1.1.1. Synonyms, structural and molecular data

Chem. Abstr. Serv. Reg. No.: 2429-74-5; replaces 51568-94-6; 95032-75-0

Chem. Abstr. Name: 3,3′[(3,3′-Dimethoxy[1,1′-biphenyl]-4,4′-diyl) bis(azo)]bis[5-amino-4-hydroxy-2,7-naphthalenedisulfonic acid], tetrasodium salt

Colour Index No.: 24400

Synonym: Direct Blue 15

1.1.2. Chemical and physical properties

• (a) Description: Dark-blue powder (US National Toxicology Program, 1992)
• (b) Melting-point: 300 °C (decomposes) (US National Toxicology Program, 1992)
• (c) Spectroscopy data: Infrared and nuclear magnetic resonance spectral data have been reported (US National Toxicology Program, 1992).
• (d) Solubility: Soluble in water; insoluble in most organic solvents (Society of Dyers and Colourists, 1971a)

1.1.3. Trade names, technical products and impurities

Some trade names are: Airedale Blue D; Aizen Direct Sky Blue 5B; Aizen Direct Sky Blue 5BH; Amanil Sky Blue; Atlantic Sky Blue A; Atul Direct Sky Blue; Azine Sky Blue 5B; Belamine Sky Blue A; Benzanil Sky Blue; Benzo Sky Blue A-CF; Benzo Sky Blue S; Cartasol Blue 2GF; Chloramine Sky Blue A; Chloramine Sky Blue 4B; Chrome Leather Pure Blue; Cresotine Pure Blue; Diacotton Sky Blue 5B; Diamine Blue 6B; Diamine Sky Blue; Diaphtamine Pure Blue; Diazol Pure Blue 4B; Diphenyl Brilliant Blue; Diphenyl Sky Blue 6B; Direct Blue 10G; Direct Blue HH; Direct Pure Blue; Direct Pure Blue M; Direct Sky Blue; Direct Sky Blue A; Direct Sky Blue 5B; Enianil Pure Blue AN; Fenamin Sky Blue; Hispamin Sky Blue 3B; Kayafect Blue Y; Kayaku Direct Sky Blue 5B; Mitsui Direct Sky Blue 5B; Naphtamine Blue 10G; Niagara Blue 4B; Niagara Sky Blue; Nippon Direct Sky Blue; Nippon Sky Blue; Nitto Direct Sky Blue 5B; Oxamine Sky Blue 5B; Paper Blue S; Phenamine Sky Blue A; Pontamine Sky Blue 5BX; Shikiso Direct Sky Blue 5B; Sky Blue 4B; Sky Blue 5B; Tertrodirect Blue F; Vondacel Blue HH.

The raw dye contains about 25% sodium chloride; a desalted preparation (containing ∼ 3% salt) contained about 50% CI Direct Blue 15 and about 35 impurities, including 3,3′-dimethoxybenzidine dihydrochloride at 836–1310 ppm (mg/kg). Benzidine was not present at the detection limit of 1 ppm (mg/kg) (US National Toxicology Program, 1992).

CI Direct Blue 15 is available at a purity of 65.5%, containing 15 ppm 3,3′-dimethoxybenzidine (ortho-dianisidine; see IARC, 1974, 1987) (Bowman et al., 1982).

1.1.4. Analysis

No data were available to the Working Group.

1.2.1. Production

CI Direct Blue 15 was first prepared in 1890 (Society of Dyers and Colourists, 1971a). It is produced by coupling 3,3 ′-dimethoxybenzidine to 1-amino-8-naphthol-3,6-disulfonic acid under alkaline conditions (US Environmental Protection Agency, 1987).

Approximate US production was 108 tonnes in 1972, 241 tonnes in 1977, 98 tonnes in 1981 and 123 tonnes in 1982 (US International Trade Commission, 1974, 1978, 1982, 1983).

