followed up for 11-31 years
Aortic aneurysm SMR = 1.27 (1.04-1.53)
Female workers (n = 8238):
87
All-cause mortality, endocrine, SMR <1.00 (p <0.05)
circulatory, respiratory and GI
disease
Leukaemia SMR = 0.86 (0.32-1.88)
Nelson et al.
National population All-cause mortality, all cancer SMR <1.00 (p <0.05)
9187 male workers employed for 6 months
(1987)
and CV, respiratory and GI
at 10 US refineries from 1970-80 and
disease
followed up for 2-12 years
All CBLS SMR = 0.60 (0.34-0.97)
10/11 deaths from skin cancer were due to
Skin cancer SMR = 2.01(1.00-3.60)
melanoma
Pukkala (1998)
National population The breast cancer cases were
All workers:
9454 workers employed for 3 months in 3
concentrated among clerical workers
Kidney cancer SIR = 1.97 (1.29-2.88)
refineries, 1 petrochemical plant and the
(SIR = 1.70 (1.08-2.56)), particularly in the
Male workers (n = 7512):
head office of an oil company in Finland
head office (SIR = 2.29 (1.25-3.84)), and
Non-Hodgkin's lymphoma SIR = 2.01 (1.00-3.59)
from 1967-82 and followed up for 13-28 years
the SIRs did not differ from those found in
other studies of Finnish women in office
Female workers (n = 1942):
jobs
Breast cancer SIR = 1.50 (1.05-2.08)
Table 11.4: Continued
Exposed population Controls Ratio (95% CI) Comments Reference
Health outcome*
Petroleum production, refining and distribution: Continued
SMR <1.00 (p <0.05) The SMR for melanoma was not elevated Rushton
All-cause mortality, stroke
34,569 men employed at 8 UK refineries for 1 Regional populations
in workers first employed before 1955, but (1993a)
all heart, respiratory and
continuous year between 1950-75 and followed
reached 2.30 (1.05-4.37) and 4.67 (2.02-
liver disease, all cancer and
up for 15-40 years
9.20) in workers first employed between
cancer of the mouth,
1955-64 and after 1965 respectively. It
pharynx, lung and pleura
varied markedly between refinery locations
SMR = 1.20 (1.07-1.35) and was higher among office staff than in
Diseases of the arteries
SMR = 0.97 (0.76-1.24) workers employed outdoors.
Leukaemia
SMR = 1.78 (1.20-2.54)
Melanoma
UK national Thorpe (1974)
SMR = 0.77 (0.41-1.13)
All leukaemias
Workers representing 383,276 man-years of In a subgroup of workers exposed for 5
employment in 1962-71 at 8 European affiliates population years to streams containing 1% benzene,
of a US oil company the SMR for leukaemia was 1.21 (0.37-
Wong & Raabe
MSMR = 1.02 (0.93-1.11) Meta-analysis of leukaemia mortality by
All leukaemias
Meta-analysis of 19 cohorts comprising 208,741 Various national and
regional populations (1995); Raabe
MSMR = 1.16 (0.81-1.61) cell type; no data on other death rates
Acute lymphocytic
refinery, production, pipeline and distribution
& Wong (1996)
leukaemia
workers ever employed in USA and UK from
MSMR = 0.96 (0.81-1.13)
Acute myeloid leukaemia
1937-1989 and followed up for 13-50 years
88
MSMR = 0.84 (0.67-1.04)
Chronic lymphocytic
leukaemia
MSMR = 0.89 (0.68-1.15)
Chronic myeloid leukaemia
Various national and Wong & Raabe
MSMR = 0.90 (0.82-0.98)
Non-Hodgkin's lymphoma
Meta-analysis of 26 cohorts comprising
regional populations (2000)
more than 308,000 refinery, production and
distribution workers ever employed in
Australia, Canada, Finland, Italy, USA
and UK from 1937-1996
Printing
National population Paganini-Hill et
SMR = 3.03 Exposed to printing inks and solvents
Kidney cancer
1361 men ever employed as rotary press
al. (1980)
SMR = 2.05 containing benzene
Liver cirrhosis
workers in Los Angeles from 1949-65 and
SMR = 2.47 No analysis for statistical significance
Leukaemia
followed up till 1980
Tyre manufacturing
National population McMichael et
SMR <1.00 No exposure assessment, but `benzene
All-cause mortality
18,903 male workers employed for 10 years
al. (1976)
SMR = 1.48/1.16/1.19 was once the most widely used organic
Stomach/colon/prostate
at 4 tyre manufacturing plants in Ohio and
solvent in the industry'
cancer
Wisconsin, USA, from 1945-1964 and
SMR = 1.31 No analysis for statistical significance
CBLS
followed up for 10 years
Priority Existing Chemical Number 21
SMR = 1.30
Leukaemia
SMR = 1.58
Lymphocytic leukaemia
SMR = 1.29
Lymphoma
Table 11.4: Continued
Benzene
Exposed population Controls Ratio (95% CI) Comments Reference
Health outcome*
Vehicle mechanics
All-cause mortality and
335 predominantly black men employed for Regional population Hunting et al.
SMR <1.00 (p <0.05) Regular contact workers used petrol to
liver and GI disease (1995)
clean engine parts and wash hands or
1 year as vehicle maintenance workers in
CBLS (all workers) SMR = 3.63 (0.75-10.63) siphoned petrol by mouth
Washington, DC, from 1977-89 and
CBLS (regular contact SMR = 9.26 (1.12-33.43)
followed up for 3-15 years
workers)
Miscellaneous industries
All-cause mortality and
74,828 male and female workers employed 35,805 unexposed Yin et al.
No difference
all cancer deaths
from 1972-87 in the painting, printing, workers (1996)
Fatal CBLS
footwear and chemical industries in RR = (2.5-)
Fatal leukaemia
China and followed up for 1-15 years RR = 2.3 (1.1-5.0)
Fatal lymphoma RR = 4.5 (1.3-28.4)
89
* CBLS = cancer of the blood and lymphatic system; CV = cardiovascular; GI = gastrointestinal; GU = genitourinary.
IC = incalculable (no observed cases); MSRM = meta-SMR; SIR = standardised incidence ratio; SMR = standardised mortality
ratio; RR = relative risk; = indefinite (no cases in the unexposed group).
Cohort studies with detailed benzene exposure assessments
There are four occupational cohort studies in which the exposure to benzene has
been assessed in detail.
The Chinese cohort
The US National Cancer Institute and the Chinese Academy of Preventive
Medicine have collaborated to follow up on a large cohort study commenced in
1982 to assess the risks of specific bone marrow disorders in relationship to
occupational benzene exposure (Hayes et al, 1997). The final cohort comprises
74,828 male and female benzene-exposed workers employed from 1972 to 1987 in
672 factories in 12 cities in China and 35,805 unexposed workers. The subjects
were followed until the end of 1987, for an average of approximately 11 years. RRs
were determined for incident cancer of the blood and lymphatic system, NHL,
leukaemia, ANLL, a diagnosis of either ANLL or MDS, and leukaemia other than
ANLL, with stratification by age and sex. Smoking or other potential confounders
were not considered. The exposed workers held permanent jobs in the painting,
printing, footwear, rubber and chemical industries. Exposure levels were estimated
from available area monitoring data, detailed production and process information,
and employee records.
There were 58 specified cancers of the blood and lymphatic system and 18 other
bone marrow disorders (2 cases of agranulocytosis, 9 of aplastic anaemia and 7 of
MDS) in the cohort, compared to 13 and 0 respectively in the control group.
When the cohort was divided into three categories according to the estimated
average benzene exposure level, the RRs for all cancer of the blood and lymphatic
system and ANLL/MDS were elevated in all categories, with a positive trend for
increasing average exposure, as shown below:
Estimated average exposure:
Cancer type/R: <10 ppm 10-24 ppm >25 ppm Trend
Blood and
lymphatic system 2.2 (1.1-4.2) 3.1 (1.5-6.5) 2.8 (1.4-5.7) p = 0.003
ANLL/MDS 3.2 (1.0-10.1) 5.8 (1.8-18.8) 4.1 (1.2-13.2) p = 0.01
The RR for NHL was 4.7 (1.2-18.1) in workers exposed to 25 ppm, but was not
elevated in the lower average exposure categories.
When the cohort was divided into three categories according to the estimated
cumulative benzene exposure level, the RR for all cancer of the blood and
lymphatic system was elevated in all categories, whereas the RRs for leukaemia
and ANLL/MDS were elevated at cumulative exposures 40 ppm-years:
Estimated cumulative exposure
Trend
Cancer type/RR <40 ppm-years 40-99 ppm-years 100 ppm-years
Blood and
lymphatic system 2.2 (1.1-4.5) 2.9 (1.3-6.5) 2.7 (1.4-5.2) p = 0.004
Leukaemia 1.9 (0.8-4.7) 3.1 (1.2-8.0) 2.7 (1.2-6.0) p = 0.04
ANLL/MDS 2.7 (0.8-9.5) 6.0 (1.8-20.6) 4.4 (1.4-13.5) p = 0.01
The RR for NHL was not elevated in any of the three cumulative exposure
categories.
Priority Existing Chemical Number 21
90
Only NHL was linked to duration of exposure. None of the RRs were related to the
year of initial employment in the study factories. ANLL/MDS was linked to recent
exposure (<10 years prior to diagnosis), whereas NHL was linked to distant
exposure (10 years prior to diagnosis).
The authors concluded that the results suggest an association between benzene
exposure and a spectrum of blood cancers and related disorders, with an increase in
cancer risk at cumulative exposures <40 ppm-years and a tendency, although not
strong, for the risk to rise with increasing levels of exposure.
It should be noted that personal monitoring in a subset of the Chinese cohort
measured current exposure levels which were reported to be `much higher than
expected' compared to the estimates that were made in the course of the main study
(Rothman et al, 1995, 1996b). As such, the historical exposure levels used to
determine the dose-response relationship may have been grossly underestimated
(Budinsky et al, 1999; EPA, 1998a; Wong, 1999).
Overall mortality rates in the Chinese cohort have been reported by Yin et al.
(1996) and are summarised in Table 11.4. The average latency period of fatal
leukaemia in benzene-exposed workers was estimated at 11-12 years, with a range
from 10 months to 50 years (Yin et al, 1987b).
The CMA cohort
The Chemical Manufacturers Association (CMA) sponsored a study of 4602 male
chemical workers who were employed for 6 months from 1946-75 at 7 US plants
(Wong, 1987a, 1987b). Two comparison groups were used: the general US
population and 3074 unexposed male workers employed at the same plants at the
same time as the cohort. Smoking or other potential confounders were not
considered. The vital status of all subjects was followed up until the end of 1987
and the findings compared to average and peak exposures as determined from
available air monitoring data and employment records obtained from the
participating companies.
There were 19 deaths from cancer of the blood and lymphatic system in the
exposed workers compared to 3 in the unexposed group. In the exposed group, 7 of
the observed cases were diagnosed with leukaemia and the remaining 12 with
lymphoma. The subjects with leukaemia comprised 1 case with acute lymphatic
leukaemia (ALL), 2 with chronic lymphatic leukaemia (CLL), 1 with unspecified
lymphatic leukaemia, 2 with chronic myeloid leukaemia (CML) and 1 with
unspecified acute leukaemia. In the unexposed workers, all 3 cases were diagnosed
with lymphoma. The SMRs for all cancers of the blood and lymphatic
system/leukaemia reached 0.91/0.97, 1.47/0.78 and 1.75/2.76 for cumulative
exposures of <180, 180-719 or 720 ppm-months respectively, but none of the
ratios was significantly different from unity. The RRs for all cancers of the blood
and lymphatic system were 2.10, 2.95 and 3.93 respectively for the same
cumulative exposure groups, with p = 0.02 for trend. The RRs for leukaemia were
indefinite as there were no cases in the unexposed workers, with p = 0.01 for trend
with cumulative exposure. There was no correlation with peak levels or duration of
exposure.
