Scheduling delegate's interim decisions and invitation for further comment: ACCS/ACMS, November 2016

Scheduling medicines and poisons

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2 February 2017

1.9 o-Toluidine and o-anisidine

Part A - Interim decisions on matters referred to an expert advisory committee (November 2016)

Advisory Committee on Chemicals Scheduling (ACCS #18)

1.9 o-Toluidine and o-anisidine

Referred scheduling proposal

An application was submitted by the National Industrial Chemicals Notification and Assessment Scheme (NICNAS) to create new Schedule 10 entries for o-toluidine and o-anisidine for use in preparations for skin colouration (including tattooing) and dyeing of hair, eyelashes or eyebrows.

Scheduling application

General application.

The applicant's proposed amendments to the Poisons Standard are as follows:

Schedule 10 - New Entries

o-TOLUIDINE in preparations for skin colouration (including tattooing) and dyeing of hair, eyelashes or eyebrows.

o-ANISIDINE in preparations for skin colouration (including tattooing) and dyeing of hair, eyelashes or eyebrows.

The applicant's reasons for the request are:

  • As part of activities undertaken by NICNAS in 2014-15, o-toluidine and o-anisidine were identified as impurities in tattoo inks in Australia. The chemicals have been detected in hair dye samples overseas.
  • The chemicals are mutagenic and carcinogenic following long-term repeated exposure. A genotoxic mode of action is suggested by the available evidence.
  • There is an increasing popularity of body adornment through tattoos or permanent make-up (PMU). Tattoos result in long term exposure to the injected chemicals.
  • There are international restrictions on the presence of these amines in cosmetic products and tattoo ink preparations.
  • The schedule 10 (Appendix C) entry for 2,4-toluenediamine (an aromatic amine with similar toxicological profile) was recently amended to include use in tattoos.
  • The inclusion in Schedule 10 is consistent with other aromatic amines with similar toxicological profiles such as 2,4-toluene diamine and would cover the presence of the chemical as an impurity.
Current scheduling status and relevant scheduling history

Neither o-anisidine nor o-toluidine are specifically scheduled. The chemicals are named as part of Schedule 7 entry for azo dyes as follows:

p-aminoazobenzene (CAS No. 60-09-3)

o-aminoazotoluene (CAS No. 97-56-3)

o-anisidine (CAS No. 90-04-0)

p-chloroaniline (CAS No. 106-47-8)

4-chloro-o-toluidine (CAS No. 95-69-2)

6-methoxy-m-toluidine (p-cresidine) (CAS No. 120-71-8)

2-naphthylamine (CAS No. 91-59-8)

5-nitro-o-toluidine (CAS No. 99-55-8)

2,4-toluenediamine (CAS No. 95-80-7)

o-toluidine (CAS No. 95-53-4)

2,4,5-trimethylaniline (CAS No. 137-17-7)

o-Toluidine and o-anisidine were first included in Schedule 7 in February 2016 as part of the azo dyes entry due to their carcinogenicity and genotoxicity (see section Current scheduling status). The toxicological data for both o-toluidine and o-anisidine were provided by NICNAS to support the application for the scheduling of azo dyes at the August 2015 Advisory Committee for Chemicals Scheduling (ACCS) meeting. The reports provided at the August 2015 meeting were the same as those provided with this application.

o-Toluidine and o-anisidine are not otherwise specifically scheduled.

Australian regulatory information

The chemicals have been detected in tattoo inks in Australia. It was not possible to identify all popular tattoo inks used in Australia. Of the 49 tattoo inks analysed:

  • 6 tattoo inks contained o-toluidine at concentrations between 20 and 89 ppm.
  • 15 tattoo inks contained o-anisidine at concentrations between 15 and 77 ppm.

The limit of detection for the assay was 5 ppm.

The data analysis conducted by NICNAS suggests that for the majority of amine-containing tattoo inks, the amines are introduced at some point during manufacture rather than as a result of the reduction of the azo colourants. The pigments used in tattoos are not produced specifically for such application and purity is on average around 70-90 % (European Commission, 2015; European Commission, 2016).

The popularity of tattoos is reported to have increased in the last decade. Based on a survey completed by a representative sample of 8656 men and women ages 16-64 years in Australia, the prevalence of tattoos was reported to be 14.5 % in men and 13.6 % in women. Tattoo prevalence in young adults may represent more than double (European Commission, 2015).

The chemicals have also been detected in tattoo inks (overseas) (European Commission, 2015, p180). The brands in which the chemicals were detected are reported to be available in Australia. Two thirds of the RAPEX notifications in Europe are reported to pertain to imported products, with the highest percentages from the United States (European Commission, 2015). Therefore it is considered likely that these tattoo inks could be imported into Australia.