1.2.2. Use

CI Direct Blue 15 is used to dye cellulose, leather, paper, cotton, silk and wool and to stain biological materials; it is also used to tint cinematographic film (Society of Dyers and Colourists, 1971b). The use pattern for CI Direct Blue 15 in the USA is 65% in textile dyeing, 30% as a paper colourant and 5% for other uses.

1.3.1. Natural occurrence

CI Direct Blue 15 is not known to occur as a natural product.

1.3.2. Occupational exposure

No data were available to the Working Group.

The US Environmental Protection Agency, the American Textile Manufacturers Institute and the Ecological and Toxicological Association of the Dyestuffs Manufacturing Industry conducted a joint survey in 1986–87 to estimate airborne concentrations of dye dust in dye weighing rooms of plants where powder dyes are used in the dyeing and printing of textiles. The survey was based on a sample of 24 sites chosen at random from among textile plants where powder dyes are weighed. Although CI Direct Blue 15 was not included in the survey, the results were considered to be representative of dye dust levels during weighing of this type of powder dye. The mean airborne concentration of total active colourant in the plants monitored was estimated to be 0.085 mg/m³ (US Environmental Protection Agency, 1990).

On the basis of a survey conducted in the USA between 1981 and 1983, the US National Institute for Occupational Safety and Health estimated that a total of 4527 workers, including 201 women, may have been exposed to CI Direct Blue 15 in seven industries (US National Library of Medicine, 1992).

1.3.3. Other

Anaerobic biodegradation of CI Direct Blue 15 gives rise to the amine metabolite, 3,3′-dimethoxybenzidine. Following incubation of 100 mg/l of dyestuff at 35 °C in the presence of anaerobic sludge inoculum, primary degradation was complete within seven days (Brown & Hamburger, 1987).

Rat

Groups of 50, 35, 65 and 50 male and 50, 35, 65 and 50 female Fischer 344/N rats, 40–47 days old, were administered 0, 630, 1250 or 2500 mg/I (ppm) CI Direct Blue 15 (purity, ∼ 50%; with ∼ 35 impurities, including 3,3′-dimethoxybenzidine) in distilled drinking-water for 96 weeks and were necropsied at 103–104 weeks of age. Survival at 22 months was 37/50, 8/35, 11/65 and 2/50 for male rats and 40/50, 13/35, 22/65 and 4/50 for females in the control, low-, mid- and high-dose groups, respectively (p < 0.001 for both males and females). The decreased survival in the treated groups was due to development of treatment-related neoplasms. As shown in Table 1, there were increased incidences of benign and malignant tumours of the skin, Zymbal gland, liver, oral cavity and small intestines and of mononuclear-cell leukaemia in male and female rats, of benign and malignant tumours of the large intestine and preputial gland in males and of the clitoral gland and uterus in females (US National Toxicology Program, 1992).

Table 1

Survival and tumour incidences in male and female Fischer 344/N rats administered CI Direct Blue 15 in the drinking-water for 96 weeks.

4.1.1. Humans

No data were available to the Working Group.

4.1.2. Experimental systems

Anaerobic biodegradation of CI Direct Blue 15 gives rise to the amine metabolite, 3,3′-dimethoxybenzidine (Brown & Hamburger, 1987). The dye was cleaved by pure cultures of anaerobic bacteria and by suspensions derived from the intestinal content of rats, with subsequent formation of the amine (Cerniglia et al., 1982).

CI Direct Blue 15 (100 mg/kg) containing 46 ppm (mg/kg) 3,3′-dimethoxybenzidine as an impurity was administered once in the diet to two female mongrel dogs weighing 15 kg, and 48-h urine was analysed for 3,3′-dimethoxybenzidine, the potential metabolic product (Lynn et al., 1980). Excretion was found to be 0.03% of the dose of dye administered, which cannot be attributed to the level of impurity. The same dose was also administered once to four male Sprague-Dawley rats by intragastric intubation; after 72 h, 0.17 ± 0.18% of the theoretical maximum was excreted as 3,3′-dimethoxybenzidine and the monoacetyl derivative, the latter constituting a substantial fraction.