Based on the RRs and their trend with cumulative exposure, the author concluded
that workers exposed to benzene exhibited a significant excess of deaths from
leukaemia as well as from the broader category of all cancers of the blood and
Benzene 91
lymphatic system when compared with workers who were not exposed to the
chemical.
Ireland et al. (1997) conducted an extended mortality study in production personnel
from one of the plants included in the CMA-sponsored study. The workers were
stratified into three categories based on cumulative exposure: <12 ppm-months (n =
666), 12-72 ppm-months (n = 378) and 72 ppm-months (n = 164). Compared to
the regional population, the SMR for leukaemia was 2.5 (0.3-8.9) in the lowest,
incalculable (0.0-5.9) in the middle, and 4.6 (0.9-13.4) in the highest exposure
category, with no clear dose-response relationship.
The Dow Chemical cohort
This cohort comprised 956 male chemical workers employed at a single site in
Michigan, USA, between 1940 and 1982. The workers were exposed to benzene in
chlorobenzene or alkylation plants which used benzene as a raw material, or in an
ethyl cellulose plant where benzene was used as a solvent (Bond et al, 1986; Ott et
al, 1978). They were followed up until the end of 1982. The average exposure
duration and length of follow-up were 7 and 26 years respectively. Each job entry
was assigned an exposure intensity level on the basis of job classification and
representative personal air monitoring data.
The analysis accounted for co-exposure to arsenic, asbestos or high levels of vinyl
chloride. Smoking or other potential confounders were not considered. There were
6 deaths from cancer of the blood and lymphatic system against 4.8 expected,
including 4 cases of myelogenous leukaemia against 0.9 expected, and 4 from skin
cancer (3 melanomas and 1 squamous cell carcinoma) against 0.9 expected, using
concurrent US white male mortality rates as reference values. The excess of
myelogenous leukaemia was statistically significant (p = 0.011; SMR and 95% CI
not stated) and the risk for skin cancer was significantly elevated (SMR = 4.41
(1.21-11.38)). There were no significant trends with either work area, cumulative
exposure or duration of exposure. Of the 6 cases of blood and lymphatic system
cancer, 4 had been exposed to <500 ppm-months and 2 to 1000 ppm-months. In
the case of myelogenous leukaemia, cumulative exposures varied from 18-4211
ppm-months. The 4 cases of skin cancer all occurred in workers with exposures
<500 ppm-months, but otherwise had no unusual or common characteristics.
The authors concluded that their study provided support for an association between
exposure to benzene and myelogenous leukaemia.
The Pliofilm cohort
An excess incidence of leukaemia in rubber workers at two Goodyear facilities in
Ohio, USA was reported in a preliminary paper by Infante et al. (1977) and in more
detail by Rinsky et al. (1981). Depending on its definition, this cohort comprises
1165-1212 male workers employed from 1936-75 in the manufacture of Pliofilm,
which is a material made from rubber hydrochloride (Paxton et al, 1994a; Rinsky et
al, 1987). The manufacturing process used large volumes of benzene as a solvent
and there was no exposure to other known carcinogenic substances. The last worker
joined the cohort in 1965 and the most recent follow-up was in 1987.
Excluding deaths before 1950, Rinsky et al. (1987) identified 15 deaths from
lymphatic and haematopoietic cancers versus 6.6 expected (SMR = 2.27 (1.27-
3.76)) and 9 deaths from leukaemia versus 2.7 expected (SMR = 3.37 (1.54-6.41)).
In a later analysis that included deaths between 1940-50, Paxton et al. (1994a)
Priority Existing Chemical Number 21
92
identified 21 deaths from lymphatic and haematopoietic cancers versus 9.51
expected (SMR = 2.21 (1.37-3.38)) and 14 deaths from leukaemia versus 3.89
expected (SMR = 3.60 (1.97-6.04)). Neither of these analyses considered smoking
or other potential confounders.
The individual exposure histories of the cohort members were reconstructed after
the plants closed in 1975, from fairly detailed monitoring and health surveillance
data and other information on record.
Based on unpublished exposure estimates, Rinsky et al. (1987) found SMRs for
leukaemia of 1.09 (0.12-3.94) at a cumulative exposure <40 ppm-years, 3.22 (0.36-
11.65) at 40-200 ppm-years, 11.86 (1.33-42.85) at 200-400 ppm-years and 66.37
(13.34-193.93) at >400 ppm-years.
Paxton et al. (1994a) recalculated the SMRs for a different set of cumulative
exposure categories and compared them with similar statistics derived from
independent, more detailed exposure estimates produced by Crump & Allen
(unpublished report prepared for the Occupational Safety and Health
Administration in 1984) and Paustenbach et al. (1992), as shown in Table 11.58.
The results reproduced in the table suggest a strong dose-response relationship of
risk increasing with cumulative exposure, no matter which estimate is used, and
indicate that there is a significantly elevated risk for leukaemia (according to 2 of
the 3 available exposure estimates) at a cumulative dose >50 ppm-years,
corresponding to a long-term average exposure of 1.25 ppm over 40 years.
Table 11.5: SMRs (95% CI) for leukaemia in the Pliofilm cohort, analysed by
cumulative exposure as estimated by Crump & Allen (1984, unpublished),
Paustenbach et al. (1992) and Rinsky et al. (1987)(from Paxton et al, 1994a)
Cumulative Exposure estimate
exposure
(ppm-years) Crump & Allen Paustenbach et al. Rinsky et al.
0-5 0.88 (0.02-4.89) 1.33 (0.03-7.43) 1.97 (0.41-5.76)
>5-50 3.25 (0.88-8.33) 1.79 (0.22-6.45) 2.29 (0.47-6.69)
6.93 (2.78-14.28)
>50-500 4.87 (1.79-10.63)* 2.80 (0.76-7.16)
10.34 (2.13-30.21) 11.86 (4.76-24.44)
>500 20.00 (0.51-111.4)
* p <0.05
p <0.01
As the SMR was not significantly different from unity at cumulative exposures 50
ppm-years for any of the three exposure estimates, the authors concluded that the
results of the analysis were consistent with a threshold model for benzene-induced
leukaemia. However, the power of the analysis was insufficient to support this
conclusion. The upper 95% confidence limits given in Table 11.5 range from 6.45-
8.33 in the >5-50 ppm-year exposure category and from 4.89-7.43 the 0-5 ppm-
year category. In either case, the upper limits are well above unity irrespective of
the exposure estimate used. Therefore, it cannot be excluded that a cumulative
exposure level 50 ppm-years is also associated with an excess mortality from
leukaemia.
8
The major distinction between the three exposure estimates is the disregard by Rinsky et al. (1987)
for the likely increase in exposure levels during and in the aftermath of World War II because of
wartime conditions and longer working hours. In addition, only Paustenbach et al. (1992) have given
consideration to the potential for dermal exposure.
Benzene 93
Wong (1995) reanalysed the findings of Paxton et al. (1994a) by cell type (AML
and multiple myeloma (MM)), using the Rinsky et al. (1987) exposure estimate
which in general is the lowest of the three. He found no relationship between
cumulative exposure and the risk of MM, whereas the SMR for AML showed a
clear dose response, as follows:
Cumulative exposure SMR (95% CI) Statistical significance
<200 ppm-years 0.91 (0.02-5.11) Not significant
200-400 ppm-years 27.21 (3.29-98.24) p <0.01
>400 ppm-years 98.37 (20.28-287.65) p <0.01
Total cohort 5.03 (1.84-10.97) p <0.01
The author concluded that there was no significant increase in the risk of AML for
cumulative exposure to benzene <200 ppm-years, above which the risk rose sharply
to a very substantial SMR of 98.37 for >400 ppm-years. However, as the 95%
upper confidence limit in the lowest exposure group was 5.11, an increase in
mortality from AML at a cumulative exposure <200 ppm-years cannot be ruled out.
In another re-analysis based on the three sets of exposure estimates referred to
above, Schnatter et al. (1996b) used the work history of each Pliofilm worker to
define each worker's maximally exposed job/department combination over time
and the long-term average benzene exposure level associated with the maximally
exposed job. They then determined the number of observed and expected cases of
leukaemia (all cell types) in subcategories of workers and person-years who were
always exposed at levels that did not exceed specific concentrations of benzene. As
shown in Table 11.6, this analysis showed that there were fewer observed than
expected deaths in all subcategories that were always exposed to benzene
concentrations 15 ppm, irrespective of the exposure estimate used. However,
because of the low number of expected cases, this finding could also be due to
chance.
Table 11.6: Observed and expected cases of leukaemia (all cell types) for
selected cut-off points for the average long-term exposure level in the
maximally exposed job (Schnatter et al, 1996b)
Long-term Exposure estimate
benzene
Crump & Allen Paustenbach et al. Rinsky et al.
exposure
(ppm) Observed Expected Observed Expected Observed Expected
1 0 0.53 0 0.07 1 1.53
5 0 1.01 0 0.10 1 1.72
10 0 1.04 0 0.11 1 2.00
15 0 1.28 0 0.15 1 2.00
20 2 1.92 0 0.21 3 2.30
25 2 2.13 1 0.80 7 2.92
30 3 2.35 1 0.90 7 3.24
40 5 2.73 1 1.33 10 4.04
50 5 2.98 3 1.96 14 4.79
100 8 3.98 5 3.55 14 4.87
200 9 4.20 14 4.70 14 4.87
260 14 4.87 14 4.87 14 4.87
Priority Existing Chemical Number 21
94
Conclusions
Cohort studies with poorly characterised benzene exposure levels
Several of the studies summarised in Table 11.4 have associated cancer of the
blood and lymphatic system (including but not limited to leukaemia) with the
obsolete practice of using benzene-containing adhesives, cleaners and solvents.
Some studies indicate a positive association with long-term employment at
petroleum refineries, in the chemical industry or in highway maintenance. There
was no excess mortality from leukaemia in three cohorts of coke plant workers.
The risk for malignant melanoma or skin cancer (mainly melanoma) was elevated
in three petroleum industry cohorts (Health Watch, 1998; Nelson et al, 1987;
Rushton, 1993a). The SIR for breast cancer was elevated in female workers in a
Finnish oil company cohort (Pukkala, 1998). However, the elevation was mainly
due to cases among clerical workers and similar in magnitude to that found in other
studies of Finnish women in office jobs.
Cohort studies with detailed benzene exposure assessments
There was an excess mortality from cancer of the blood and lymphatic system in all
four cohorts for which detailed benzene exposure assessments are available and a
significant trend with cumulative exposure in all but the smallest cohort (the Dow
Chemical cohort). As such, it is widely accepted that these studies provide
sufficient evidence of a clear dose-response relationship between benzene exposure
and the broad category of all cancers of the blood and lymphatic system (ATSDR,
1997; OECD, 2000; IARC, 1982a; IPCS, 1993; USEPA, 1998a). In terms of
specific cancer categories, the relationship is primarily due to the risk for AML
(ANLL).
In the CMA cohort, the SMR for leukaemia was elevated (2.6) in workers with a
cumulative exposure of 720 ppm-months (that is, 60 ppm-years), but it was not
significantly different from unity and therefore could have been due to chance. In a
subset of the CMA cohort, the SMR for leukaemia was 4.6 in workers with a
cumulative exposure of 72 ppm-months (6 ppm-years), but again did not differ
significantly from unity. There was no clear dose-response relationship in the Dow
Chemical cohort and there is doubt about the true exposures in the Chinese cohort.
As such, the Pliofilm cohort is the most suitable for the determination of the
carcinogenic potency of benzene. In addition, the Pliofilm cohort has the advantage
of limited if any co-exposure to other potentially carcinogenic compounds and a
very long follow-up period. However, it suffers from uncertainty about actual
exposure levels, particularly prior to 1950, which is important as there are no cases
of leukaemia in workers first employed after that year (USEPA, 1998a).
