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IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Some Aromatic Amines, Organic Dyes, and Related Exposures. Lyon (FR): International Agency for Research on Cancer; 2010. (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, No. 99.)

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Some Aromatic Amines, Organic Dyes, and Related Exposures.

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3Studies of Cancer in Experimental Animals

3.1. Occupational use of hair dyes: Hair-dye formulations and some of their components

3.1.1. Skin application

(a) Mouse

Groups of 50 male and 50 female Swiss Webster mice, 6–8 weeks of age, received applications of one of three oxidation (permanent) hair-dye formulations, PP-7588, PP-7586 or PP-7585 (all three contained 2,5-toluenediamine sulfate, para-phenylenediamine and resorcinol; PP-7586 also contained 2,4-diaminoanisole sulfate, PP-7585 contained meta-phenylenediamine, and PP-7588 contained 2,4-toluene-diamine) [see reference for additional details on composition], mixed with an equal volume of 6% hydrogen peroxide just before use; 0.05 mL of the mixture in acetone was applied to the shaved skin of the interscapular region. Controls were given acetone or were left untreated. For each formulation and for the vehicle control, one group was treated once weekly and another group once every other week for 18 months. Survival at 18 months varied from 58% to 80%. No sign of systemic toxicity was found in any of the dye-treated groups. Average body weights were comparable in all groups throughout the study. The incidence of tumours at all sites, including lung tumours, was not statistically different between treated and control groups. No skin tumours were observed at the site of application (Burnett et al., 1975).

The chronic toxicity of 2,4-toluenediamine (2,4-TDA) alone [purity not specified] or in combination with a hair-dye complex (2,5-toluenediamine, para-phenylenediamine, and resorcinol) [see reference for additional details on composition] was studied in groups of 28 male and 28 female Swiss Webster mice, 4–7 weeks of age, and weighing 15–20 g by use of a skin-painting technique, whereby a 6% solution of the study materials in a water/isopropanol solvent were mixed with equal volumes of either 6% peroxide or distilled water at doses of 50, 150 or 1500 µg 2,4-TDA per week. Additional groups of vehicle and untreated controls were used. No information on survival was provided. The predominant neoplasms seen in these mice were primary pulmonary adenomas and adenocarcinomas. Skin neoplasms were seen in most groups of mice, including untreated controls. Statistical analysis of the incidences of skin neoplasms among the various groups of mice did not show any significant differences. The 2,4-TDA, alone or mixed with the hair-dye complex, did not produce any abnormal proliferation and maturation of the squamous epithelium of the skin (Giles et al., 1976). [The Working Group noted the lack of information on survival, and the use of water as the skin-painting solvent.]

Groups of 26 male and 22 female DBAf and 26 male and 26 female strain-A mice, 6–7 weeks of age, received skin applications of 0.4 mL (reduced to 0.2 mL at 24 weeks for DBAf mice) of a 10% solution of a commercially available semi-permanent hair dye (‘GS’), containing, among other constituents [unspecified], 1,4-diamino-2-nitrobenzene (2-nitro-para-phenylenediamine) and 1,2-diamino-4-nitrobenzene (4-nitro-ortho-phenylenediamine) in 50% aqueous acetone twice a week on the clipped dorsal skin. Groups of 16 male and 16 female control mice of each strain received applications of acetone alone. When the experiment was terminated at 80 weeks, four (18%) lymphomas and six (27%) tumours of the reproductive tract (four ovarian cystadenomas and two uterine fibrosarcomas) had developed in the 22 treated female DBAf mice within 37–80 weeks, and one (4%) lymphoma had developed by week 26 among the 26 treated males. In control DBAf mice, 1 lymphoma and 1 lung adenoma were found in females and 1 hepatoma in males. No difference was observed in the incidence of lymphomas or liver or lung tumours between treated and control strain-A mice. No skin tumour at the site of application was observed in either strain. Of the treated animals, 27 DBAf mice and 32 strain-A mice survived 60–80 weeks without tumours (Searle & Jones, 1977). [The Working Group noted that this study was conducted with a low concentration of the formulation used in this study.]

In the same study, groups of 17 male and 15 female DBAf and 16 male and 16 female strain-A mice, 6–7 weeks of age, received skin applications of 0.4 mL (reduced to 0.2 mL at 24 weeks for DBAf mice) of a 10% solution of a commercially available semi-permanent hair dye (“RB”), containing, among other constituents [unspecified], 4-amino-2-nitrophenol and CI Acid Black 107, in 50% aqueous acetone twice a week on the clipped dorsal skin. The experiment was terminated at 80 weeks. No significant difference was observed in the incidence of tumours at any site between treated and control animals of either strain, and no skin tumour at the site of application was observed in either strain (Searle & Jones, 1977). [The Working Group noted the low concentration of the formulation used in this study.]

