Benzenamine, 2-methyl-6-nitro-: Human health tier II assessment

25 November 2016

CAS Number: 570-24-1

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This assessment was carried out by staff of the National Industrial Chemicals Notification and Assessment Scheme (NICNAS) using the Inventory Multi-tiered Assessment and Prioritisation (IMAP) framework.

The IMAP framework addresses the human health and environmental impacts of previously unassessed industrial chemicals listed on the Australian Inventory of Chemical Substances (the Inventory).

The framework was developed with significant input from stakeholders and provides a more rapid, flexible and transparent approach for the assessment of chemicals listed on the Inventory.

Stage One of the implementation of this framework, which lasted four years from 1 July 2012, examined 3000 chemicals meeting characteristics identified by stakeholders as needing priority assessment. This included chemicals for which NICNAS already held exposure information, chemicals identified as a concern or for which regulatory action had been taken overseas, and chemicals detected in international studies analysing chemicals present in babies’ umbilical cord blood.

Stage Two of IMAP began in July 2016. We are continuing to assess chemicals on the Inventory, including chemicals identified as a concern for which action has been taken overseas and chemicals that can be rapidly identified and assessed by using Stage One information. We are also continuing to publish information for chemicals on the Inventory that pose a low risk to human health or the environment or both. This work provides efficiencies and enables us to identify higher risk chemicals requiring assessment.

The IMAP framework is a science and risk-based model designed to align the assessment effort with the human health and environmental impacts of chemicals. It has three tiers of assessment, with the assessment effort increasing with each tier. The Tier I assessment is a high throughput approach using tabulated electronic data. The Tier II assessment is an evaluation of risk on a substance-by-substance or chemical category-by-category basis. Tier III assessments are conducted to address specific concerns that could not be resolved during the Tier II assessment.

These assessments are carried out by staff employed by the Australian Government Department of Health and the Australian Government Department of the Environment and Energy. The human health and environment risk assessments are conducted and published separately, using information available at the time, and may be undertaken at different tiers.

This chemical or group of chemicals are being assessed at Tier II because the Tier I assessment indicated that it needed further investigation.

For more detail on this program please


NICNAS has made every effort to assure the quality of information available in this report. However, before relying on it for a specific purpose, users should obtain advice relevant to their particular circumstances. This report has been prepared by NICNAS using a range of sources, including information from databases maintained by third parties, which include data supplied by industry. NICNAS has not verified and cannot guarantee the correctness of all information obtained from those databases. Reproduction or further distribution of this information may be subject to copyright protection. Use of this information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner. NICNAS does not take any responsibility whatsoever for any copyright or other infringements that may be caused by using this information.

Acronyms & Abbreviations

Chemical Identity

Synonyms 6-nitro-o-toluidene

Structural Formula Structural formula of Benzenamine, 2-methyl-6-nitro-
Molecular Formula C7H8N2O2
Molecular Weight (g/mol) 152.152
Appearance and Odour (where available) Dark red crystalline powder
SMILES c1(N(=O)=O)c(N)c(C)ccc1


The chemical is on the 'List of chemicals used as dyes in permanent and semi-permanent hair dyes in Australia' (NICNAS, 2007), indicating its use in semi-permanent hair dye preparations.

NICNAS was informed through an industry consultation that the chemical is no longer used in hair dyes in Australia (2016), largely due to the inclusion of the substance in Annex II of the EU Cosmetics Regulations.


The following international uses have been identified through Galleria Chemica; the European Commission Cosmetic Ingredients and Substances (CosIng) database; United States (US) Personal Care Product Council International Nomenclature of Cosmetic Ingredients (INCI) dictionary.

The chemical has reported cosmetic use in semi-permanent hair dye preparations. The maximum concentration in semi-permanent hair dye formulations was indicated as 0.35 % (SCCNFP, 2003).

The chemical has reported site-limited use as an intermediate.


No known restrictions have been identified.


The chemical is listed on the following (Galleria Chemica):

  • European Union (EU) Cosmetics Regulation 1223/2009 Annex II—List of substances prohibited in cosmetic products;
  • EU Commission banned hair dye substances;
  • New Zealand Cosmetic Products Group Standard—Schedule 4: Components cosmetic products must not contain; and
  • Association of Southeast Asian Nations (ASEAN) Cosmetic Directive Annex II (Part 1)—List of substances which must not form part of the composition of cosmetic products.