Based on an unpublished assessment of individual exposures in the Pliofilm cohort,
Rinsky et al. (1987) determined SMRs for leukaemia that increased exponentially
with cumulative exposure, starting from near unity at a cumulative exposure <40
ppm-years. More recent dose-response analyses that include other, more
comprehensive exposure assessments indicate that the risk for leukaemia is
significantly elevated at a cumulative exposure level above, but not below 50 ppm-
years, corresponding to an average exposure level of 1.25 ppm over 40 years
(Paxton, 1994b). Moreover, whatever exposure estimate was used, the number of
observed cases of leukaemia was consistently below the expected number in all
workers whose long-term exposure never exceeded 15 ppm (Schnatter et al,
1996b). However, because of the limited statistical power resulting from the size of
Benzene 95
the Pliofilm cohort, these results do not rule out the possibility of an increased risk
of leukaemia at exposure levels lower than those cited above.
In the Dow Chemical cohort, there was an association between benzene exposure
and skin cancer. However, all cases occurred in the lowest cumulative dose group
(<500 ppm-months) and there was no trend with either level or duration of
exposure.
11.6.2 Case-control studies
The case-control studies reviewed below have been divided by organ system. They
comprise studies based in a specific industry, such as petroleum refining, and
studies conducted in a community population. Limitations in statistical power and
study quality, particularly in relation to exposure assessment and/or control for
potential confounders, pervade all of the studies reviewed.
Cancer of the blood and lymphatic system
Industry-based studies
A study nested within a cohort of male workers at a large tyre manufacturing plant
in Ohio, USA included 11 cases of lymphocytic leukaemia and 1350 controls
(Checkoway et al, 1984). The OR for direct exposure through routine use or
handling of benzene or benzene-containing solutions was 4.50 (95% CI not stated),
but did not reach statistical significance (p = 0.22). The ORs for exposure to
acetone, carbon disulfide, carbon tetrachloride, ethyl acetate, hexane or methanol
ranged from 4.3-18 (95% CIs not stated) and were all statistically significant.
Austin et al. (1986) compared 14 cases of leukaemia in white male workers at a US
refinery, including 8 cases of AML, with 50 controls. Neither job category,
department nor length of employment was a significant risk factor.
In an exploratory study of cancer mortality at a transformer assembly facility in
Massachusetts, USA, where benzene was used for general cleaning purposes until
1950, benzene exposure was not a significant risk factor for leukaemia (OR = 1.4
(0.64-3.2)) (Greenland et al, 1994).
Sathiakumar et al. (1995) studied 69 workers with leukaemia, predominantly AML
or CLL (numbers not specified), and 284 controls who had worked for the same
US-based petroleum company for 1 year from 1976-90. Forty-four risk factors
tested for included site of work, involvement in production, job category, duration
and year of employment. The only risk factors identified were for AML and
included length of employment, with an OR = 8.7 (2.0-37.2) in workers employed
for >30 years (trend: p = 0.01), and upstream employment in crude oil production
or maintenance (OR = 3.2 (1.1-9.2)).
Schnatter et al. (1996a) compared 7 cases of NHL, 7 cases of MM and 55 controls
drawn from the cohort of Canadian petroleum distribution workers described by
Lewis et al. (2000). Tests included several measures for benzene exposure. The
only risk factors identified were a family history of cancer and cigarette smoking,
with cumulative benzene exposure showing no additional risk.
A study nested in the cohort of British petroleum distribution and marketing
workers described by Rushton (1993b) compared 91 cases of leukaemia,
predominantly ANLL (31) and CLL (31), with 364 controls (Rushton & Romaniuk,
1997). Risk factors tested for included cumulative, mean and maximum airborne
Priority Existing Chemical Number 21
96
and potential skin exposure to benzene, duration of employment, date of hire,
employment as driver, socio-economic status, and age at and years from start of
work. For ANLL, none of the ORs differed from unity. For CLL, the risk factors
identified included duration of employment, white-collar status and years of work,
but not exposure to benzene.
The case-control study nested within the cohort of Australian petroleum industry
workers currently comprises 63 cases with lympho-haematopoietic cancers, mainly
NHL, MM, AML and CLL, and 315 controls (Health Watch, 1998). In the analysis,
the OR was used to compare groups with different levels of exposure to various
potential causative agents, relative to the least exposed or baseline group.
Compared to the baseline of the rate in refineries, the OR was marginally elevated
for work in terminals (1.8 (1.0-3.5)). Length of employment and period of first
employment were not significant risk factors. Past exposure to benzene was ranked
on a scale from 1-5, depending on AIP jobcode, the company site where the job
was carried out and length of service in any job. When cases and controls were
compared to the highest benzene rank of any job ever held (ranks 4-5), the OR was
7.9 (1.6-39) times higher than for rank 1 (the baseline). When compared to the
benzene rank of the job held longest, the OR was 3.2 (1.1-9.4) times higher than
baseline for rank 3 and 6.6 (1.4-30) times higher for rank 4 (the highest rank in this
test). The authors concluded that a relatively higher exposure to benzene might be
the significant factor leading to an increased risk of leukaemia and MM in the
cohort study.
Nilsson et al. (1998) conducted a nested case-control study of Swedish seamen with
two study bases. These comprised a total of 92 men who were registered as seamen
at the national censuses in Sweden in 1960 and 1970 respectively and recorded in
the Swedish National Cancer Register with cancer of the blood and lymphatic
system from 1971-88. The controls were 291 age-matched men registered as
seamen at the same censuses. NHL (37) and leukaemia (30) accounted for most of
the cases. There were no increased risks for the 1960 cohort, in which few cases
were exposed to benzene or petrol. In the 1970 cohort, the OR was increased for
cancer of the blood and lymphatic system (OR = 2.6 (1.1-5.9)) and for NHL (OR =
3.3 (1.1-10.6)) in seamen who had worked on deck on chemical or petroleum
product tankers, but not on crude oil tankers.
Wong et al. (1999) studied 59 cases of leukaemia, including unspecified leukaemia
(35), AML (13) and MM (11), and 220 controls drawn from a US-based cohort
study of 18,135 petrol distribution workers (Wong et al, 1993). Test variables
included duration of employment, duration of exposure, job category, cumulative
exposure to hydrocarbons, cumulative frequency of peak exposure to hydrocarbons,
and year of first exposure. None of these was identified as a risk factor for any of
the study diagnoses.
In a study nested within the Pliofilm cohort described above, Finkelstein (2000)
examined the temporal variation of leukaemia risk following exposure to benzene.
Each leukaemia case was matched with 6-333 control subjects and the exposure of
cases and controls were then assessed according to Rinsky et al. (1987) and
compared at various times before the death of the case subject. As expected,
leukaemia risk was significantly associated with cumulative exposure (p = 0.024).
However, exposures incurred in the previous 10 years were found to account for
most of the risk and there was no significant difference in the benzene exposure of
cases and controls 15 or more years prior to the death of the case subject.
Benzene 97
Community-based studies
Exposure to benzene and/or toluene was investigated in 401 cases of various
serious blood disorders and 124 controls sampled from the same general hospital in
Lyon, France (Girard & Revol, 1970). The prevalence of exposure was
significantly higher among patients with acute leukaemia, CLL and aplastic
anaemia than in the comparison group. The majority of the exposed patients had
worked in small workshops where the main sources of exposure were reported to
be cleaning fluids and paint and glue thinners.
Ishimaru et al. (1971) interviewed 303 matched pairs of controls and cases of
leukaemia (not further specified) with onset from 1945-67 in Hiroshima or
Nagasaki in Japan. The OR was 2.5 (p <0.01) among those with a history of any of
11 occupations with the potential for frequent exposure to benzene or x-rays and
showed a positive trend with the length of time in those occupations.
Eighteen (36%) out of 50 working men with ANLL seen at a hospital in Lund,
Sweden, were occupationally exposed to petroleum products or vehicle exhaust
fumes through occupation as petrol stations attendants, drivers or operators of
excavators or power saws (Brandt et al, 1978). By comparison, similar exposure
patterns occurred in only 10% of three outpatient control groups (p = 0.0002),
including a group of male patients with CML or CLL (p = 0.006), or 10-11% of the
general male population in the region.
Linos et al. (1980) compared 138 cases of acute or chronic leukaemia (not further
specified) that occurred in residents in a county in Minnesota, USA between 1955-
74 with 276 controls, with regard to past occupational and chemical exposure. The
OR for exposure to benzene was not significantly elevated (3.34 (0.60-27.60)).
In a study of 131 cases of MM, 111 cases of CLL and 431 controls resident in a
rural woodland district in central Sweden, Flodin et al. (1987, 1988) observed an
association with occupational exposure to exhaust fumes from diesel and petrol
engines, including tractors and chainsaws. The OR was 2.1 (1.2-3.9) for MM and
2.2 (1.2-4.2) for CLL.
Another Swedish study of 125 cases of acute leukaemia (including 97 AML and 24
ALL cases) and an equal number of controls found a large excess risk for
professional painters exposed to solvents that would have contained benzene as an
impurity (OR = 13 (2-554) (Lindquist et al, 1987). There was also an excess risk
among professional drivers, with an OR = 3.0 (1.1-9.2). The OR reached 5.0 (95%
CI not stated; p <0.05) for those who had been drivers for >5 years in their lifetime
or >1 year during the 5-20-year period prior to diagnosis and remained after
adjustments for exposure to organic solvents, smoking and therapeutic x-ray
treatment (Lindquist et al, 1991).
A study of 475 cases of lymphoma, leukaemia and MM in white male residents in
Missouri, USA, and 1425 controls found an elevated risk of leukaemia in
mechanics (OR = 4.79 (1.42-16.18)) (Brownson & Reif, 1988).
Richardson et al. (1992) conducted an interview study of occupational risk factors
of acute leukaemia in French adults, based on 31 cases of ALL, 154 cases of AML
and 513 controls. A significant relationship was observed between AML and high
or medium exposure to benzene (OR = 3.6 (1.7-7.7)). For ALL and AML
combined, the OR for any exposure to benzene was 1.3 (0.8-2.3), whereas it was
2.8 (1.3-5.9) for high or medium exposure.
Priority Existing Chemical Number 21
98
In an interview study of 622 white males with NHL and 1245 controls drawn from
the general population in Iowa and Minnesota, USA during 1980-83, there were no
indications that industrial exposures were a major determinant for NHL (Blair et al,
1993). The OR for benzene exposure was close to unity, but did increase slightly
with intensity of exposure (lower intensity: OR = 1.1 (0.8-1.4); higher intensity:
OR = 1.5 (0.7-3.1)).
In a study of 86 cases of AML, CML or MDS in residents in Turin, Italy, there was
a marginally elevated risk of leukaemia/MDS in vehicle mechanics (OR = 2.7
(0.97-7.6)) and truck and other drivers (OR = 2.7 (0.8-9.6)), but no association with
exposure to benzene (Ciccone et al, 1993).
A French, hospital-based case-control study of 226 male cases of hairy cell
leukaemia and 425 matched controls found no association between occupational
exposure to benzene and this rare B-lymphoid chronic leukaemia (Clavel et al,
1996).
A recent review by Savitz & Andrews (1997) identified 12 additional community-
based case-control studies of benzene and cancer of the blood and lymphatic
system, none of which reported any association between the two.
Childhood leukaemia
Leukaemia is the most common cancer in children under the age of 15 (Shu, 1997).
A number of case-control studies have explored the potential relationship between
childhood leukaemia and parental exposure to agents that might be toxic to the
unborn or breast-fed baby and/or the germ cells of the parents.
Some of these studies have suggested a link between childhood leukaemia and pre-
conceptional occupational exposure of the father to solvents, petroleum products,
motor vehicle exhaust fumes, benzene, or plastic monomers or polymers (Buckley
et al, 1989; Fabia & Thuy, 1974; McKinney et al, 1991; Shu et al, 1999; Vianna et
al, 1984). Others have found a weak association with maternal employment in jobs
with the potential for exposure to various chemicals including benzene, petrol,
solvents and thinners, paints and/or plastic monomers or polymers (Shu et al, 1988,
1999; van Steensel-Moll et al, 1985).
A study of 123 cases of childhood leukaemia and an equal number of matched
controls found a significant, dose-related elevation in the risk of leukaemia for
children whose parents burned incense in the house during pregnancy or lactation
(Lowengart et al, 1987). Incense stick has been reported to emit the same quantity
of benzene in smoke as tobacco and herbal cigarettes (Löfroth et al, 1991).
