SCCNFP/0782/04 OPINION OF THE SCIENTIFIC COMMITTEE ON COSMETIC PRODUCTS AND NON-FOOD PRODUCTS INTENDED FOR CONSUMERS CONCERNING ACID BLUE 62 COLIPA n° C67 Adopted by the SCCNFP during the 28th plenary meeting of 25 May 2004 SCCNFP/0782/04 Evaluation and opinion on Acid Blue 62 _____________________________________________________________________________________________ 1. Terms of Reference 1.1 Context of the question The adaptation to technical progress of the Annexes to Council Directive 76/768/EEC of 27 July 1976 on the approximation of the laws of the Member States relating to cosmetic products. 1.2 Request to the SCCNFP The SCCNFP is requested to answer the following questions: * Is Acid Blue 62 safe for use in cosmetic products as a hair dye ingredient? * Does the SCCNFP propose any restrictions or conditions for its use in cosmetic products? 1.3 Statement on the toxicological evaluation The SCCNFP is the scientific advisory body to the European Commission in matters of consumer protection with respect to cosmetics and non-food products intended for consumers. The Commission’s general policy regarding research on animals supports the development of alternative methods to replace or to reduce animal testing when possible. In this context, the SCCNFP has a specific working group on alternatives to animal testing which, in co-operation with other Commission services such as ECVAM (European Centre for Validation of Alternative Methods), evaluates these methods. The extent to which these validated methods are applicable to cosmetic products and its ingredients is a matter of the SCCNFP. SCCNFP opinions include evaluations of experiments using laboratory animals; such tests are conducted in accordance with all legal provisions and preferably under chemical law regulations. Only in cases where no alternative method is available will such tests be evaluated and the resulting data accepted, in order to meet the fundamental requirements of the protection of consumer health. 2 SCCNFP/0782/04 Evaluation and opinion on Acid Blue 62 _____________________________________________________________________________________________ 2. Toxicological Evaluation and Characterisation 2.1. General Acid blue 62 is listed as CI 62045 in Annex IV, part 1 – list of colouring agents allowed for use in cosmetic products – to Directive 76/768/EEC on cosmetic products; field of application 4: colouring agents allowed exclusively in cosmetic products intended to come into contact only briefly with the skin. 2.1.1. Primary name Acid Blue 62 (INCI) 2.1.2. Chemical names Sodium 1-amino-4-(cyclohexylamino)-9,10-dihydro-9,10- dioxaanthracene-2-sulfonate (IUPAC); 2-Anthracenesulfonic acid, 1-amino-4-(cyclohexylamino)-9,10-dihydro-9,10-dioxo-, monosodium salt; 1-Amino-4-cyclohexylamino-anthraquinone-2-sulfonic acid, sodium salt 2.1.3. Trade names and abbreviations Trade name : / COLIPA n° : C67 2.1.4. CAS no. and EINECS no. CAS n° : 4368-56-3 EINECS n° : 224-460-9 Colour Index no. : CI 62045 2.1.5. Structural formula 2.1.6. Empirical formula Emp. Formula : C20H19N2NaO5S Mol weight : 422.4 3 SCCNFP/0782/04 Evaluation and opinion on Acid Blue 62 _____________________________________________________________________________________________ 2.1.7. Purity, composition and substance codes Purity Batch No. 91100013 (Acid Blue 62 with dispersing agent) Content of Acid Blue 62 determined by HPLC using batch 112 (considered as 100%) : 53.4 % Loss on drying : 3.8% Sulphated ash : 38.5% Sulphate ions (as Na2SO4) : 4.2% Chloride ions (as NaCl) : 22.9% Residual solvents : <100 ppm (methanol, ethanol, isopropanol, n-propanol, acetone, ethylacetate, cyclohexane, methylethyl ketone and monochlorobenzene together) evaluated by Headspace-GC, FID Batch No. 01515-200P-0 (Acid Blue 62 without dispersing agent) Content of Acid Blue 62 determined by HPLC using batch 112 (considered as 100%) : 98.4 % Impurity (reagents and intermediate products) : HPLC profile Retention Batch No. Batch No. λmax Comments Time (min) 911003 01515-200P-0 1.35 A A 563 nm and 603 nm 1.51 E - Trace amount 1.75 B B 536 nm 1.90 C - Trace amount 3.00 D 281 nm and 525 nm Significant amount 7.94 F 530 nm 9.43 G 250 nm and 281 nm 12.70 H 245 nm and 281 nm Chemical compounds could not be characterised by LC-MS 2.1.8. Physical properties Acid Blue 62 is described as a black powder containing 2-Anthracenesulfonic acid, 1-amino-4- (cyclohexylamino)-9,10-dihydro-9,10-dioxo-, monosodium salt (approximately 60%) together with dispersing agents (sodium salts) Log Pow : -1.14 Batch No. 01515-200P-0 (Acid Blue 62 without dispersing agent) Appearance : Dark Blue Powder, odourless Melting point : / Boiling point : / 4 SCCNFP/0782/04 Evaluation and opinion on Acid Blue 62 _____________________________________________________________________________________________ Density (bulk density) : 400-600 kg/m3 Rel. vap. Density : / Vapour pressure : / Log Pow : 0.5 (calculated) 2.1.9. Solubility Batch number 91100013 (Acid Blue 62 with