Existing Work Health and Safety Controls

Hazard Classification

The chemical is not specifically listed on the Hazardous Substances Information System (HSIS) (Safe Work Australia).

However, there is a group entry in the HSIS, which is applicable for this chemical,

'Nitrotoluidines, with the exception of those specified elsewhere in HSIS'. Based on this, the following risk phrases are applicable for this chemical:

  • T; R23/24/25 (acute toxicity)
  • R33 (cumulative effects)

No specific exposure standards are available.


No specific exposure standards are available.


Acute and repeated dose animal toxicity studies indicate that the chemical can be absorbed via the oral and dermal routes. When the amino group of the chemical is oxidised, 2,3-dinitrotoluene will be formed, which is then metabolised in the liver by cytochrome P450 to form dinitrobenzyl alcohol glucuronide. Excretion occurs in the bile or urine. The nitro group of the chemical can be reduced to 2,3-diaminotoluene (SCCNFP, 2003).

The dermal absorption of the chemical at 0.69 % (in a formulation) was tested in human epidermis using the Franz diffusion cell system. The epidermal surface (2 cm2) was subjected to 20 mg/cm2 of the chemical for 30 minutes and then left to diffuse for a further 24 hours. The absorbed amounts (in epidermis, dermis and receptor fluid) were calculated to be 2.73 ± 1.44 % of the applied dose (3.65 ± 2.00 µg/cm2) (SCCNFP, 2003).


The chemical has low acute oral toxicity in animal tests. Therefore, the hazard classification recommended for nitrotoluidines in the HSIS is not applicable for this chemical.

In an acute oral toxicity study (conducted similarly to the Organisation for Economic Cooperation and Development (OECD) Test Guideline (TG) 401), Sprague Dawley (SD) rats (n = 5/sex) were administered (by gavage) the chemical once at 2000 mg/kg bw and observed for 14 days. On day one post-dosing, there were three mortalities (1/5 male and 2/5 females). Observed sublethal effects for the surviving animals included sedation, hypokinesia and dyspnoea. These were reversible by day four. The acute median lethal dose (LD50) was reported to be >2000 mg/kg bw (SCCNFP, 2003).


No data are available for the chemical. However, nitrotoluidines are classified as hazardous with the risk phrase 'Toxic in contact with skin’ (Xn; R24) in the HSIS. Until data on the chemical become available, this group classification is considered applicable for the chemical.


No data are available for the chemical. However, nitrotoluidines are classified as hazardous with the risk phrase 'Toxic by inhalation’ (Xn; R23) in the HSIS. Until data on the chemical become available, this group classification is considered applicable for the chemical.

Skin Irritation

Only limited data are available. The chemical at a 3 % concentration was slightly irritating to the skin of rabbits.

In a skin irritation study (OECD TG 404), the chemical at 3 % (suspended in 1,2-propanediol) was applied (0.5 mL) to the intact skin of three New Zealand male rabbits for four hours. Slight erythema was observed in 2/3 animals after one hour, which persisted for up to 48 hours. Well-defined erythema was observed in the third animal, which persisted until day six, accompanied by dryness on days 4–6. No other effects were reported (SCCNFP, 2003). The irritation scores were not available.

Eye Irritation

Only limited data are available. The chemical at a 3 % concentration was slightly irritating to the eyes of rabbits.

In an eye irritation study (OECD TG 405), the chemical at 3 % concentration suspended in 1,2-propanediol was applied (0.1 mL) to the left eye of three New Zealand male rabbits. Observations were made at 1, 24, 48 and 72 hours following application. After one hour, slight conjunctival reactions were observed in all animals, which persisted for up to 24 hours in 2/3 animals (irritation scores were not available). No iridial or corneal reactions were observed (SCCNFP, 2003).

Skin Sensitisation

The chemical is not expected to be a skin sensitiser.

In a Magnusson and Kligman sensitisation study, groups of Dunkin Hartley guinea pigs (n = 10/sex) were induced with three intradermal injections of Freund’s complete adjuvant (FCA), the chemical at 1 % and a mixture of the two. On day six following the induction phase, 10 % of lauryl sulphate was applied (0.5 mL) to the injection site to induce skin irritation. On day seven, a single topical dose of the chemical at a 3 % concentration was applied (0.5 mL) for 48 hours. During the challenge phase (day 14), animals were exposed to a 3 % concentration of the chemical (0.5 mL) on the flank for 24 hours. Observations were made at 24 and 48 hours after exposure ceased. Irritation was observed at the intradermal injection site at the end of the induction period, but no skin reactions were observed at 48 hours after the challenge. The test substance was not a skin sensitiser under the test conditions used (SCCNFP, 2003).