In a study of 97 cases of childhood leukaemia (78 of whom had ALL) and 259
matched controls from Denver, USA, Pearson et al. (2000) found an association
between childhood leukaemia and proximal high traffic streets with traffic counts
20,000 vehicles per day (OR = 8.28 (2.09-32.80)).
Skin cancer
In a study of 307 cases of non-melanoma skin cancer (basal and/or squamous cell
carcinoma) and 229 controls resident in Texas, USA, the most important risk
factors were red hair, fair skin, outdoor sun exposure, and a family history of skin
cancer (Gamble et al, 1996). Employment at any time in the petroleum industry
was associated with a slightly elevated risk of developing concurrent basal and
squamous cell carcinomas (OR = 2.10 (1.08-4.09)).
Benzene 99
In a Dutch study of 140 cases with non-metastasised melanoma and 181 controls
with other types of malignancy, increased risks were found for subjects ever
employed in the electronics, metal and transport and communication industries
(Nelemans et al, 1993). However, they were not statistically significant and there
were no trends for duration of employment or latency. Also, there was no increase
in risk for workers in the chemical industry.
The American Cancer Society enrolled 1.2 million randomly selected people in a
study of life style and environmental factors in relation to cancer mortality, 2780 of
whom had a history of or developed melanoma during the 6-year study follow-up
period (Pion et al, 1995). These cases were compared with controls selected from
the remaining people enrolled on a 1:3 basis and matched for age, sex, race, and
geographic location. In men, the risk of melanoma was elevated in high-paying
versus low-paying jobs (OR = 1.58; p <0.001) and in white-collar versus blue-
collar jobs (OR = 1.33; p <0.001), but unrelated to outdoor versus indoor
occupations. In women, the findings were inconclusive. The only specific work-
related risk factor was exposure to x-rays. Other large community-based studies in
Australia, Britain and Sweden came to similar results (Burnley, 1997; Vågerö et al,
1990).
Other cancers
Gérin et al. (1998) conducted a community-based case-control study of 19 specific
cancers excluding leukaemia in 3730 men and 533 controls aged 35-70 years and
resident in Montreal, Canada. Their exposure to various workplace chemicals
including benzene, toluene, xylenes and styrene was estimated through interviews
and from workplace records. There were 737 subjects, mainly mechanics, service
station attendants and shoe workers, who had been exposed to benzene, usually
with concomitant exposure to toluene and xylenes. However, there was no evidence
that the risks of common cancers such as those of the gastrointestinal tract, lungs,
prostate, bladder or kidney were related to exposure to any of the chemicals under
investigation. For NHL (215 cases) and melanoma (103 cases), the ORs for
benzene exposure were <1.00.
The industry-based study by Wong et al. (1999) mentioned above under cancer of
the blood and lymphatic system also included 12 cases of kidney cancer. There was
no difference between cases and controls with regard to duration of exposure or to
cumulative or peak exposures to hydrocarbons.
Petralia et al. (1999) studied the relationship between the risk of pre-menopausal
breast cancer and exposure to benzene or polycyclic aromatic hydrocarbons in 301
cases and 316 controls sampled from two counties in New York State between
1986-91. There were 55 breast cancer cases and 35 controls who had been exposed
to benzene, mainly through employment as laboratory technicians, painters,
sculptors, craft-artists, or assemblers in the motor vehicle industry. Following
adjustment for age, years of education, age at first birth, age at menarche, history of
benign breast disease, history of breast cancer in a first-degree relative, body mass
index, and months of lactation, four variables relating to benzene had ORs that
reached or approached statistical significance. These were duration of exposure 4
years (2.57 (1.23-4.73)); medium-to-high probability of exposure (1.95 (1.14-
3.33)); low average exposure intensity (2.36 (1.30-4.30)); and medium-to-high
cumulative exposure (1.93 (1.00-3.72)).
In Denmark, Hansen (2000) conducted a nationwide register-based case-control
study on primary breast cancer in men, which included 230 cases and 12,880
Priority Existing Chemical Number 21
100
controls. Allowing for a lag time 10 years and after adjustment for socio-
economic status, the OR was 2.5 (1.3-4.5) in all men with >3 months of
employment as car mechanic or petrol station worker and 5.4 (2.4-11.9) in men
who were <40 years old when first employed in those occupations. Exposure to
benzene was not assessed.
Conclusions
While the case-control studies reviewed above have limitations in statistical power
and study quality, there are several which indicate that occupation and/or benzene
exposure is associated with an increased risk of cancer of the blood and lymphatic
system, including, but not limited to AML and other leukaemias. Positively
identified risk factors include employment in upstream petroleum production, at
petroleum terminals, on deck on chemical or petroleum product tankers, and as a
mechanic, machinist, chemical worker, chemist, painter, driver or logger. The
studies by Health Watch (1998) and Richardson (1992) found that the risk was
significantly elevated at relatively high, but not at lower levels of exposure to
benzene.
There are some indications that parental exposure to benzene and in particular
maternal exposure during pregnancy may be linked to childhood leukaemia, but the
overall evidence for this association is limited at present. Other tentative findings
suggest a relationship between the risk of breast cancer and exposure of female
workers to benzene on the one hand and between male breast cancer and exposure
to petrol and vehicle exhaust on the other.
11.6.3 Ecological studies
Leukaemia and car traffic variables
Robinson (1982, 1991) found a strong relationship between leukaemia mortality
and vehicle usage (as monitored by the annual rate of vehicle fatalities) in
Australia, France, Germany, Italy, Japan, The Netherlands, UK and USA. In a
study of all incident childhood cancer in Denver, Colerado, USA, between 1976-
83, Savitz & Feingold (1989) found a statistically significant association between
traffic density at the place of residence at the time of diagnosis and the risk for all
leukaemia combined. In a sample of 22 British counties, Wolff (1992) found a
significant correlation coefficient between the incidence of AML, lymphoma, ALL,
CLL and low-grade NHL for the years 1984-88 and the number of cars per
household reported in the 1981 Great Britain Population Census.
Swaen & Slangen (1995) found a non-significant, inverse relationship between
leukaemia mortality and petrol consumption in 19 European countries, but a weak
positive association between the incidence of myeloid leukaemia and the
consumption of petrol per km2. However, both findings could be due to unrelated
factors such as changes in prognosis or country differences in leukaemia case
ascertainment. As such, the authors concluded that their study did not support an
association between petrol consumption and leukaemia incidence or mortality.
Nordlinder & Järvholm (1997) compared the 1975 car density in Swedish local
government areas with the 1975-1985 cumulative incidence of ALL, AML, CML
and NHL in persons under 25. None of these showed a significant correlation with
car density, although the combined group of areas with >5 cars/km2 had a higher
rate of AML than those with <5 cars/km2 (95% CI for the difference: 0.1-4.0
cases/106 person-years).
Benzene 101
Leukaemia and industry emission variables
There was an excess mortality rate between 1950-69 from childhood leukaemia and
young adult Hodgkin's disease and lymphoma among residents in the heavily
industrialised New Jersey-New York-Philadelphia Metropolitan Region compared
to USA as a whole (Greenberg et al, 1980). However, other early studies of
populations residing in the vicinity of petroleum refineries and chemical plants
have not suggested links with cancers of the blood and lymphatic system (Blot et
al, 1977; Hearey et al, 1980; Hoover et al, 1975; Kaldor et al, 1984).
A more recent study of an area within a radius of 3.0 km of a large petrochemical
plant in South Wales, UK, compared the 1974-91 incidence of leukaemia and
lymphoma with onset before age 25 among study area residents with those in the
regional population (Lyons et al, 1995). There were no statistically significant
differences, although the number of observed cases was higher than expected for all
disease types except myeloid leukaemia. Data from ambient monitoring for
benzene around the site showed monthly peak values varying from 4-16 ppb.
11.7 The Illawarra leukaemia cluster
The Illawarra leukaemia cluster refers to a group of 12 cases of leukaemia that
occurred in 1989-96 in three contiguous suburbs bordering the Port Kembla
steelworks near Wollongong, New South Wales (Westley-Wise et al, 1999). There
were 3 cases of AML, 3 of CML and 6 of ALL. All cases were under 44 years of
age at the time of diagnosis and nine were 20 years of age. Four attended the same
local high school in the late 1980s, three of them in the same school year. Using the
rest of the region as reference population, only 3.49 cases were expected,
corresponding to a SIR of 3.44 (1.42-6.92).
The regional health authority launched an investigation which examined a wide
range of possible explanations for the cluster, including benzene emissions from
the coking ovens and coal gas by-product plant at the steelworks. It was estimated
that ambient air levels of benzene had averaged up to 3 ppb since 1970, although
the mean levels measured in 1996 did not exceed 1 ppb within 1.6 km from the
plant. As this is less than one-thousandth of the level at which leukaemia risk has
been identified in occupational epidemiological studies, the authors concluded that
the cause of the cluster was uncertain, although an association with chemical
exposures could not be totally excluded. The odds that it was due to chance were
calculated at 1 in 4-8000 (Westley-Wise & Hogan, 1997).
11.8 Summary and conclusions
There is anecdotal evidence that acute exposure to benzene vapours causes
dizziness and other CNS effects at concentrations above 25 ppm and eye, mucous
membrane and skin irritation at levels above 30-60 ppm. Furthermore, aspiration of
liquid benzene has been observed to cause lung oedema and bleeding. Deaths from
cardio-respiratory arrest have occurred following short-term inhalation of 20,000
ppm benzene, or from ingestion of a single dose of 125 mg benzene per kg BW.
Several studies demonstrate that repeated exposure to benzene may induce bone
marrow depression, cause damage to genetic material and induce leukaemia,
specifically AML. Some studies also point to an association between benzene
exposure and the risk for lymphoma, specifically NHL and MM. For bone marrow
depression, the best estimate for a LOAEL is 7.6 ppm (TWA8), based on current
Priority Existing Chemical Number 21
102
data. An appropriate NOAEL has not been determined, although studies with
various limitations indicate that it is likely to be >0.5 ppm (TWA8). Dose-related
structural or numerical chromosome aberrations have been detected in peripheral
LC of workers exposed to benzene levels above 10 ppm (TWA8), but a threshold
level has not been identified. The risk of developing leukaemia increases with
exposure and has been shown to be significantly elevated above, but not below, a
cumulative exposure of 50 ppm-years, corresponding to an average occupational
exposure of 1.25 ppm (TWA8) over 40 years. However, this finding derives from a
single cohort study with insufficient statistical power to rule out the possibility of
some increase in leukaemia risk at lower exposures.
In addition, some studies suggest an association between repeated exposure to
benzene or benzene-containing products and several other adverse health effects,
including menstruation disorders, spontaneous abortions, melanoma and breast
cancer in adults and reduced birth weight and leukaemia in the children of exposed
parents. However, considering the multiple exposure circumstances in most studies
and the limited consistency of the findings reviewed above, the human database
does not in itself suffice to establish a causal relationship between these effects and
benzene exposure.
Benzene 103
12. Modes of Action
While a general overview of benzene metabolism has been presented in Section 9,
this section presents a review of the evidence for the molecular basis of the action
of benzene metabolites. Several reviews of benzene metabolism and the proposed
mechanisms of toxicity have been published (Ross, 1996; Snyder, 2000; Snyder et
al, 1993; Snyder & Hedli, 1996; Yardley-Jones et al, 1991).