The chemicals have also been detected in permanent hair dyes and commercial henna samples (colours not specified) overseas. In the absence of specific Australian data, this is considered to be representative of Australian use.

International regulations

The chemicals are restricted under Annex XVII to REACH Regulations. 'The chemical cannot be used in substances and preparations placed on the market for sale to the general public in individual concentrations ≥0.1 %' (European Parliament and Council 1999; European Parliament and Council 2006; European Parliament and Council 2008). The chemicals are also included as part of 22 aromatic amines listed in Appendix 8 which places restrictions on their presence in leather or textile articles.

The chemicals are on the candidate list of substances of very high concern (SVHC) for eventual inclusion in Annex XIV (ECHA, 2013). In the EU, companies could have legal obligations if the chemical that they produce, supply or use is included on the candidate list whether on its own, in mixtures, or present in articles.

ResAP (2008)1 specifies requirements for the composition, labelling, uses and risk evaluation of tattoo inks in the European Union. ResAP (2008)1 lists 27 aromatic amines (including o-toluidine and o-anisidine) that should not be present in tattoo inks or released from azo-colourants in concentrations that are technically avoidable. The non-binding ResAP are the reference for the national legislation in several European countries and New Zealand (NZ EPA, 2012).

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

  • EU Cosmetics Regulation 1223/2009 Annex II-List of substances prohibited in cosmetic products.
  • New Zealand Cosmetic Products Group Standard-Schedule 4: Components cosmetic products must not contain.
  • In addition o-toluidine is listed in the Health Canada List of prohibited and restricted cosmetic ingredients (The Cosmetic Ingredient "Hotlist") under the entry 'Toluidines, their isomers, salts and halogenated and sulfonated derivatives'.
Substance summary

Chemical structures of <em>o</em>-toluidine (A) and <em>o</em>-anisidine (B)

Figure 1.9: Chemical structures of o-toluidine (A) and o-anisidine (B)

Table 1.9A: Substance summary
Property o-Toluidine o-Anisidine
CAS name benzenamine, 2-methyl- benzenamine, 2-methoxy-
CAS number 95-53-4 90-04-0
IUPAC and/or common and/or other names o-toluidine o-anisidine
Molecular formula C7H9N C7H9NO
Molecular weight 107.16 g/mol 123.15 g/mol

The following toxicology information was extracted from the NICNAS assessment report for o-toluidine and o-anisidine.

Table 1.9B: Acute toxicity end-points for o-toluidine
Toxicity Species Outcome SPF (2015) Classification
Acute oral toxicity LD50 (mg/kg bw) Rats 750* Schedule 6
Acute dermal toxicity LD50 (mg/kg bw) Rabbit 3250 Schedule 5
Acute inhalational toxicity LC50 (mg/m3/4h) Rats 3800* Schedule 5/6
Skin irritation Rabbit Slight irritant Schedule 5
Eye irritation Rabbit Irritant Schedule 6
Skin sensitisation Limited data N/A

* Methaemoglobinaemia has been observed in cats and humans at relatively low doses

Table 1.9C: Acute toxicity end-points for o-anisidine
Toxicity Species Outcome SPF (2015) Classification
Acute oral toxicity LD50 (mg/kg bw) Rats 1505-1890* Schedule 6
Acute dermal toxicity LD50 (mg/kg bw) Rats >2000 Schedule 5
Acute inhalational toxicity LC50 (mg/m3/4h) Rats >3870 Schedule 5
Skin irritation Rabbits Slight irritant Schedule 5
Eye irritation Rabbits Slight irritant Schedule 5
Skin sensitisation ocal lymph node assay (LLNA) and guinea pig maximisation test (GPMT) Equivocal N/A

* Methaemoglobinaemia has been observed in cats and other species at relatively low doses

Acute toxicity, skin irritation and sensitisation

Refer to tables 1.9B and 1.9C.

Repeat-dose toxicity

The toxic effects of the o-toluidine have been investigated in several sub-chronic and chronic repeated dose oral toxicity studies. The results demonstrated not only chemically-induced development of tumours (see carcinogenicity section), but also non-cancer effects. The sub-chronic studies, whilst limited by poor documentation or a single dose test concentration, demonstrated consistent effects with the targets for toxicity being the blood, liver, kidney, spleen and urinary bladder. A no observed adverse effect level (NOAEL) value could not be established with a lowest observed adverse effect level (LOAEL) for effects on the blood of 24 mg/kg bw/day in a 14-day study.