Based on the limited data available, the chemical is considered to cause serious damage to health from repeated oral exposure.

Nitrotoluidines are classified as hazardous with the risk phrase 'Danger of cumulative effects' (Xn; R33) in the HSIS. The available information is not sufficient to determine whether the effects observed in the short-term studies are the result of the chemical accumulating in the body. The liver weight changes observed at the 20 mg/kg bw/day dose administered for 30 days are more consistent with classification with the risk phrase 'Danger of serious damage to health by prolonged exposure' (Xn; R48).

No 90-day studies are available, but there are two 29–30 days studies available in SD rats. In the first study (OECD TG 407), groups of SD rats (n = 10/sex/dose) were administered the chemical by oral gavage at doses of 0, 20, 100 or 500 mg/kg bw/day, seven days/week, for 29 or 30 days. There were no mortalities. Rats that received the highest dose showed hypersalivation, dyspnoea, decreased spontaneous activity and weight gain. Corneal dystrophy was observed in one male at 20 mg/kg bw/day, one male at 100 mg/kg bw/day and two males and four females at 500 mg/kg bw/day. Dose-related liver enlargement ('with an accentuated lobular pattern') was observed in both sexes. Increased liver weights were statistically significant at 100 and 500 mg/kg bw/day. Increased spleen and ovarian weights were observed in high dose females. A dose-related increase in alanine aminotransferase level in males was reported (statistically significant at 100 and 500 mg/kg bw/day). Histopathological changes in the liver included centrilobular or diffuse hepatic cell hypertrophy (with centrilobular vacuolisation and multifocal hepatic cell degeneration in most animals) at =100 mg/kg bw/day. A few males (up to three) showed centrilobular cytoplasmic vacuolation at 20 mg/kg bw/day. Tubular dilatation, tubular epithelial cell vacuolisation and/or degeneration and necrosis in the kidneys were observed at 500 mg/kg bw day in all rats, and in one female at 100 mg/kg bw/day. One male at 20 mg/kg bw/day had a dilated kidney pelvis. A no observed adverse effect level (NOAEL) could not be established in this study (SCCNFP, 2003).

In the second study (OECD TG 407), groups of SD rats (n = 10/sex/dose) were administered the chemical by oral gavage doses of 0, 4, 9 or 20 mg/kg bw/day for 29–30 days. No mortalities or clinical signs of toxicity were observed. Minor eye conditions were observed on day one of the study (no details), but were reported to be resolved by the end of the study. Dose-related increases in liver weights were statistically significant at 20 mg/kg bw/day (>100 % increase compared with 10 % increase at 9 mg/kg bw/day). In a few cases, histopathological changes in the liver and lungs were seen at the highest dose (limited or not investigated in low and mid dose groups). Although the study authors reported a NOAEL of 20 mg/kg bw/day, the SCCS concluded the NOAEL to be 9 mg/kg bw/day, based on increased liver weights (SCCNFP, 2003).


No data are available.


No data are available.


Based on the available data, the chemical is not considered to be genotoxic.

The chemical gave mixed results in several in vitro assays (SCCNFP, 2003):

  • positive results in a bacterial reverse mutation assay with Salmonella typhimurium strain TA 98, with or without metabolic activation and strain TA 100 without metabolic activation, at concentrations of 156–2500 mg/plate;
  • negative results were reported in S. typhimurium strains TA 1535, 1537 and Escherichia coli strain WP2uvrA, with or without metabolic activation;  
  • negative results in L5178Y mouse lymphoma cell forward mutation assay, with or without metabolic activation; and
  • positive results for inducing chromosomal aberrations in Chinese hamster ovary (CHO) cells at 500 and 2500 mg/ml without metabolic activation.