Exposure to benzene can result in haematotoxicity, immunotoxicity and
carcinogenicity in humans and animals. Haematotoxicity resulting from chronic
benzene exposure can present as anaemia, aplastic anaemia, leukopenia,
lymphocytopenia, thrombocytopenia, or pancytopenia (Aksoy, 1989). The principal
carcinogenic response in humans to chronic benzene exposure is leukaemia while
other animals tend to produce solid tumours in specific organs. While the liver is
the initial site for the biotransformation of benzene, hepatotoxicity is not a
consequence of benzene exposure. However, a number of studies have shown that
for benzene to produce haematotoxicity in animals it must first be metabolised by
the liver (Andrews et al, 1977; Sammett et al, 1979). Subsequent accumulation of
the major hepatic metabolites, phenol, hydroquinone and catechol, occurs in the
bone marrow where they are known to persist for varying durations after exposure
to benzene ceases (Rickert et al, 1979). Longacre et al. (1981) observed that strains
of mice that exhibit greater sensitivity towards benzene accumulate more benzene
metabolites (water-soluble and covalently bound) in bone marrow compared to less
sensitive strains. However, administration of specific benzene metabolites to test
animals has failed to reproduce the characteristic toxicity of benzene although co-
administration of phenol and hydroquinone has been shown to mimic its
haematotoxic effects (Eastmond et al, 1987). These data suggest that phenolic
metabolites of benzene in combination and not the parent molecule are responsible
for the haematotoxicity of benzene. The data further suggest that subsequent
biotransformation of the hepatic metabolites to reactive intermediates is required
and that this occurs within the bone marrow and those animal organs exhibiting
solid tumours.
12.1 Activation of benzene metabolites
In order for the phenolic metabolites of benzene to exert their toxic effect on bone
marrow, they must undergo activation to their oxidised forms. Once activated, they
can participate in covalent binding reactions with macromolecules. Several studies
have identified the presence of peroxidase enzymes in bone marrow and other
tissues as the primary mechanism by which activation of benzene phenolic
metabolites is achieved (Eastmond et al, 1986; Lévay et al, 1993). The peroxidases
are a diverse class of enzymes that catalyse the general reaction:
Donor + H2O2 oxidised donor + 2H2O.
While peroxidases generally act to detoxify peroxides, including hydrogen
peroxide, that form within cells as a result of several metabolic reactions, a number
of specialised peroxidases with other functions have evolved. In particular, the
leukocytes of several species, including humans, have been shown to possess large
amounts of a specific form, myeloperoxidase (Bainton et al, 1971; Himmelhoch et
al, 1969). In concert with the leukocyte nicotinamide adenine dinucleotide
Priority Existing Chemical Number 21
104
phosphate (NADPH) oxidase system that causes hydrogen peroxide to be produced
(Patriarca et al, 1971), myeloperoxidase plays a crucial role in the host defence
system by producing a potent microbicidal oxidant that protects the host from
microorganisms. Immature leukocytes in the bone marrow generally have higher
levels of myeloperoxidase than circulating mature cells (Bainton et al, 1971).
Consequently, bone marrow has considerable capacity to metabolise suitable
electron donors including the benzene metabolites, phenol, hydroquinone, catechol
and 1,2,4-trihydroxybenzene, to reactive species.
12.1.1 Activation of phenol
Incubation of phenol with human leukocyte lysates, which contain
myeloperoxidase, have demonstrated the formation of reactive intermediates that
covalently bind to macromolecules in the presence of hydrogen peroxide as a co-
oxidant (Eastmond et al, 1986; Smith et al, 1989). Eastmond et al. (1986)
concluded that although 4,4'-biphenol and diphenoquinone were identifiable
reaction products derived from the oxidation of phenol, only 6% of the covalent
binding could be attributed to diphenoquinone with most of the covalent binding
observed due to other reactive species, possibly the phenoxy radical or oxidation
products of 2,2'-biphenol or 4,4'-biphenol. However, in the presence of
hydroquinone, phenol appears to undergo a recycling process such that the initial
phenoxy radical is reduced to phenol by transferring an electron to hydroquinone
(Smith et al, 1989), thus limiting the formation of biphenol derivatives.
12.1.2 Activation of hydroquinone and catechol
Under physiological conditions, hydroquinone, catechol and 1,2,4-
trihydroxybenzene can undergo autoxidation to their respective semiquinone and
quinone forms (Brunmark & Cadenas, 1989) or their oxidation can be facilitated by
the presence of a peroxidase and hydrogen peroxide (Sadler et al, 1988; Schlosser
et al, 1989; Smith et al, 1989). Quinones are chemically reactive species capable of
depleting intracellular glutathione, promoting lipid peroxidation and forming
covalent adducts with macromolecules (Bolton et al, 2000; Irons, 1985; Monks et
al, 1992). Several studies have shown that hydroquinone and catechol are readily
oxidised by human myeloperoxidase (Eastmond et al, 1986; Sadler, et al, 1988) and
it has been observed that the oxidation of hydroquinone to benzoquinone by
peroxidase enzymes is enhanced by the presence of excess phenol which acts as a
co-oxidant obviating the need for hydrogen peroxide to drive the reaction (Smith et
al, 1989; Subrahmanyam, et al, 1990). Hydroquinone was found to be metabolised
by activated human neutrophils to covalent-binding species and the amount of
binding could be increased by approximately 70% by the addition of phenol
(Eastmond et al, 1987). Subrahmanyam et al. (1990) reported that the presence of
phenol enhanced the covalent binding of [3H]-hydroquinone metabolites to
macromolecules of mouse blood and bone marrow, but not to the kidneys or liver.
It was further noted that hydroquinone enhanced binding of [3H]-phenol
metabolites in blood, bone marrow and the kidneys but inhibited binding in the
liver. In contrast, catechol did not enhance [3H]-hydroquinone metabolite binding.
Sadler et al. (1988) observed that the oxidation of catechol by human neutrophil
peroxidases (myeloperoxidase) resulted in the formation of 1,2-benzosemiquinone
and 1,2-benzoquinone. Bhat et al. (1988) found that the addition of [14C]-catechol
to rat or human bone marrow cells resulted in the formation of a glutathione-
conjugate and covalent binding of radiolabel to protein. Both conjugate formation
and the binding of radiolabel were substantially increased by the presence of
Benzene 105
hydrogen peroxide or phenol, however, protein binding could be markedly
decreased by the presence of exogenous glutathione (GSH) or hydroquinone.
12.1.3 Role of cyclooxygenase
In addition to activation by peroxidases, phenol and hydroquinones can be
activated by prostaglandin H synthase (cyclooxygenase), an enzyme with
oxygenase and endoperoxidase activity (Markey et al, 1987; Schlosser et al, 1989).
Acting as an endoperoxidase, the enzyme requires an oxidant as a co-substrate
which phenol or hydroquinones can replace (Markey et al, 1987). Prostaglandin H
synthase is present in a number of bone marrow-derived cells including
monocyte/macrophage populations and platelets and converts arachidonic acid to
several prostaglandins including prostaglandin E2 (PGE2). PGE2 plays a major role
in the inhibition of progenitor cell proliferation and differentiation (Gentile and
Pelus, 1987). In vitro, both phenol and hydroquinone are activated by cell lysates
containing prostaglandin H synthase or by the purified enzyme and in the presence
of arachidonic acid or hydrogen peroxide (Schlosser et al, 1989).
The role of prostaglandin H synthase activity has been demonstrated in a number of
mouse strains (B6C3F1, DBA/2 and C57BL/6) where it was shown that bone
marrow toxicity could be reduced by prior treatment of the animals with non-
steroidal anti-inflammatory drugs (aspirin, indomethacin or meclofenamate) that
inhibit prostaglandin H synthase activity (Gaido and Wierda, 1987; Kalf et al,
1989). Pirozzi et al. (1989) further demonstrated that benzene-induced bone
marrow depression and micronucleus formation in erythrocytes of C57B1/6 mice
could be prevented by the co-administration of indomethacin and that protection
was achieved at doses that did not inhibit cytochrome P450 or myeloperoxidase
activity.
The bio-activation of catechol by prostaglandin H synthase activity in rat bone
marrow appears to be limited as the addition of arachidonic acid provided only a
small but significant (p<0.05) increase in covalent binding which was of limited
duration (Bhat et al, 1988).
12.1.4 Formation of reactive oxygen species
In addition to the formation of semiquinone and quinone species, the oxidation of
hydroquinones results in the formation of reactive oxygen species. Initially,
molecular oxygen is reduced to superoxide anion which, by dismutation, is
converted to hydrogen peroxide (Figure 12.1). In the presence of transition metal
ions (for example, iron) the very reactive hydroxyl radical can form. These reactive
species can promote the oxidation of protein and DNA bases, induction of
chromosomal aberrations, lipid peroxidation and the modulation of cellular
functions.
While hydroquinone and its semiquinone radical can both reduce molecular oxygen
to superoxide, Sadler et al. (1988) found superoxide production by catechol to be
limited to the first one electron reduction step to 1,2-benzosemiquinone with
molecular oxygen being unable to effect the subsequent oxidation of the
semiquinone to the quinone form. In contrast, 1,2,4-benzenetriol undergoes rapid
autoxidation to yield hydrogen peroxide (Brunmark & Cadenas, 1988).
Priority Existing Chemical Number 21
106
Figure 12.1. Redox cycle of hydroquinone with formation of reactive oxygen
species and biological effects
NQO1
NADPH-Cytochrome
NADPH-Cytochrome
reductase
reductase or
Disproportionation
.
OH O O
Autoxidation
Autoxidation
/Peroxidase
/Peroxidase
Hydroquinone 1,4-Benzoquinone
.-
.-
OH OH O
O2 O
O
O2 2
2
Dismutation/
DNA adducts
Superoxide dismutase H2O2
DNA oxidation
3+
Fe
Protein oxidation
Lipid peroxidation
.
Modulation of cellular function
OH
The detoxification of quinones can be achieved by a two-electron reduction to their
fully reduced forms. Two enzymes involved in the reduction of quinones are
NADPH-cytochrome reductase and NAD(P)H:quinone oxidoreductase (NQO1;
DT-diaphorase; Lind et al, 1982). In the case of NADPH-cytochrome reductase,
reduction of the quinone to a semiquinone is achieved by a one-electron transfer to
give a semiquinone and a second electron is transferred to molecular oxygen to
yield the superoxide anion. The resulting semiquinone is then free to autoxidise to
the quinone producing more superoxide. However, it has been proposed that redox
cycling of the semiquinone could not be maintained by NADPH-cytochrome
reductase at physiological pH due to protonation of the semiquinone thus
minimising superoxide production (Boersma et al, 1994). In the case of NQO1,
reduction to the hydroquinone is achieved by a simultaneous two-electron transfer
to the quinone with no reduction of molecular oxygen. The redox cycle for
hydroquinone, the role of NADPH-cytochrome reductase and NQO1 and the
biological effects of these processes are illustrated in Figure 12.1.
In cells that possess both peroxidase and NQO1 activities, the ratio of the two
enzymes may determine the extent to which reactive metabolites form. Thus a high
intracellular myeloperoxidase/NQO1 ratio, such as occurs in human stroma and
CD34+ bone marrow progenitor cells, may result in a greater risk of benzene-
induced cellular toxicity (Ross et al, 1996b). Although characterisation of NQO1
activity in primary cultures of mouse bone marrow stromal cells was found to be
low, the enzyme was shown to be inducible and induction of the enzyme conferred
protection against hydroquinone-induced toxicity (Twerdok et al, 1992).
Conjugation reactions, for example with GSH, can enhance the ability of
hydroquinones to autoxidise. Glutathionyl hydroquinone, identified as a urinary
benzene metabolite (Nerland & Pierce, 1990), was found by Brunmark & Cadenas
(1988) to autoxidise at a rate 8-fold faster than hydroquinone. Rao (1996)
Benzene 107
concluded that, in vitro, glutathionyl hydroquinone acted as a potent pro-oxidant
based on its ability to degrade DNA.
Reactive oxygen species may also form due to the reduction of molecular oxygen
by the action of cytochromes P450. Johannson & Ingelman-Sundberg (1983)
observed that benzene could be directly oxidized to phenol by hydroxyl radicals
derived from the reduction of molecular oxygen by microsomal cytochrome P450
activity or reconstituted enzyme systems. Similarly, Kahn et al. (1990) detected the
presence of hydroxyl radicals during the NADPH-dependent metabolism of
benzene by rat bone marrow microsomal preparations.