In an interim report of the Searle and Jones (1977) study mentioned above, it was noted that the tumours arose consistently earlier in the treated than in the control mice, notably in the DBAf strain, in which the first lymphoma was detected after 26 weeks. Apart from the early appearance, there was also an increased tumour incidence in this strain, due mainly to uterine and ovarian tumours that were not seen in the control group (Venitt & Searle, 1976).

Twelve groups of 50 male and 50 female random-bred Swiss Webster mice, 6–8 weeks of age, were exposed dermally, once per week for 21–23 months, to different hair-dye formulations by placing a 0.05-mL sample on a 1-cm2 area of the interscapular region, which had been clipped free of hair 24 hours before treatment. Nine oxidative hair dyes (7401, 7402, 7403, 7404, 7405, 7406, P-21, P-25, and P-26) were mixed with an equal volume of 6% hydrogen peroxide and 0.025 ml was applied within 15 minutes. Three semi-permanent hair-dye formulations (P-22, P-23, and P-24) were applied in 0.05 ml within 30 minutes after opening the bottle. In an earlier study, the composition of these hair-dye formulations was given in an extensive table (Burnett et al., 1976). Three groups of 50 males and 50 females served as controls that had their backs shaved but were not further treated. After seven and nine months, groups of 10 females and 10 males were randomly selected from each group, necropsied and examined by histopathology. The experiment was terminated after 21–23 months. The treatments had no effect on survival. The incidences of skin tumours and liver and lung lymphomas were not greater than in control mice (Burnett et al., 1980).

Groups of 60 male and 60 female Swiss Webster mice, eight weeks of age, received topical applications of two oxidative and 12 non-oxidative hair-dye formulations supplied by five cosmetic companies. The composition of the hair-dye formulations was described. Two groups of 60 male and 60 female Swiss Webster mice served as controls. Each oxidative formulation was applied at 0.05 mL/mouse once weekly for 20 months. Each non-oxidative dye was applied at a dose of 0.05 mL per mouse three times weekly for 20 months. The mice were shaved 24 hours before treatment as needed. Control animals were shaved only and received no treatment. The application of hair dyes did not have an adverse effect on average body-weight gain or survival of any group. There was no significant increase in the incidence of malignant lymphomas in male mice or in liver hemangiomas or lung adenomas of both sexes in these studies. Significant increases in numbers of malignant lymphomas over those in one of the untreated control groups were observed in female mice in the hair dye-formulation groups treated with formulation 7602A (19/60 (32%) treated vs 7/60 (12%) control, P < 0.01), formulation 7605 (18/60 (30%) treated vs 7/60 (12%) control, P<0.05) and formulation 7610 (23/60 (38%) treated vs 7/60 (12%) control, P <0.01). The authors pointed out that the incidence in each treated group is not significantly different from the incidence in the second control group in this study, and is within the range of control values previously reported for this strain of mouse. They concluded that these tumours were probably not treatment-related (Jacobs et al., 1984).

(b) Rat

Groups of 50 male and 50 female Sprague-Dawley rats, approximately 14 weeks of age, received topical applications of 0.5 mL of permanent hair-dye mixtures containing [purity unspecified] either 4% para-toluenediamine or 3% para-toluenediamine, 0.75% resorcinol and 0.75% meta-diaminoanisole in vehicle solution (4% Tylose HT, 0.5% sodium sulfite, 8.5–13% ammonia (25%), 3.7% ammonium sulfate or as formed by neutralization and deionized water to 100.0%), with 6% hydrogen peroxide added immediately before use on a 3-cm2 area of shaved dorsal skin twice a week for two years. The animals were then observed for a further six months. Control groups of 25 males and 25 females of the same strain and age received topical applications of 0.5 mL vehicle alone, to which 6% hydrogen peroxide was added immediately before use. Another group of 50 males and 50 females of the same strain served as untreated controls. No difference in survival was observed between treated, vehicle control and untreated control groups. The skin at the application site, and the liver, kidney, lung and gross lesions were studied histologically. No skin tumour was observed at the site of application, and there was no significant difference in the incidence of tumours, including those of the skin, between treated, vehicle control and untreated control groups (Kinkel & Holzmann, 1973).

Groups of 10 male and 10 female Wistar rats weighing 120–140 g received topical applications of 0.5 mL oxidized para-phenylenediamine [purity unspecified] (1:1 mixture of 5% para-phenylenediamine and 2% ammonium hydroxide) and 6% hydrogen peroxide on shaved dorsal skin once a week for 18 months. control rats were shaved and treated with the corresponding vehicle. Treated and control groups did not differ significantly in body weight gain or survival. All surviving rats were killed after 21 months. Treated rats had a significantly increased incidence of mammary tumours (5/10 (50%); P < 0.05 [incidental tumour test]) in comparison with female vehicle-controls (0/9). The first mammary tumour observed was a fibrosarcoma, which occurred at week 47; the others were three adenomas and one fibroadenoma. No skin tumour was observed at the site of application (Rojanapo et al., 1986).