Two in vivo assays with the chemical gave negative results (SCCNFP, 2003):

  • in a mammalian erythrocyte micronucleus test (OECD TG 474) no induction of micronucliei in bone marrow cells was found in Swiss mice that received the chemical by two oral gavage doses (24 hours apart) at up to 1600 mg/kg bw; the test confirmed that the chemical reached the bone marrow cells of mice at the highest dose; and
  • in an unscheduled DNA synthesis (UDS) assay (draft OECD TG 475—1991), Wistar rats (three males per dose) that received the chemical by an oral gavage dose of 100 or 1000 mg/kg bw showed no difference in the viability of isolated hepatocytes 16 hours after the treatment.


No carcinogenicity data are available on the chemical. However, based on the information provided below (from SCCNFP, 2003), the chemical may be carcinogenic, warranting hazard classification.

The chemical can be metabolised into 2,3-dinitrotoluene (CAS No. 602-01-7) and 2,3-diaminotoluene (CAS No. 2687-25-4) (see Toxicokinetics). The SCCNFP report (2003) stated that 2,3-dinitrotoluene is classified as a category 2 carcinogen and a category 3 mutagen by the EU, and the chemical may give rise to the same aminonitrobenzyl alcohol as 2,3-dinitrotoluene (SCCNFP, 2003).

The SCCNFP report (2003) further stated that:

  • 'The aminonitrobenzyl alcohol is reabsorbed and transported back to the liver by enterohepatic circulation';
  • 'The amine group is subsequently N-hydroxylated by cytochrome P450 and conjugated with sulfate';
  • 'The sulfate conjugate is unstable and can be decomposed to form a carbonium or nitrenium ion that can be bound to macromolecules; this ostensibly leads to mutations and the formation of tumours';
  • 'In agreement with this it is found that 5-nitro-o-toluidine is carcinogenic (Classified as carcinogen category 3 by EU and category 2 by the MAK-Committee in Germany)';
  • 'It should also be noted that if the nitro-group of 6-nitro-o-toluidine is reduced 2,3-diaminotoluene is formed';
  • 'The EU has classified diaminotoluenes as carcinogen category 2'; and
  • 'In the absence of data on metabolic fate in vivo, 6-nitro-o-toluidine may be carcinogenic'.

Chemicals acting by a nitrenium ion mechanism are commonly found to give positive results in Ames tests in certain S. typhimurium strains such as TA 98, with metabolic activation (Benigni & Bossa, 2011). These positive results were seen for the chemical, consistent with the postulated mechanism.

The single study available using the chemical does not indicate any reproductive or developmental toxicity effects in rats dosed at up to 90 mg/kg bw/day.

In a teratogenicity study (OECD TG 414), pregnant female SD rats (n = 25) were administered the chemical by oral gavage doses of 0, 10, 30 and 90 mg/kg bw/day during gestation days (GD) 6–15 and euthanised on day 20. There were no premature deaths. At the highest dose, reduced weight gain and food consumption were observed from days 6–15. There were no treatment-related observations in the dams and all other parameters (such as 'mean numbers of corpora lutea, implantation sites, post-implantation loss, live foetuses, sex distribution and the mean foetal bodyweights') were reported to be similar between the treatment and control groups. No dose-related increases in foetal abnormalities or malformations were reported (SCCNFP, 2003).  

Critical Health Effects

The critical health effects for risk characterisation include systemic long-term effects (carcinogenicity).

The chemical can also cause adverse effects following repeated oral exposure. Acute effects by the dermal and inhalation routes cannot be excluded.

Public Risk Characterisation

Although the chemical was reported to be used in semi-permanent hair dye preparations in Australia in 2007 (NICNAS, 2007), an industry consultation confirmed that the chemical is no longer used in hair dyes in Australia. Therefore, it is unlikely that the public will be exposed to this chemical and no public health risks are expected.

Occupational Risk Characterisation

Given the critical health effects, the chemical may pose an unreasonable risk to workers unless adequate control measures to minimise exposure to the chemical are implemented. The chemical should be appropriately classified and labelled to ensure that a person conducting a business or undertaking (PCBU) at a workplace (such as an employer) has adequate information to determine appropriate controls.

NICNAS Recommendation

Current risk management measures are considered adequate to protect public and workers’ health and safety, provided that all requirements are met under workplace health and safety, and poisons legislation as adopted by the relevant state or territory. No further assessment is required.

Work Health and Safety

The chemical is recommended for classification and labelling under the current approved criteria and adopted GHS as below. This assessment does not consider classification of physical hazards and environmental hazards.