When [¹⁴C-biphenyl]CI Direct Blue 15 was given as a single dose of 12 mg/kg to six-week-old male Fischer 344 rats by gavage, 74.4% of the dose was excreted in the faeces and 18.8% in urine within 192 h. Only 12% of the dose appeared as intact dye in the faeces within 48 h, the remainder being unidentified metabolic products. Excretion of the free diamine, 3,3′-dimethoxybenzidine, and of its mono- and diacetyl derivatives in urine was determined to be 0.22, 0.27 and 0.22% of the dose, respectively. An equivalent dose of ¹⁴C-labelled 3,3′-dimethoxybenzidine was administered for comparison: 52% of the dose appeared in the faeces and 35% in the urine, indicating that the free amine is metabolized to a greater extent than the dye. Only 1.5% of the dose in faeces could be attributed to the free amine fraction, including the acetylated metabolites; in urine, 1.18% of the dose was excreted as the parent compound, 0.35% as the monoacetyl derivative and 0.93% as the diacetyl derivative. Radiolabel was found in all tissues examined from rats dosed with ¹⁴C-CI Direct Blue 15. The levels peaked at 4 and 8 h and were highest in the gastrointestinal tract, liver, kidney and lung (Bowman et al., 1982).

4.2.1. Humans

No data were available to the Working Group.

4.2.2. Experimental animals

CI Direct Blue 15 binds to albumin, α₁-lipoprotein, ß-lipoprotein, haemopexin, prealbumin and α₁-antichymotrypsin, to alter their mobility in crossed immunoelectrophoresis and to degrade C₃ globulin (Emmet et al., 1985). The importance of these findings in vivo remains to be established, as it is not known how much unchanged dye is absorbed and transported within the body.

CI Direct Blue 15 was tested for subchronic toxicity in male and female Fischer 344 rats (Morgan et al., 1989; US National Toxicology Program, 1992). Groups of 10 animals of each sex received the dye in drinking-water for 13 weeks at concentrations of 0,0.063,0.125,0.25, 0.50 and 1.0% for females and 0, 0.125, 0.25, 0.50,1.0 and 3.0% for males. Seven male rats died in the highest-dose group; the first death occurred after three weeks and the last after 13 weeks. Groups given 1% CI Direct Blue 15 gained 17% less body weight than controls, and males treated with 3% of the dye gained 43% less weight than controls. Absolute and relative kidney weights increased in a dose-related manner in males and females. Changes in haematology and clinical chemistry were not observed. Histopathology showed renal and hepatic toxicity in high-dose males that died before termination of the study. In addition to necrosis of hepatocytes and fatty metamorphosis, blue pigment was observed in Kupffer cells. Focal necrosis occurred in proximal tubular epithelial cells. Mild chronic nephropathy was observed in male and female rats given 1% of dye.

4.4.1. Humans

No data were available to the Working Group.

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

Table 2

Genetic and related effects of CI Direct Blue 15.

Technical-grade CI Direct Blue 15 was not mutagenic to Salmonella typhimurium in standard protocols, but it was mutagenic under conditions favouring azo reduction, which would generate 3,3′-dimethoxybenzidine, a known mutagen. CI Direct Blue 15 was reported in an abstract to be mutagenic at the tk locus in mouse lymphoma L5178Y cells. It did not induce unscheduled DNA synthesis in rat hepatocytes (abstract) or sister chromatid exchange or chromosomal aberrations in Chinese hamster ovary cells in vitro.

Activated ras genes were found in 21/34 tumours induced in rats by CI Direct Blue 15 (US National Toxicology Program, 1992) and in 1/38 spontaneous tumours tested (Reynolds et al., 1990; Table 3).

Table 3

Activating ras mutations in tumours induced in Fischer 344 rats by CI Direct Blue 15 and in untreated animals.