1.1.1. Synonyms, structural and molecular data

Chem. Abstr. Serv. Reg. No.: 33229-34-4

Chem. Abstr. Name: 2,2′-([4-([2-Hydroxyethyl]amino)-3-nitrophenyl]imino)bis(etha-nol)

Synonyms: HC Blue 2; HC Blue Number 2; 2,2′-([4-([2-hydroxyethyl]amino)-3-nitro- phenyl]imino)di(ethanol); N¹,N⁴,N⁴-tris(2-hydroxyethyl)-2-nitro-para-phenylenedia- mine

1.1.2. Chemical and physical properties of the substance

From US National Toxicology Program (1985)

• (a) Description: Dark-blue microcrystalline (75% pure) or blackish-blue amorphous powder (98% pure), with a copper cast
• (b) Melting-point: 93–98 °C (98% pure)
• (c) Spectroscopy data: Infrared, ultraviolet and nuclear magnetic resonance spectral data have been reported.
• (d) Solubility: Soluble in water, ethanol, methanol and acetone
• (e) Octanol/water partition coefficient (P): 1.7

1.1.3. Trade names, technical products and impurities

HC Blue No. 2 is available commercially with a purity ≥ 95% with possible impurities, including methylamine (≤ 1%), 1,2-dihydroxyethane (≤ 1%) and water (≤ 1%). It is also available in a technical grade containing 55–90% dye, 10–30% inorganic salts and 2,2-[(4-amino-3-nitrophenyl)imino]bis(ethanol) (≤ 7%) as a possible impurity.

1.1.4. Analysis

No data were available to the Working Group.

1.2.1. Production

The form of HC Blue No. 2 that is ≥ 95% pure is produced commercially by the reaction of 4-fluoro-3-nitrobenzenamine with ethylene oxide (see IARC, 1985, 1987) to form 2,2-[(4-fluoro-3-nitrophenyl)imino]bis(ethanol); this intermediate is reacted with mono-ethanolamine to form HC Blue No. 2. The technical grade is produced by reaction of 2-nitro-para-phenylenediamine (see IARC, 1978) with ethylene oxide or 2-chloroethanol.

Production and use of HC Blue No. 2 began in the late 1950s. Approximately 9–11 tonnes are used annually in the USA according to industry estimates.

1.2.2. Use

HC Blue No. 2 is used exclusively as a dye in semi-permanent hair colouring products. These products are generally shampooed into the hair, lathered and then allowed to remain in contact with the hair and scalp for 30–45 min. The concentration of HC Blue No. 2 used in these preparations ranges from 1.6 to 2% (Frenkel & Brody, 1973; US National Toxicology Program, 1985).

1.3.1. Natural occurrence

HC Blue No. 2 is not known to occur as a natural product.

1.3.2. Occupational exposure

No data were available to the Working Group.

3.1.1. Mouse

Groups of 50 male and 50 female B6C3F₁ mice, seven weeks of age, were fed 0,5000 or 10 000 mg/kg (ppm) (males) or 0,10 000 or 20 000 ppm (females) HC Blue No. 2 (about 98% pure) in the diet for 104 weeks and were killed at 112–113 weeks of age. Final mean body weights of treated males were similar to those of controls; however, those of treated females were 15% (low-dose) and 22% (high-dose) lower than those of controls. Survival at the end of the study was: males—control, 24/50; low-dose, 24/50; and high-dose, 34/50; females-control, 35/50; low-dose, 28/50; and high-dose, 20/50 (p < 0.005, life table test). The reduced survival of female mice was attributed to infection of the ovaries and uteri with Klebsiella sp. No significant increase in the incidence in any tumour was observed (US National Toxicology Program, 1985; Kari et al., 1989a). [The Working Group noted that the reduced survival of females precluded adequate evaluation of the study]

3.1.2. Rat

Groups of 50 male and 50 female Fischer 344/N rats, six to seven weeks of age, were fed 0, 5000 or 10 000 mg/kg of diet (ppm) (males) or 0, 10 000 or 20 000 mg/kg ppm (females) HC Blue No. 2 (∼ 98% pure) in the diet for 103 weeks and were killed at 110–112 weeks of age. Final mean body weights were depressed by less than 10% in treated males but by 13% (low-dose) and 22% (high-dose) in females. No adverse effect on survival was observed in males (control, 32/50; low-dose, 38/50; high-dose, 42/50) or females (control, 41/50; low-dose, 40/50; high-dose, 39/50). The incidence of C-cell adenomas of the thyroid in males was 7/50 control, 2/50 low-dose and 5/49 high-dose; the incidence of C-cell carcinomas was 0/50 control, 3/50 low-dose and 5/49 high-dose (p = 0.029, incidental tumour trend test). The trend in combined incidence (7/50, 5/50 and 10/49) was not significant. There was no excess of thyroid tumours in females. No other significant increase in the incidence of tumours was reported; however, malignant mixed mesenchymal tumours of the kidney were detected in 2/50 high-dose female rats and none of 1863 historical controls. A negative trend in the incidence of fibroadenomas of the mammary gland was seen in female rats (control, 20/50; low-dose, 10/50; high-dose, 4/50 [p < 0.001, Cochran Armitage test for trend]) (US National Toxicology Program, 1985; Kari et al., 1989a).