Groups of 60 male and 60 female Sprague-Dawley rats (aged 6–8 weeks) received topical applications of an oxidative hair-dye formulation (7406) containing 0.5% 2-amino-5-nitrophenol, 4.0% para-phenylenediamine, 0.7% para-aminophenol, 2.0% 4-chlororesorcinol, 5.0% oleic acid, 15.0% isopropanol, 0.2% sodium sulfite, 6.0% ammonia and water to 100%. The formulation was diluted in an equal volume of 6% hydrogen peroxide before application, and 0.5 mL were applied to a shaved area of the back (approx. 2.5 cm in diameter) twice a week up to week 117. Three separate, similarly treated, concurrent control groups of 60 rats received applications of the vehicle alone. Mean body weights and survival were similar in treated and control groups. No skin tumours were observed. The incidence of pituitary adenomas was increased in females in comparison with all three control groups (45/51 (88%) vs 34/50 (65%), 36/51 (71%) and 35/50 (70%); P < 0.05, χ2-test). The incidence of mammary gland adenomas was increased in comparison with one control group (6/53 (11%) vs 0/49; P < 0.05, χ2-test). The authors concluded that the tumours were probably not treatment-related (Burnett & Goldenthal, 1988).

In the same study, groups of 60 male and female Sprague-Dawley rats, 6–8 weeks of age, received topical applications of an oxidative hair-dye formulation (7405) containing 0.4% 2-amino-4-nitrophenol, 6.0% 2,5-diaminoanisole sulfate, 2.0% resorcinol, 0.3% ortho-aminophenol, 5.0% oleic acid, 3.0% isopropanol, 0.2% sodium sulfite, 6.0% ammonia (29%) and water to 100%. The formulation was diluted in an equal volume of 6% hydrogen peroxide, and 0.5 mL was applied to a shaved area of the back (approx. 2.5 cm in diameter) twice a week up to week 117. Mean body weights and survival were similar in treated and control groups. No skin tumours were observed. The incidence of pituitary adenomas was increased in males in comparison with one of the control groups 35/52 (67%) vs 14/49; P < 0.01; χ2-test), and no increase in the incidence of tumours at any other site was observed in treated rats compared with control animals. The authors concluded that the tumours were probably not treatment-related (Burnett & Goldenthal, 1988).

In the same study, groups of 60 male and female Sprague-Dawley rats, 6–8 weeks of age, received topical applications of an oxidative hair-dye formulation (7403) containing 6.0% para-toluenediamine sulfate, 0.7% meta-aminophenol, 1.0% para-aminophenol, 0.25% 4-nitro-ortho-phenylene diamine, 0.50% 1-naphtol, 15% oleic acid, 10% isopropanol, 4.5% glycerine, 9.0% propylene glycol, 0.2% sodium sulphite, 9.0% ammonia and water to 100%. The formulation was diluted in an equal volume of 6% hydrogen peroxide before application, and 0.5 mL was applied to a shaved area of the back (approx. 2.5 cm in diameter) twice a week until week 117. Mean body weights and survival were similar in treated and control groups. The incidence of mammary gland adenomas was increased in comparison with one control group (10/52 (19%) vs 0/49; P < 0.01, χ2-test). There was no significant increase in the incidence of tumours at any site, and no skin tumour was observed. The authors concluded that the tumours were probably not treatment-related (Burnett & Goldenthal, 1988). [The Working Group wondered why the tumours were assumed not to be treatment-related.]

3.1.2. Subcutaneous injection

(a) Rat

Groups of 10 male and 10 female rats weighing 120–140 g received subcutaneous injections of 0.5 mL oxidized para-phenylenediamine (5% para-phenylenediamine in 2% ammonium hydroxide and 1.8% sodium chloride) in an equal volume of 6% hydrogen peroxide in the hip area every other week for 18 months. Controls were injected similarly with vehicle only. There was no significant difference between treated and control groups in body-weight gain or survival. All survivors were killed after 21 months. The incidence of mammary lesions [duct ectasia or adenosis] was significantly increased (4/7 (57%); P < 0.05 incidental tumour test) in females in comparison with vehicle controls (0/10). Two uterine tumours, an adenocarcinoma and an endometrial polyp were observed in females; no such tumours were observed in controls. Two sarcomas [not otherwise classified] at the injection site and two lipomas were also observed in treated animals (Rojanapo et al., 1986). [The Working Group noted the small number of animals used in this study.]

3.2. Personal use of hair dyes

No data were available to the Working Group

©International Agency for Research on Cancer, 2010.
Bookshelf ID: NBK385467

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