The toxic effects of o-anisidine from repeated oral exposure were investigated in a number of well-conducted studies (OECD TG 407) in Fischer 344, Wistar rats, and B6C3F1 mice. The results demonstrated not only chemically-induced development of tumours (see Carcinogenicity section), but also non-cancerous effects. The lowest observed adverse effect level (LOAEL) available from the 28 day study in rats was 80 mg/kg bw/day. The targets for toxicity are the bone marrow and spleen.

Genotoxicity

o-Toluidine is considered mutagenic based on the weight of evidence from available, well-documented in vitro and in vivo studies. The chemical was recommended for classification as a category 2 (1B for GHS) mutagenic substance). The chemical has been reported to cause mutations, DNA and chromosomal damage and cell transformation. o-Toluidine was reported to induce DNA lesions in different organs of exposed rats (liver, bladder) and mice (liver, bladder and brain. Furthermore, inhibited testicular DNA synthesis was reported in male mice following oral intubation. Renal DNA synthesis was also inhibited after a single intraperitoneal injection of the lethal doses of the chemical in the suckling mice.

o-Anisidine classified or as hazardous (Category 3 mutagenic substance) with the risk phrase 'Possible risk of irreversible effects' (Xn; R68) The genotoxic and mutagenic potentials of the chemicals were evaluated in several well-conducted in vitro and in vivo studies. DNA adducts derived from the N hydroxylated metabolite have been detected in both in vitro and in vivo (i.p. route of administration) studies.

Carcinogenicity

o-Toluidine

o-Toluidine is currently classified as hazardous (Category 2 carcinogen) with the risk phrase 'May cause cancer' (T; R45) in HSIS (Safe Work Australia). The available epidemiological data support an amendment to this classification to a Category 1 carcinogen.

The carcinogenicity of the hydrochloride salt (not listed on the AICS) was investigated in a number of well-conducted OECD TG 452-compliant oral studies (feeding) in male and female F344 and CD-1 rats and mice (B6C3F1 and CD-1). In addition to reduced bodyweight and survival, significant increases in the incidence of multi-organ benign and/or malignant tumours were reported in mice and rats exposed to 3000-16000 ppm of the chemical over three months to two years. The equivalent intake doses were calculated at 130-800 mg/kg bw/day. In rats, tumours were found in the spleen, scrotum, urinary bladder, mammary glands, liver, skin, abdominal cavity and blood vessels of the chemically-exposed animals. In mice, hepatocellular carcinomas, adenomas, haemangiosarcomas, abdominal haemangiosarcomas and haemangiomas were observed.

In humans, findings derived from several cohort studies of workers provide strong evidence for an increased risk of urinary bladder cancer associated with long-term occupational exposure to o-toluidine. The chemical is listed in the National Toxicology Program (NTP) Report on Carcinogens (Twelfth Edition) as 'reasonably anticipated to be a human carcinogen' (NTP, 2011). The International Agency for Research on Cancer (IARC) has reviewed epidemiological data and subsequently concluded that it is 'carcinogenic to humans' (Group 1) (IARC, 2010; IARC, 2012).

The mechanism of action underlying the carcinogenicity of o-toluidine is not fully understood, although it is considered to involve metabolic activation, DNA adduct formation and induction of DNA-damaging effects. A mechanism which includes genotoxicity is suspected.

o-Anisidine

o-Anisidine is currently classified as hazardous (Category 2 carcinogen) with the risk phrase 'May cause cancer' (T; R45) in HSIS (Safe Work Australia). The available data support this classification.

Data from long-term carcinogenicity studies indicated that chronic exposure to o-anisidine in its hydrochloride form, CAS No.134-29-2 (not listed on the Australian Inventory of Chemical Substances) produced malignant and benign tumours, particularly in the urinary bladder of rats and mice.

Chronic oral exposure of F344 rats and B6C3F1 mice to o-anisidine and its hydrochloride salt for two years resulted in transitional-cell papillomas and carcinomas of the urinary bladder in both species. The doses tested were 5000 or 10000 ppm (average of 333 or 666 mg/kg bw/day) for rats and 2500 or 5000 ppm for mice (average of 214 or 428 mg/kg bw/day). A very rare form of tumour in the rat, leiomyosarcoma, was also observed in the urinary bladder of high dose animals. Kidney and thyroid gland cancer, including transitional cell carcinoma of the renal pelvis, follicular-cell adenoma and carcinoma, papillary cystadenoma, and cystadenocarcinoma, were also identified in rats. Cancer-related deaths occurred within 83-88 weeks in animals treated with 5000 or 10000 ppm of the chemical. Another study has indicated the tumour-promoting activity of the chemical.