4.1.1. Humans

About 85 g of a commercial semi-permanent hair dye formulation containing 1.77% HC Blue No. 2 enriched with 378.6 µCi/mg [ring-¹⁴C]-labelled HC Blue No. 2 was applied to the hair of human volunteers, worked in gently for 5–8 min and allowed to remain in contact with the hair and scalp for an additional 30 min. After 30 days, radiolabel accounting for less than 0.1% of that applied was detected in the urine; half was excreted in urine after 52 h (Wolfram & Maibach, 1985). [The Working Group noted that urinary metabolites were not identified, so that the metabolic fate of HC Blue No. 2 in humans remains unknown.]

4.1.2. Experimental systems

Up to 40% of radiolabel was recovered in the urine of B6C3F₁ mice and Fischer 344/N rats after oral administration by gavage of 100 mg/kg bw [ring-¹⁴C]-labelled HC Blue No. 2 (0.1 mCi/mmol [35 µCi/mg]; 98% pure). Metabolism of HC Blue No. 2 (200 µM [57 mg]) by hepatocytes isolated from mice and rats yielded profiles similar to those seen in vivo. High-performance liquid chromatography separation showed that HC Blue No. 2 is metabolized extensively in mice to one major metabolite, which has not been characterized (Kari et al., 1988, 1989b, 1990a,b).

4.2.1. Humans

No data were available to the Working Group.

4.2.2. Experimental systems

Concentrations of 0, 5000 or 10 000 ppm (mg/kg) HC Blue No. 2 (98% pure) were administered in the diet to male Fischer 344/N rats for 103 weeks and to male B6C3F₁ mice for 104 weeks and concentrations of 0, 10 000 or 20 000 ppm (mg/kg) to female rats for 103 weeks and to female mice for 104 weeks. The calculated average daily doses were 194 and 391 mg/kg bw for male rats, 465 and 1001 mg/kg bw for female rats, 1321 and 2243 mg/kg bw for male mice, and 2362 and 5609 mg/kg for female mice. A dose-related increase in the incidence of hyperostosis of the skull was observed in male and female rats (US National Toxicology Program, 1985; Kari et al., 1989a).

HC Blue No. 2 was present at low concentrations in semi-permanent hair colouring formulations evaluated in a 13-week study of dermal toxicity in rabbits (1.7%) (Burnett et al., 1976) and in a two-year feeding study in dogs (1.63%) (Wernick et al., 1975), described in detail on p. 97. No treatment-related adverse effect was detected. [The Working Group noted that the dose of each component of the formulation was very low and unlikely to have been toxic]

4.3.1. Humans

No data were available to the Working Group.

4.3.2. Experimental systems

No data were available to the Working Group on the reproductive and developmental effects of HC Blue No. 2 alone. The compound was present at low concentrations in semipermanent hair colouring formulations evaluated in a study of fertility and reproductive performance in rats (Wernick et al., 1975, 1.63%; see p. 99) and in studies of teratogenesis in rats (Wernick et al., 1975, 1.63%; Burnett et al., 1976, 1.70%) and rabbits (Wernick et al., 1975, 1.63%) (see p. 100). No treatment-related adverse effect was detected. [The Working Group noted that the dose of each component of the formulations was very low and unlikely to have been toxic]

4.4.1. Humans

No data were available to the Working Group.

4.4.2. Experimental systems (see also Table 1 and Appendices 1 and 2)

Table 1

Genetic and related effects of HC Blue No. 2.

Although two different lots of HC Blue No. 2 have been used in most of the short-term tests (98.5% and 99.8% purity), they appear to induce similar responses when tested in the same assay system.

HC Blue No. 2 induced mutation in Salmonella typhimurium but did not induce reverse mutation in Escherichia coli. It induced mutations at the tk locus of mouse lymphoma L5178Y cells and induced unscheduled DNA synthesis in cultures of primary hepatocytes from mice, rats, Syrian hamsters and rabbits, but not in those from monkeys. HC Blue No. 2 induced sister chromatid exchange in cultured Chinese hamster ovary cells and, weakly, in female B6C3F₁ mouse primary hepatocytes. Chromosomal aberrations were not induced in Chinese hamster ovary cells or in primary cultures of rat or mouse hepatocytes.

HC Blue No. 2 did not inhibit gap-junctional communication in Chinese hamster lung V79 cells, but it elicited a slight, dose-dependent increase in communication at non-cytotoxic concentrations.

HC Blue No. 2 did not induce micronuclei in mouse bone-marrow in three assays (one of which was with the lower purity sample).

As reported in two abstracts, HC Blue No. 2 did not increase proliferation of hepatocytes from rats and mice treated in vivo (Mirsalis et al., 1986, 1988), but it enhanced S-phase DNA synthesis in male (but not female) mouse hepatocytes. It did not induce unscheduled DNA synthesis in the livers of mice or rats in feeding studies.