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Gene toxicity in vitro:

Various peer reviewed publications for the target chemical and its read across have been summarized below to determine the mutagenic nature of the test compound D and C orange no 5:

Some lipsticks and their dyes were tested by Green et al (1980) for mutagenicity using S. typhimurium strain TA98. One of these dyes was the test chemical D & C Orange no. 5 (CAS no 596 -03 -2). The standard quantitative mutagenicity assay in which the bacteria are incorporated into an agar overlay with the test agent was performed. 2-Nitrofluorene was used as a positive control in the absence of S-9. DMBA was used as a positive control in the presence of S-9. DMSO was used as a solvent control. D&C Orange Nos. 5 was mutagenic without S-9. Dose responses in terms of the numbers of His+ revertants were observed for this dye in three experiments. The mutagenic effect was diminished or lost in the presence of S-9. The test material D & C Orange no. 5 is mutagenic in the tester strainSalmonella typhimuriumTA98 in the absence of S9 metabolic activation system. However, the mutagenic response with this dye was observed only at and above the 100µg level. However, this fluorescent dye may also contain other fluorescent dyes that have been shown to damage DNA of other microbial systems as a result of photodynamic action. It is possible, therefore, that these or other impurities may account their results. D & C Orange no. 5 is non-mutagenic in the tester strain Salmonella typhimurium TA98 in the presence of S9 metabolic activation system.

Salmonella/mammalian microsome assay was performed by Brown et al (1979) for the test material D and C Orange No. 5 (CAS no 596 -03 -2) using the Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538, and TA98. The dye was dissolved in DMSO and up to 0.2 ml was introduced into 2.5 ml of the tempered top agar together with 0.1 ml Salmonella typhimurium broth suspension and 0.25 ml Aroclor 1254 induced rat liver S9. The mixtures was plated on 20 ml of Vogel-Bonner E bottom agar in the usual fashion and incubated for 3 days at 35°C. The test material failed to induce any mutation in the strain. Gene toxicity in vitro result for the test compound D and C Orange No. 5 is negative in the Salmonella typhimurium strains TA1535, TA100, TA1537, TA1538 and TA98 by theSalmonella/mammalian microsome assay performed.

Salmonella/ mammalian-microsome test was performed by Muzall and Cook (1979) to evaluate the mutagenic nature of the test compound D & C Orange no. 5. The 2 ml of liquid top agar was cooled to 45°C and 0.1 ml of a broth culture of microorganism and test substance in volumes of ≤ 0.4 ml of DMSO was added prior to placing on minimal agar plates. After 48 h incubation at 37°C, the colonies which reverted to the prototroph were counted and compared to counts on the control plate (containing no test substance) to demonstrate mutagenicity or toxicity. Materials which caused a 2-fold increase of revertants, as compared to the number of spontaneous revertants on the control plates, were denoted as mutagens. Those which reduced the number of revertants were considered inhibitory. The test compound D & C Orange no. 5 failed to induce mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 and hence is negative for gene mutation in vitro.

In the same study by Muzall and Cook (1979) (Spot test) was performed to evaluate the mutagenic nature of the test compound D & C Orange no. 5.The spot test was used to screen the test material for potential mutagenicity. The test material was placed in the center of the plate. The test compound was tested with and without the S9 mix. Inhibition of the bacterium was indicated by a clearing of the background lawn in a zone surrounding the test material. Mutagenicity was indicated by a clustering of revertant colonies directly around the test material or at the edge of the inhibitory zone. A known mutagen, Captan, was used as a reference mutagen. The test compound D & C Orange no. 5 failed to induce mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535) in the spot test performed and hence is negative for gene mutation in vitro..

Based on the available data summarized, the test chemical D and C orange no 5 is not likely to classify for gene mutation in vitro.

 

Justification for classification or non-classification

Based on the available data summarized, the test chemical D and C orange no 5 is not likely to classify for gene mutation in vitro.