No human case reports or epidemiological studies are available. The International Agency for Research on Cancer (IARC) overall evaluation is that o-anisidine is 'possibly carcinogenic to humans' (Group 2B).

The mechanism of action for carcinogenicity of the animal is not completely understood; although, both genotoxic and non-genotoxic modes of action are considered plausible. The formation of reactive species that are capable of binding to DNA has been observed. Results from a two-year carcinogenicity study suggested that the observed increased incidence of follicular-cell tumours in male Fischer 344 rats is a potential consequence of the inhibition of thyroid hormone formation, which is catalysed by the thyroid peroxidase.

Reproduction and developmental toxicity

Limited data are available

Observation in humans

o-Toluidine

o-Toluidine is highly toxic to humans and can be absorbed via inhalation and through dermal contact. Exposed individuals have been reported to exhibit clinical signs of toxicity including methaemoglobinaemia, haematuria, renal and bladder irritation, physiological and psychological deficits. Severe intoxication was also observed in individuals exposed to 40 ppm of the chemical for 60 minutes.

Results from a patch test study in 40 dermatitis patients, known to be hypersensitive to p-phenylenediamine, indicated positive reactions in 25% of participants following exposure to o-toluidine in yellow paraffin.

Workers with chronic exposure to the o-toluidine have been reported to display anaemia, weight loss, anorexia, cyanosis, methaemoglobinaemia, skin lesions, and disturbance in the central nervous system such as headache, dizziness and confusion.

Additionally, haemoglobin adducts were found in the urine of workers exposed to the chemical, where higher levels were identified in those with existing impaired skin condition. These adducts were also present in the blood of children with exposure to the chemical and in hairdressers with chronic exposure to hair dyes containing the chemical.

o-Anisidine

Complaints of headache and vertigo, increased sulfhaemoglobin and methaemoglobin, and frequent occurrence of Heinz bodies were reported in workers exposed to o-anisidine (0.4 ppm (2 mg/m3)).

Pre-meeting public submissions

One (1) pre-meeting submission was received in support. The main points were that the new schedule entry would be in alignment with international regulators in the EU, ASEAN and NZ.

Summary of ACCS advice to the delegate

The committee advised that new Schedule 10 entries be created for o-toluidine and o-anisidine for use in preparations for skin colouration (including tattooing) and dyeing of hair, eyelashes or eyebrows as follows:

Schedule 10 - New Entries

o-TOLUIDINE in preparations for skin colouration (including tattooing) and dyeing of hair, eyelashes or eyebrows.

o-ANISIDINE in preparations for skin colouration (including tattooing) and dyeing of hair, eyelashes or eyebrows.

The committee also recommended an implementation date of 1 June 2017.

Members agreed that the relevant matters under Section 52E(1) of the Therapeutic Goods Act 1989 included: (b) the purpose for which a substance is to be used and the and extent of use; and (c) the toxicity of a substance.

The reasons for the advice comprised the following:

  • o-Toluidine and o-anisidine are used in tattoo inks and hair colourants.
  • o-Toluidine and o-anisidine are genotoxic and carcinogenic.
Delegate's considerations

The delegate considered the following in regards to this proposal:

  • Scheduling proposal
  • ACCS advice
  • Public Submissions received
  • Section 52E of the Therapeutic Goods Act 1989
  • Scheduling Policy Framework (SPF 2015)
  • Other relevant information.
Delegate's interim decision

The delegate's interim decision is to create new Schedule 10 entries for o-toluidine and o-anisidine for use in preparations for skin colouration (including tattooing) and dyeing of hair, eyelashes or eyebrows.

The proposed Schedule entry is as follows:

Schedule 10 - New Entries

o-TOLUIDINE in preparations for skin colouration (including tattooing) and dyeing of hair, eyelashes or eyebrows.

o-ANISIDINE in preparations for skin colouration (including tattooing) and dyeing of hair, eyelashes or eyebrows.

The proposed implementation date is 1 June 2017.

The matters under subsection 52E (1) of the Therapeutic Goods Act 1989 considered relevant by the delegate included: b) the purposes for which a substance is to be used and the extent of use of a substance; and c) the toxicity of the substance.

The reasons for the recommendation comprised the following:

  • The delegate acknowledges the committee's advice.
  • o-Toluidine and o-anisidine are used in tattoo inks and hair colourants.
  • o-Toluidine and o-anisidine are genotoxic and carcinogenic.
  • Earliest possible implementation date.

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