Hazard Approved Criteria (HSIS)a GHS Classification (HCIS)b
Acute Toxicity Toxic in contact with skin (T; R24)* Toxic by inhalation (T; R23)* Toxic in contact with skin - Cat. 3 (H311) Toxic if inhaled - Cat. 3 (H331)
Repeat Dose Toxicity Harmful: danger of serious damage to health by prolonged exposure if swallowed (Xn; R48/22) May cause damage to organs through prolonged or repeated exposure - Cat. 2 (H373)
Carcinogenicity Carc. Cat 3 - Limited evidence of a carcinogenic effect (Xn; R40) Suspected of causing cancer - Cat. 2 (H351)

a Approved Criteria for Classifying Hazardous Substances [NOHSC:1008(2004)].

b Globally Harmonized System of Classification and Labelling of Chemicals (GHS) United Nations, 2009. Third Edition.

* Existing Hazard Classification. No change recommended to this classification

Advice for industry

Control measures

Control measures to minimise the risk from oral, dermal and inhalation exposure to the chemical should be implemented in accordance with the hierarchy of controls. Approaches to minimise risk include substitution, isolation and engineering controls. Measures required to eliminate, or minimise risk arising from storing, handling and using a hazardous chemical depend on the physical form and the manner in which the chemical is used. Examples of control measures which could minimise the risk include, but are not limited to:

  • using closed systems or isolating operations;
  • using local exhaust ventilation to prevent the chemical from entering the breathing zone of any worker;  
  • health monitoring for any worker who is at risk of exposure to the chemical, if valid techniques are available to monitor the effect on the worker’s health;
  • minimising manual processes and work tasks through automating processes;
  • work procedures that minimise splashes and spills;
  • regularly cleaning equipment and work areas; and
  • using protective equipment that is designed, constructed, and operated to ensure that the worker does not come into contact with the chemical.

Guidance on managing risks from hazardous chemicals are provided in the Managing risks of hazardous chemicals in the workplace—Code of practice available on the Safe Work Australia website.

Personal protective equipment should not solely be relied upon to control risk and should only be used when all other reasonably practicable control measures do not eliminate or sufficiently minimise risk. Guidance in selecting personal protective equipment can be obtained from Australian, Australian/New Zealand or other approved standards.

Obligations under workplace health and safety legislation

Information in this report should be taken into account to help meet obligations under workplace health and safety legislation as adopted by the relevant state or territory. This includes, but is not limited to:

  • ensuring that hazardous chemicals are correctly classified and labelled;
  • ensuring that (material) safety data sheets ((M)SDS) containing accurate information about the hazards (relating to both health hazards and physicochemical (physical) hazards) of the chemical are prepared; and
  • managing risks arising from storing, handling and using a hazardous chemical.

Your work health and safety regulator should be contacted for information on the work health and safety laws in your jurisdiction.

Information on how to prepare an (M)SDS and how to label containers of hazardous chemicals are provided in relevant codes of practice such as the Preparation of safety data sheets for hazardous chemicals—Code of practice and Labelling of workplace hazardous chemicals—Code of practice, respectively. These codes of practice are available from the Safe Work Australia website.

A review of the physical hazards of the chemical has not been undertaken as part of this assessment.


Benigni R and Bossa C 2011. Mechanisms of Chemical Carcinogenicity and Mutagenicity: A Review with Implications for Predictive Toxicology. Chem. Rev. 2011, 111, 2507-2536.

ChemIDPlus, CAS No. 570-24-1. Accessed August 2014 at

Cosmetic Directive (CosIng). 6-Nitro-o-toluidine (570-24-1). Accessed August 2014 at

Galleria Chemica. Accessed August 2014 at

National Industrial Chemicals Notification and Assessment Scheme (NICNAS) 2007. List of Chemicals used as Dyes in Permanent and Semi-Permanent Hair Dyes in Australia.

Personal Care Products Council (INCI Dictionary). Accessed Auguest 2014 at

Scientific Committee on Consumer Products and Non-Food Products Intended for Consumers (SCCNFP) 2003. Opinion of the Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers concerning 6-nitro-o-toluidine. Adopted by the SCCNFP during the 24th plenary meeting of 24-25 June 2003. Accessed August 2014 at

Scientific Committee on Consumer Safety (SCCS) 2012. Opinion on Nitrosamines and Secondary Amines in Cosmetic Products. Adopted at its 14th plenary meeting of 27 March 2012. Accessed at

Last update 25 November 2016