12.2 Reactivity of benzene metabolites
Results derived from in vivo and in vitro studies indicate that a number of
mechanisms contribute to the cytotoxicity, genotoxicity and carcinogenicity of
benzene metabolites. Cytotoxicity can arise due to depletion of intracellular GSH
and changes in intracellular redox status (Ludewig et al, 1989; Rao & Snyder,
1995; Witz, 1985) and covalent binding of benzene metabolites to macromolecules
(Latriano et al, 1989; Lutz and Schlatter, 1977; Mazzullo et al, 1989; Snyder et al,
1987). Metabolites suspected of contributing to the genotoxicity and
carcinogenicity of benzene include benzene oxide, hydroquinone, catechol, 1,2,4-
trihydroxybenzene and trans,trans-muconaldehyde. The effects induced by these
metabolites comprise DNA base alterations, chromosome structural aberrations and
aneuploidy. However, the metabolite concentrations at which many of these effects
have been shown to occur in vitro are higher than those expected to occur in vivo.
12.2.1 Genotoxicity
Several studies have demonstrated the formation of DNA adducts after incubation
of benzene metabolites with purified DNA. Hydroquinone when incubated with
calf thymus DNA resulted in the formation of deoxycytidine (Pongracz et al, 1990),
deoxyadenosine (Pongracz & Bodell, 1991) and deoxyguanosine adducts (Jowa et
al, 1990). Gut et al. (1996) were able to demonstrate the formation of the N-7
guanine adduct on exposure of calf thymus DNA to benzene oxide under in vitro
conditions while Latriano et al. (1989) found trans,trans-muconaldehyde to form
adducts with deoxyguanosine. In addition to nuclear DNA, in vitro studies have
shown mitochondrial DNA, derived from bone marrow mitoplasts, to undergo
alkylation by benzene metabolites (Kalf et al, 1985; Snyder et al, 1987).
Under cellular conditions, a comparison of the ability of benzene metabolites to
induce DNA adduct formation in HL-60 cells, a promyelocytic leukemia cell line,
found hydroquinone to be 7-9 times more effective at inducing such adducts than
catechol or 1,2,4-trihydroxybenzene and that a correlation existed between adduct
formation and cytotoxicity. Co-incubation of hydroquinone with either catechol or
1,2,4-trihydroxybenzene produced a synergistic effect that was 3-6 times greater
than the added effects of each metabolite. It was further observed that DNA
adducts form in the presence of benzene metabolite mixtures which are not
observed when cells are incubated with the individual metabolites, leading the
authors to suggest that other processes leading to adduct formation may be
involved (Lévay & Bodell, 1992; Lévay et al, 1991). Chenna et al. (1995)
subsequently identified an enzyme with glycosylase activity that excises
deoxycytidine and deoxyadenosine adducts of benzoquinone from DNA.
Priority Existing Chemical Number 21
108
It is noteworthy that transfected HL-60 cells expressing a high level of NQO1
activity exhibited lower levels of DNA adduct formation when exposed to
hydroquinone compared to non-transfected HL60 cells which are deficient in
NQO1 activity. Similarly, C15 cells, a myeloperoxidase-deficient HL-60 subline,
produced lower levels of DNA adducts with hydroquinone compared to HL-60
cells which normally express high levels of myeloperoxidase (Wiemels et al, 1999).
The ability of hydroquinone species to induce mutations has been demonstrated by
Joseph et al. (1998). In a series of in vitro experiments, it was demonstrated that
sequence-specific frame shift mutations could be caused by hydroquinone, but not
semiquinone, benzoquinone or reactive oxygen species, in the supF tRNA gene. It
was further demonstrated that BALBc/3T3 cells undergo transformation by
hydroquinone (15 µM) and that the frequency of transformation could be increased
by a tumour promoter. Such initiated cells produced tumours with 100% frequency
when injected into severe combined immunodeficient (SCID) mice. Sakai et al.
(1995) previously had shown that benzoquinone caused initiation in a two-stage
model of carcinogenesis using BALB/3T3 cells.
Mueller et al. (1987) detected the alkylation product of benzene oxide, N-7-
phenylguanine, in the urine of rats exposed to benzene (500 ppm) for 8 h while
Norpoth et al. (1996) detected several benzene-derived urinary guanine adducts
following the administration of benzene to rats. However, it should be noted that
the presence of N-7-phenylguanine in the urine does not provide sufficient
evidence that the adduct is derived from DNA excision-repair activities. The
presence of benzene-induced DNA adducts has been detected in the tissues of rats
dosed with [14C]-benzene (Lutz and Schlatter, 1977; Mazzullo et al, 1989) although
the nature of the adducts was not investigated. However, Reddy et al. (1989b)
found only equivocal evidence for the in vivo formation of aromatic DNA adducts
in the bone marrow, liver, kidney and mammary gland of benzene-treated female
Sprague-Dawley rats. DNA isolated from Zymbal glands was found to contain 4
adducts per 109 DNA nucleotides, although the adducts did not correspond to major
adducts described in in vitro studies. Thus it was concluded that DNA-quinone
adduct formation in the rat is not extensive, possibly due to the efficient elimination
of quinones by other mechanisms. In addition to forming DNA adducts in the bone
marrow of experimental animals, benzene exposure produced DNA adducts in the
livers of male mice (Lutz and Schlatter, 1977); however, liver tumours were not
observed in 2-year carcinogenicity studies of these animals (NTP, 1986).
The mutation frequency of V75 Chinese hamster cells increased in a dose-
dependent manner after treatment for 1 h with benzoquinone, an effect that was
found to be independent of intracellular GSH status and observed at low (< 10 µM)
concentrations. The frequency of micronucleated cells was also increased by
benzoquinone but only at concentrations greater than 20 µM. In contrast,
benzoquinone did not induce sister chromatid exchanges at any concentration up to
100 µM (Ludewig et al, 1989).
As described in Sections 10.6 and 11.5, several studies have demonstrated
chromosomal aberrations in experimental animals and humans following exposure
to benzene. While the administration of benzene to CD-1 mice resulted in
micronuclei formation, treatment with either phenol, hydroquinone or catechol
failed to induce micronuclei (Gad-el-Karim et al, 1985). Barale et al. (1990)
demonstrated, in vivo, a synergistic effect on micronuclei formation in CD-1 mice
bone marrow cells by the concurrent administration of hydroquinone and phenol.
Benzene 109
Lewis et al. (1988) reported that hydroquinone, under in vitro conditions, caused
DNA to form single- and double-strand breaks by a mechanism that was
independent of reactive oxygen species. In contrast, catechol did not induce DNA
damage. These investigators further observed that DNA could be degraded by
1,2,4-trihydroxybenzene, an effect inhibited by scavengers of reactive oxygen
species. When tested together in vitro, hydroquinone and catechol produced a
synergistic effect on micronuclei formation in human lymphocytes, possibly by
interfering with mitotic spindle function and disturbing chromosome segregation
(Robertson et al, 1991). Benzoquinone has also been reported to interfere with
microtubule assembly by blocking a thiol-sensitive binding site (Irons et al, 1981).
12.2.2 Oxidative stress
The formation of reactive oxygen species is a normal part of cellular biochemistry
and is considered to be an important component of intracellular signalling
processes, including mediating signal transduction within haematopoietic cells
initiated by growth factor signals (Sattler et al, 1999). However, exposure of
biological systems to excessive levels of reactive oxygen species results in the
induction of oxidative stress. Oxidative stress can induce oxidative modification of
DNA bases and chromosomal abnormalities, depletion of intracellular GSH,
changes in intracellular redox status, peroxidation of lipids, oxidation of proteins
and modulation of cellular functions. The role of oxidative stress in benzene-
mediated toxicity has been extensively reviewed by Subrahmanyam et al. (1991).
Rao & Snyder (1995) examined the effects of hydroquinone, benzoquinone and
1,2,4-trihydroxybenzene (50 µM) on several parameters of antioxidant defence
function of HL-60 cells. The three metabolites did not induce the cells to generate
superoxide anion or nitric oxide but did produce detectable levels of hydrogen
peroxide. Intracellular GSH levels were depleted by hydroquinone and 1,2,4-
trihydroxybenzene but not benzoquinone.
The presence of lipid peroxidation products was found to increase in rat tissues
following the administration of benzene (Khan et al, 1984) while urinary levels of
malondialdehyde, a biomarker of lipid peroxidation, were elevated in rats receiving
hydroquinone (Ekström et al, 1988). The presence of intracellular peroxidation
products has been detected in HL60 cells following treatment with either 1 µM
benzoquinone or 10 µM hydroquinone (Hiraku & Kawanishi, 1996). Several
studies have identified the presence of 8-hydroxydeoxyguanosine (8-OHdG) as a
sensitive biomarker of DNA damage due to oxidative stress (Kasai & Nishimura,
1984, 1986; Shigenaga et al, 1989). In two occupational studies in workers known
to have benzene exposure, a dose-response relationship was demonstrated between
the exposure level and urinary 8-OHdG levels (Lagorio et al, 1994b, Nilsson et al,
1996). However, the presence of 8-OHdG in the urine is not conclusive evidence of
DNA excision-repair activities in response to oxidative modification of DNA.
Benzoquinone species and trans,trans-muconaldehyde readily react with GSH
(Brunmark & Cadenas, 1988; Rao et al, 1982) which can lead to depletion of
intracellular GSH levels and changes in intracellular redox status. Treatment of
V79 Chinese hamster cells with benzoquinone for 1 h resulted in decreased GSH,
NADPH and nicotinamide adenine dinucleotide levels but only at cytotoxic
concentrations at or above 100 µM (Ludewig et al, 1989). Ekström et al. (1988)
found rat hepatic GSH levels to be depleted after administration of hydroquinone
by gavage while the administration of trans,trans-muconaldehyde to mice for 10 or
16 days resulted in decreased hepatic sulfhydryl levels (Witz et al, 1985).
Priority Existing Chemical Number 21
110
The growth of HL-60 cells was found to be stimulated by the presence of
hydroquinone within the range of 10-40 µM. Similarly, the incorporation of [3H]-
thymidine was enhanced by hydroquinone, benzoquinone or 1,2,4-benzenetriol.
However, the effects produced by the three metabolites could be eliminated if the
cells were pre-incubated with catalase, an antioxidant specific for hydrogen
peroxide. The effects of hydroquinone and benzoquinone could be mimicked by
reactive oxygen species produced by a xanthine/xanthine oxidase system. It was
further observed that while hydroquinone or benzoquinone did not reduce the cell
cycle time they did increase the number of cells entering into S-phase from G0/G1
phase (Wiemels & Smith, 1999). The role of reactive oxygen species, and in
particular hydrogen peroxide, has been further explored by Sattler et al. (1999).
These investigators found haemopoietic growth factors to induce increased levels
of reactive oxygen species in MO7e cells, a growth factor-dependent human
megakaryocytic cell line. Treatment of these cells with either growth factors or
hydrogen peroxide resulted in increased tyrosine phosphorylation of cellular
proteins, a key step in intracellular signalling processes.
12.2.3 Modulation of cellular function
The mature macrophage produces interleukin-1 (IL-1), a cytokine essential for stem
cell maturation. However, it has been observed that macrophages treated with
hydroquinone (10 µM) produce less IL-1 than control cells (Thomas et al, 1989).
This is due to the inhibition of calpain, a protease required for the conversion of
pre-IL-1 to its active form, by hydroquinone (Renz & Kalf, 1991; Kalf et al, 1996).
Treatment of isolated mouse bone marrow-derived macrophages with non-
cytotoxic doses of hydroquinone (10 µM) resulted in a 10 to 30% reduction in total
calpain activity (Miller et al, 1994). In contrast, the production of PGE2 by bone
marrow cells, in vivo, was enhanced by benzene exposure (Gaido & Wierda, 1987;
Kalf et al, 1989) although it is uncertain how the release of arachidonic acid, the
precursor of prostaglandins, from phospholipid stores is initiated under these
conditions. However, hydroquinone and catechol have been shown to regulate
protein kinase C (PKC) activity by producing a short term cytosol-to-membrane
translocation of PKC (Gopalakrishna et al, 1994), a key step in the mobilisation of
arachidonic acid. Da Silva et al. (1989) have further shown that benzene can
directly activate PKC. PGE2 has been identified as an inhibitor of
granulocyte/macrophage progenitor cell proliferation (Gentile & Pelus, 1987).