dispersing agent) Water : >0.1% (w/v) Ethanol : <0.1% (w/v) Batch number 01515-200P-0 (Acid Blue 62 without dispersing agent) Water : Approximately 50 g/l at 20°C 2.1.10 Stability It is reported that Acid blue 62 is stable for 7 years, when stored in a well-closed container protected from light and moisture. Stability in aqueous solutions (23 mg/ml and 236 mg/ml) of Acid Blue 62: approximately 5% degradation in 8 days, measured by HPLC, detection at 230 nm. General comments on analytical and physico-chemical characterisation * It is considered that Acid Blue 62 containing about 50 % active ingredient will be used in hair dyeing formulations. The dispersing agents to be used in the dye are not described. * The use of the dispersing agent is not clear because of the high water solubility of the compound. * Acid Blue 62 is a secondary amine, and thus, it is prone to nitrosation. Nitrosamine content of the dye is not reported * Stability of the dye in prototype formulations is not known. Inappropriate wavelength has been selected for checking the stability of the dye in aqueous solution by HPLC. The appropriate wavelengths for the detection are 575 nm or 637 nm. * The purity of the dye used in some tests is not described. 2.2. Function and uses Acid Blue 62 is used in semi-permanent and temporary hair colouring products at concentrations up to 0.5% 5 SCCNFP/0782/04 Evaluation and opinion on Acid Blue 62 _____________________________________________________________________________________________ TOXICOLOGICAL CHARACTERISATION 2.3. Toxicity 2.3.1. Acute oral toxicity Guideline : / Species/strain : Sprague Dawley rat Group size : 10 animals (5 males & 5 females) Observation period : 14 days Test substance : Acid Blue 62 Batch no : 6120088 Purity of test substance : 40% Dose level : 500, 1000, and 2000 mg/kg bw (in distilled water) GLP statement : signed statement, no date A preliminary study was performed using three groups of two male and female rats. The doses of 500, 1000 and 2000 mg/kg bw were administered. Because no deaths were noted during this study, the dose of 2000 mg/kg bw was chosen for the principal study. The purity of the dye used in these studies was unknown. A single dose of 2000 mg/kg bw Acid Blue 62 (purity 40%) was administered in the principal study. Animals were observed 15 minutes, 1, 2 and 4 hours after compound administration, and daily thereafter for 14 days. Body weights were recorded on the day before treatment, day 1 prior to treatment, and days 8 and 15. Moribund animals were killed during the study and autopsied. All surviving animals were killed at the end of the study and examined grossly. The LD50 was calculated using the method of Bliss and that of Litchfield and Wilcoxon. Results One female rat died on day 2 of the study; no other compound-related mortalities were observed. Neither the cause of death, nor the possible relationship with substance administration, is described in the study report. Blue coloration of urine and faeces was observed in all animals, from 2h after administration, till day 2. Conclusion Based on the results of this study, the performing laboratory concludes that the acute oral LD50 of Acid Blue 62 in rats was > 2000 mg/kg bw (equivalent to 800 mg active dye). Ref.: 1 2.3.2. Acute dermal toxicity No data 2.3.3. Acute inhalation toxicity No data 6 SCCNFP/0782/04 Evaluation and opinion on Acid Blue 62 _____________________________________________________________________________________________ 2.3.4. Repeated dose oral toxicity Guideline : OECD 407 (1981) Species/strain : Sprague Dawley rat Group size : 12 animals (6 males & 6 females) / dose level Observation period : 14 days (no recovery group included) Test substance : Acid Blue 62 Batch no : 9110003 Purity of test substance : not stated in study report, 53.4% according to the summary (dye with dispersing agents) Dose levels : 0, 25, 100, 400 mg/kg bw/day (in water) GLP statement : / Three groups of six male and six female Sprague-Dawley rats received the test substance, Acid Blue 62 (53.4% pure), daily by oral gavage at doses of 25, 100 or 400 mg/kg bw/day (equivalent to 13, 53 or 214 mg/kg bw/day active dye) for 15 days. An additional group of six male and six female rats received the vehicle alone (water for injections) and served as the control. Animals were observed twice daily for mortality/morbidity and once daily for clinical abnormalities. Individual animal weights were recorded weekly. Body weight and food consumption were recorded twice weekly. Haematology, clinical chemistry and urinalysis evaluations were performed at the end of the study. At the end of the treatment period, all animals were killed and grossly examined. Selected organs were weighed. All animals were submitted to a complete macroscopic examination. Selected tissues and macroscopic lesions from animals in the control and high-dose groups were evaluated microscopically; only macroscopic lesions were evaluated in the low and intermediate dose groups.