The growth of granulocyte/macrophage colonies was stimulated in a synergistic
manner by co-treatment of mouse bone marrow cells with low concentrations of
hydroquinone (10-8 to 10-5 M) and recombinant granulocyte/macrophage colony
stimulating factor (GM-CSF) compared to cells treated with GM-CSF alone. The
maximal response was achieved with 1 µM hydroquinone but no effect was
observed when phenol, catechol or trans,trans-muconaldehyde were substituted for
hydroquinone (Irons et al, 1992). While treatment of HL-60 cells with
hydroquinone at concentrations between 10-40 µM resulted in an increase in cell
proliferation, at a concentration greater than 50 µM hydroquinone caused a
decrease in cell viability (Wiemels & Smith, 1999). In a further study, Wiemels et
al. (1999) demonstrated that 12 h after treatment with hydroquinone (50 µM)
approximately 40% of HL-60 cells were apoptotic as determined by the terminal
deoxynucleotidyl-transferase (TdT) assay. Apoptosis is a form of physiological cell
death characterised by altered cell morphology including condensation of the
cytoplasmic and nuclear compartments and internucleosomal DNA fragmentation.
Apoptosis has been observed to occur in a dose-dependent manner when HL-60
Benzene 111
cells are treated with hydroquinone and catechol (25 to 100 µM) but not by phenol.
Similarly, hydroquinone or catechol induces CD34+ human bone marrow
progenitor cells to undergo apoptosis (Moran et al, 1996). These reports support an
earlier study in which weak internucleosomal cleavage was observed in HL-60
cells following incubation for 4 h with 20 µM hydroquinone and 5 µM
benzoquinone and pronounced cleavage observed at 50 µM hydroquinone and 10
µM benzoquinone using pulse-field gel electrophoresis (Hiraku and Kawanishi,
1996).
12.3 Critical biological effects
12.3.1 Bone marrow toxicity
The critical biological effect of benzene in all experimental species is bone marrow
toxicity characterised by a reduction in bone marrow cellularity. Bone marrow
consists of stromal cells (composed of macrophage and fibroblastoid cell
populations) along with stem and progenitor cell populations that form a complex
matrix within which are produced a number of essential regulatory growth factors.
Stromal cells regulate stem and progenitor cell proliferation, differentiation and
maturation by producing both inducers (colony stimulating factors (CSFs) and
interleukins, particularly IL-1) and inhibitors (PGE2) of cell growth. PGE2 inhibits
cell growth by suppressing the production of CSFs and IL-1. Benzene metabolites
appear to disrupt the balance of these regulatory factors by inhibiting production of
CSFs and IL-1 and increasing PGE2 production, although low levels of
hydroquinone can replace or augment, in vitro, the effects of growth factors
(Wiemels & Smith, 1999). Evidence to support this hypothesis has been provided
by experiments in which the co-administration of IL-1 abrogates the effects of
benzene treatment. Similarly, if non-steroidal anti-inflammatory agents, which
inhibit PGE2 production and the cyclooxygenase-dependent oxidation of phenol
and hydroquinone, are co-administered with benzene, haematotoxicity is not
observed. Although increased apoptosis has been observed by exposing bone
marrow cells to various benzene metabolites, these effects appear to occur only at
high metabolite concentrations.
12.3.2 Leukaemia
Leukaemia is the progressive proliferation of abnormal and usually monoclonal
leukocytes in hemopoietic tissues. Benzene-induced leukaemias are typically
myelogenous in nature rather than lymphocytic. Currently, the mechanism(s) by
which benzene induces leukaemia in susceptible individuals remains obscure.
Clinical studies of therapy-related myelodysplastic syndromes and acute myeloid
leukaemia have shown an increase in chromosomal aberrations particularly
aneuploidy, long-arm deletions and translocations involving chromosomes 5, 7 and
8 (Pedersen-Bjergaard et al, 1995). Individuals with chronic exposure to benzene
tend to exhibit similar changes in chromosomes 5 and 7 of peripheral blood
lymphocytes (Zhang et al, 1998).
Chromosomal aberrations involving chromosomes 5, 7 and 8 of various cell lines,
including blood CD34+ progenitor cells, have been reported to occur, in vitro, in
response to low-dose hydroquinone exposure (Smith et al, 2000; Stillman et al,
1997). In particular, CD34+ bone marrow cells were observed to lose chromosome
7 accompanied by selective deletion of the long-arm of chromosome 5 (5q31) but
no changes in chromosome 8 (Stillman et al, 2000). Stillman et al. (1999) have also
Priority Existing Chemical Number 21
112
reported that while catechol does not alter cellular cytogenetics, a dose-dependent
synergistic effect is observed between hydroquinone and catechol. The
combination of metabolites induces changes in chromosome 5 not seen with
hydroquinone alone, a result analogous to the synergistic effect on micronuclei
formation in human lymphocytes described by Robertson et al. (1991).
A study of patients with therapy-related AML identified a close correlation
between the use of drugs with DNA-topoisomerase inhibitor activity and
aberrations in chromosomes 5, 7 and 8 (Super et al, 1993). Topoisomerases are a
class of nuclear proteins (endonucleases) that convert one topological version of
DNA into another by catalyzing the breakage and reformation of DNA
phosphodiester linkages. They are involved in DNA replication and transcription,
DNA repair, chromosome segregation and maintain genomic stability. Due to the
sulfhydryl-dependent nature of topoisomerases and the ability of several benzene
metabolites to modify sulfhydryl groups, inhibition of topoisomerase activity by
benzene metabolites has been proposed as a mechanism for leukaemia formation.
Chen & Eastmond (1995) found no evidence for topoisomerase I inhibition by
phenol, catechol, hydroquinone, benzoquinone or 1,2,4-benzenetriol at
concentrations up to 1000 µM. Similarly, topoisomerase II activity was not
inhibited by benzene metabolites at concentrations less than 500 µM with the
exception of 1,2,4-benzenetriol which was inhibitory at 250 µM. The activation of
phenol by a peroxidase and hydrogen peroxide system resulted in inhibition at 50
µM and the products of this reaction, 2,2'-biphenol and 4,4'-biphenol were found
to be inhibitory at 500 µM, whereas the peroxidase activation products of these
compounds were inhibitory at 100 and 10 µM respectively. However, Parke and
Williams (1953) failed to find any evidence for the in vivo formation of biphenol
products after benzene exposure and there is evidence that these metabolites do not
readily form in the presence of hydroquinone (Smith et al, 1989). In contrast, Hutt
and Kalf (1996) found topoisomerase II activity to be inhibited by hydroquinone or
benzoquinone at 6 and 3 µM respectively.
In addition to modulation of topoisomerase activity, benzene metabolites can
modify other nuclear proteins including tubulin (Pfeiffer & Metzler, 1996) and
produce DNA-protein cross-links (Schoenfeld & Witz, 1999) which may contribute
to chromosomal aberrations and the development of leukaemia. It has been
postulated that chromosomal aberrations could result in inactivation of tumour
suppressor genes, such as p53, activation of proto-oncogenes and altered
expression of growth-factor and growth-factor receptor genes on the aberrant
chromosomes (Irons and Stillman, 1996; Smith, 1996). Similarly, the formation of
apurinic sites due to depurination by N-7 guanine adducts of benzene oxide (Gut et
al, 1996) could result in misreplication of DNA and contribute to the development
of leukaemia, as could oxidative DNA base lesions due to benzene-induced
oxidative stress.
12.3.3 Tumours in Zymbal, Harderian, lacrimal and mammary glands
In addition to haematopoietic abnormalities, rodents exposed to benzene develop
solid tumours in the Zymbal, Harderian, lacrimal and mammary glands, although
other organs and tissues may also be involved (Huff et al, 1989). The mechanisms
by which benzene induces tumours in these glands have not been extensively
investigated. Biochemical characterisation has revealed the presence of high levels
of peroxidase enzymes which can activate phenolic metabolites of benzene to
reactive species capable of modifying DNA and altering cellular functions as
described above. Humans lack an anatomical equivalent of the Zymbal gland and
Benzene 113
the human Harderian gland is only of rudimentary development and has not been
characterised with respect to peroxidase activity.
Studies by Low et al. (1989) indicate that neither benzene nor its metabolites
accumulate in the rat Zymbal gland, a sebaceous gland of the external ear duct of
rodents. However, examination of Zymbal gland tissue after oral administration of
benzene revealed phenol and hydroquinone to constitute 3% and 30% respectively
of unconjugated metabolites. Phenyl glucuronide accounted for 35% of conjugated
metabolites but phenylsulfate could not be detected. The absence of phenylsulfate
was attributed to a lack of sulfotransferase activity in this tissue. In contrast,
Osborne et al. (1980) found the Zymbal gland to exhibit a high level of peroxidase
activity indicating that activation of phenolic benzene metabolites could occur in
this organ. Reddy et al. (1989a) subsequently identified DNA adducts in excised
Zymbal glands after incubation with benzene or its metabolites. The combination
of low sulfotransferase and high peroxidase activity would appear to be conducive
to the formation of reactive metabolites in the Zymbal gland, thus facilitating
tumour formation.
Both lacrimal glands and the accessory lacrimal glands, the Harderian glands,
develop tumours in response to benzene exposure. Biochemical characterisation of
these glands has demonstrated the presence of high constitutive levels of
lactoperoxidase (Morrison & Allen, 1966), which can activate phenolic metabolites
of benzene to reactive species in the same manner as myeloperoxidase.
Mammary gland tumours have been observed in rodents in response to benzene
(Huff et al, 1989) and limited epidemiological evidence suggests an association
between exposure to benzene or benzene-containing products and mammary
tumours in humans (Hansen, 2000; Petralia et al, 1999; see Section 11.6.2). The
mechanism for the formation of these mammary tumours is uncertain. Reddy et al.
(1989b) failed to detect DNA adducts associated with the mammary gland of
female rats after 10 weeks of benzene exposure, suggesting an epigenetic
mechanism may be involved. However, as stated in Section 9, Low et al. (1989)
found the distribution of radiolabel in female rats (Sprague-Dawley) to vary
depending on the dose of [14C]-benzene administered. When comparing doses of
benzene at 0.15, 1.5 and 15 mg/kg bw, the highest dose resulted in a substantial
increase in the amount of radiolabel associated with the mammary gland and bone
marrow compared to other tissues at the lesser doses. Mammary tissue is richly
perfused with blood and has a high fat content which allows for the accumulation
of benzene metabolites. It also contains lactoperoxidase which has been shown to
metabolise phenolic compounds to reactive species (Monzani et al, 1997).
Consequently, benzene metabolites may become activated within mammary tissue
resulting in altered cellular function and carcinogenesis.
12.4 Interindividual variations in susceptibility
12.4.1 Gender effects
While several studies have reported gender-dependent differences in the
metabolism and/or toxicity of benzene in mice, there are no reliable data to indicate
that there are gender differences in humans with respect to either the metabolism of
benzene or susceptibility to benzene toxicity.
Male Swiss (CD-1) mice exposed to benzene exhibited more severe benzene-
related toxicity, including genotoxic effects, than females (Meyne and Legator,
Priority Existing Chemical Number 21
114
1980; Ward et al, 1985). Similarly, suppression of bone marrow cellularity in male
DBA/2 mice was greater than females after exposure to benzene (Luke et al,
1988a). Corti and Snyder (1996) found, using Swiss Webster mice exposed to
benzene (10 ppm) for 6 h over 10 days, that the number of erythoid colony forming
units (CFU-E) had decreased in the bone marrow of adult-exposed males, in utero-
exposed males and foetal male livers compared to female adults and foetuses. It
was further shown by Corti and Snyder (1998), in vitro, that isolated CFU-E
derived from male mice were more susceptible to individual benzene metabolites
then female isolates.
A marked gender-related difference was observed in the hepatic glutathione-S-
transferase (GST) activity of CD-1 mice with the isoform exhibiting
approximately 25% greater activity towards trans,trans-muconaldehyde in the
females compared to males (Goon et al, 1993). Investigations of gender-related
differences in benzene metabolism by mouse bone marrow have not, as yet, been
undertaken. Hu et al. (1993) observed that microsomes prepared from the kidneys
(but not livers) of male mice (C3H/HeJ) possessed CYP2E1 activity up to 50-fold
higher for acetaminophen metabolism compared to female mice. It was further
observed that the administration of testosterone to female mice increased the
CYP2E1 activity in the kidneys of females. Supporting evidence for the role of
metabolic gender-differences was provided by Kenyon et al. (1995) who found that
male mice (B6C3F1) excreted more hydroquinone glucuronide when dosed with
phenol compared to female mice. In a subsequent study, Kenyon et al. (1998)
found bone marrow levels of phenol and hydroquinone to be higher in male mice
(B6C3F1) compared to female mice after exposure to benzene.
Attempts to demonstrate gender differences in benzene metabolism in humans by
comparing urinary metabolites (trans,trans-muconic acid and phenylmercapturic
acid) to benzene exposure levels have produced negative results (Inoue et al, 1989;
Inoue, 2000).
12.4.2 Genetic polymorphisms
It has been observed that different strains of male mice (DBA/2, C57B1/B6 and
B6C3F1) exhibit differing sensitivities to benzene when exposed under identical
conditions (Luke et al, 1988b; Pirozzi et al, 1989) and that the metabolic profile of
urinary benzene metabolites is strain-dependent (Longacre et al, 1981). These data
suggest that individual responses to benzene may be genetically determined.
Johnson & Lucier (1992) concluded from an analysis of trans,trans-muconic acid
biomarker assays in humans that genetic variability may account, in part, for the
variance between benzene exposure and urinary trans,trans-muconic acid
concentrations. Subsequently, it has been postulated that the presence of genetically
determined differences in enzyme expression or activity, genetic polymorphisms,
may partially account for the toxicity associated with benzene exposure (Aksoy,
1985; Moran et al, 1999; Rothman et al, 1997). Studies of cases involving familial
susceptibility to benzene (Aksoy, 1985) tend to support this view. Genetic
differences involved in the metabolism of benzene can modify its rate of
metabolism, the profile of metabolites produced and metabolite
activation/detoxification pathways. Such changes have been quantified by analysis
of the urinary metabolites of benzene, phenylmercapturic acid and trans,trans-
muconic acid (Rossi et al, 1999).
Benzene 115
CYP2E1
The metabolism of benzene by hepatic CYP2E1 is the critical first step in the
development of benzene toxicity as the enzyme is responsible for the formation of
phenol and its secondary metabolism to hydroquinone (Guengerich et al, 1991).
Genetic polymorphisms associated with CYP2E1 from different racial groups have
been identified, with frequencies ranging from 2-27% (Kato et al, 1992) and
changes in transcriptional activity of the enzyme in response to mutations have
been described (Hayashi et al, 1991). Seaton et al. (1994) observed that the
CYP2E1 activity of microsomes prepared from the livers of trauma victims varied
13-fold with respect to benzene metabolism. However, as DNA analysis was not
undertaken, it is not known if the differences were genetically determined.
Rothman et al. (1997) showed in a study of 50 workers exposed to benzene that
CYP2E1 genetic polymorphisms were not associated with benzene toxicity. In
another study of 59 workers exposed to benzene, although considerable variation in
urinary metabolite markers was observed, the subjects did not exhibit
polymorphisms associated with CYP2E1 (Rossi et al, 1999).
A cross-species analysis of the expression of CYP2E1 in the bone marrow of mice,
rats and rabbits and human CD34+ stem cells found the enzyme to be present in all
species tested. While the intra- and interspecies variability between mice and rats
was small with relatively low enzyme activities, rabbits exhibited enzyme activities
an order of magnitude greater (Bernauer et al, 2000).
Glutathione-S-transferase
The enzymatic conjugation of GSH to a number of benzene metabolites,
particularly benzene oxide, trans,trans-muconaldehyde and quinones, occurs via
the action of glutathione-S-transferase (GST) (Goon et al, 1993b; Jerina et al,
1968). It has been postulated that GST genetic polymorphisms are positively
correlated with increased risk of oxidative stress (Hayes & Strange, 1995) and
cancer (Strange et al, 1998). Xu et al. (1998b) found a significant association (p
<0.05) between benzene exposure (0.71 ppm TWA), sister chromatid exchanges
and the GSTT1 genotype in a study of 23 workers. Hsieh et al. (1999) examined the
role of GST polymorphism in workers exposed to benzene and found that those
with the GSTT1 and GSTM1 variants of the enzyme, which exhibit reduced
enzymatic activity, were more likely (p = 0.046) to have reduced white blood cell
counts on exposure to high levels of benzene.
Epoxide hydrolase
Epoxide hydrolase, which converts benzene oxide to benzene dihydrodiol, has the
potential to regulate the formation of trans,trans-muconaldehyde. Analysis of 40
transplant-quality human liver samples for interindividual variation in epoxide
hydrolase activity revealed an approximately 8-fold difference in enzymatic
activity and microsomal epoxide hydrolase protein levels were highly correlated
with that activity. In contrast, neither enzymatic activity nor microsomal epoxide
hydrolase protein levels correlated with microsomal epoxide hydrolase RNA levels
which varied by 49-fold. Polymorphisms in amino acid loci of epoxide hydrolase
accounted, in part, for the differences in enzyme activity (Hassett et al, 1997).
NAD(P)H:quinone oxidoreductase (NQO1)
NQO1 catalyzes the two-electron reduction of quinones to their corresponding
hydroquinone form (Lind et al, 1982). Twerdok et al. (1992) reported considerable
Priority Existing Chemical Number 21
116
strain differences in the basal and inducible levels of NQO1 between C57B1/6 and
DBA/2-derived mouse bone marrow stromal cells. The basal and maximal
inducible activity of NQO1 in C57B1/6-derived stromal cells was approximately 3-
and 5-fold greater respectively than that of DBA/2-derived cells.
Traver et al. (1992) identified a point mutation in the human NQO1 gene (609CT)
that results in loss of enzymatic activity in the protein. Thus individuals
homozygous for the mutation possess no NQO1 activity, while heterozygous
individuals exhibit reduced enzymatic activity. It has been estimated, using a
reference population, that the frequency of the mutation is 13% (Rosvold et al,
1995). Analysis of several ethnic groups has shown that homozygous individuals
range between 5-22% and heterozygous individuals from 34-52% of the population
(Kelsey et al, 1997). The study of Rothman et al. (1997) demonstrated a correlation
between NQO1 genetic polymorphism and benzene toxicity among 50 workers
exposed to benzene. Rossi et al. (1999) identified a high frequency of NQO1
genetic polymorphism (42.7% reduced activity and 8.3% no activity) amongst 59
workers exposed to benzene and urinary excretion of S-phenylmercapturic acid was
significantly lower in individuals lacking NQO1 activity. An increased prevalence
of the 609CT mutation has been found in a study of 104 patients diagnosed with
myeloid leukemias (Larson et al, 1999). However, a study of a group of six related
individuals predisposed to cancer showed that the NQO1 609CT transversion did
not correlate with NQO1 activity in heterozygous individuals, suggesting that either
the 609CT transversion has no effect on NQO1 activity or that post-transcriptional
regulation alters the activity of the modified enzyme (Kuehl et al, 1995).
Further investigations, in vitro, have found NQO1 to be inducible in wild-type
(C/C) human bone marrow cells on exposure to the benzene metabolites
hydroquinone and catechol. In contrast, cells homozygous for the 609CT mutation
(T/T) did not express NQO1 in response to hydroquinone treatment whereas
heterozygous cells (C/T) exhibited intermediate induction (Moran et al, 1999).
12.4.3 Environmental influences
In addition to genetic influences, the susceptibility of an individual to benzene
toxicity may also be influenced by environmental or lifestyle factors. Generally, the
role of environmental factors in modifying benzene toxicity have not been
adequately studied. Reviews of environmental influences on solvent toxicity,
including benzene, have been published (Medinsky et al, 1994; Sato, 1991).
Alcohol
As discussed in Section 9, CYP2E1 is the initial enzyme responsible for the
metabolism of benzene to phenolic metabolites. Studies have shown a number of
substances including alcohol (ethanol) to induce hepatic CYP2E1 activity
(Johansson & Ingelman-Sundberg, 1988; Koop et al, 1989). Alcohol consumption
by rats and rabbits resulted in increased microsomal metabolism of benzene
(Johansson & Ingelman-Sundberg, 1988; Nakajima et al, 1985) and increased
benzene-mediated myelotoxicity in rats (Nakajima et al, 1985). Consequently,
alcohol consumption by individuals exposed to benzene may result in enhanced
metabolite formation and increased risk of myelotoxicity.
Toluene
Toluene acts as a substrate for CYP2E1 (Nakajima et al, 1992) and thus, in the
presence of benzene, can act as an inhibitor of benzene metabolism. Andrews et al.
Benzene 117
(1977) demonstrated the inhibition of benzene metabolism by the co-administration
of toluene to male Swiss mice. Urinary benzene metabolites were significantly
decreased (p <0.01) and the amount of exhaled benzene increased in toluene- and
benzene-treated animals compared to control benzene-treated animals. While the
tissue concentration of benzene did not alter with toluene treatment, the
concentration of total benzene metabolites was significantly reduced (p <0.05) in
various tissues including blood and bone marrow. Inoue et al. (1989) showed that
workers exposed concurrently to benzene and toluene produced significantly less (p
<0.01) urinary trans,trans-muconic acid compared to workers exposed only to
benzene. However, in a study in which urinary phenylmercapturic acid was used as
a biomarker for workers exposed to benzene, no correlation with toluene exposure
was found (Inoue et al, 2000).
Non-steroidal anti-inflammatory drugs
Cyclooxygenase activity within bone marrow contributes to benzene-mediated
bone marrow toxicity by participating in the oxidation of phenolic metabolites to
reactive species and by the conversion of macrophage-derived arachidonic acid to
PGE2, an inhibitor of stem cell proliferation. Non-steroidal anti-inflammatory drugs
(NSAIDs) such as aspirin and indomethacin are potent inhibitors of
cyclooxygenase activity (Randall et al, 1980; Roth et al, 1975). The administration
of NSAIDs, prior to benzene exposure, has been shown to diminish the bone
marrow toxicity associated with benzene in mice (Kalf et al, 1989; Pirozzi et al,
1989). The routine use of NSAIDs may confer some protection from the effects of
benzene exposure.
12.5 Summary
Exposure to benzene can result in bone marrow toxicity in several species in
addition to leukaemia in humans and solid tumours in other animal species. In
order for bone marrow toxicity to occur, benzene must first be metabolised by the
liver to intermediate metabolites. These metabolites become localised within the
bone marrow where they undergo activation by peroxidase enzymes, particularly
myeloperoxidase which is found in large amounts in bone marrow, and, to a lesser
extent, by cyclooxygenase. While individual benzene metabolites appear not to
induce bone marrow toxicity, the combination of phenol and hydroquinone have
been shown to induce the same effects on bone marrow as benzene. This effect
appears to be due to the ability of phenol to act as a co-oxidant in the activation of
hydroquinone to the semiquinone and benzoquinone by myeloperoxidase.
Subsequent changes in cellular function result in altered growth factor production
with inhibition of bone marrow stem cell proliferation, differentiation and
maturation. The oxidation of hydroquinone also results in the formation of reactive
oxygen species. Damage to cells by these species can result from DNA adduct
formation, DNA base modification, chromosomal aberrations and changes to
intracellular redox status, particularly depletion of glutathione and oxidation of
protein sulfhydryl groups. Damaged cells not deleted by apoptosis and which
possess activated oncogenes or damaged tumour suppressor genes may begin to
proliferate as clonal lines, which may result in leukaemia in humans or solid
tumours in animals.
While a number of gender-related differences have been described in the response
of rodents to benzene exposure, there is no evidence for such differences in the
response of humans. However, humans do exhibit differences in the expression and
Priority Existing Chemical Number 21