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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 36002 was found to be non mutagenic in a Salmonella reverse mutation assay. Read across substance, FAT 36038/J TE was tested for mutagenic effects in bacteria and eucaryontic cells in vitro and for clastogenic effects in eucaryontic cells in vitro. While both Ames tests conducted gave a positive response an additional HPRT test conducted in vitro gave negative results. Therefore the test substance is considered to be non-mutagenic in eucaryonic cells. In addition an chromosomal aberration test according to OECD 473 has been conducted and was was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells. Hence, the read across substance FAT 36038 as well as the target substance FAT 36002 (using the principles of read across) may be considered as non-genotoxic and further testing is not considered.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In a Salmonella reverse mutation assay, FAT 36002 was tested using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 at the concentrations ranging from 2 to 300 micrograms per plate with and without rat liver preparation. However, no mutagenic activity was detected with the test substance. Hence FAT 36002 was considered to be not mutagenic in this Salmonella reverse mutation assay.


A key study was performed to determine the detection of gene mutations induced by the test material FAT 36038/F or its metabolites in histidine-requiring strains of Salmonella typhimurium. FAT 36038/F was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 61.7 to 5000 µg/plate. In the experiment without and with metabolic activation no toxic effect of the test material on the growth of the bacteria was observed.

In the first experiment carried out with metabolic activation, treatment of strain TA 1537 with Terasil Violett BL roh feucht (FAT 36038/F) led to a slight increase in the number of revertant colonies at the highest concentration. No effects were observed with the other strains. In the experiment performed without activation, a slight increase in the number of back-mutants occurred on strains TA 98 and TA 1535 at the highest concentration. No effects were observed with the other strains.

In the confirmatory experiment carried out with metabolic activation, treatment of strain TA 1537 with Terasil Violett BL roh feucht (FAT 36038/F), led to a slight increase in the number of revertant colonies at the highest concentration. No effects were observed with the other strains. In the experiment performed without activation, a slight increase in the number of back-mutants occurred on strains TA 98, TA 1535 and TA 1537 at the highest concentration. No effects were observed with the other strains.

In the mutagenicity tests without metabolic activation performed on strain TA 102, a slight decline in the number of revertant colonies was registered. The test substance exerted a weak inhibiting effect on the growth of this bacterial strain.

Based on the results of these experiments and on standard evaluation criteria, it is concluded that Terasil Violet BL roh feucht (FAT 36038/F) exerted a weak mutagenic action on strains S. typhimurium TA 98 and TA 1535. The metabolites of the test material were weakly mutagenic with strain TA 1537.

 

In another supporting study, FAT 36038/C was tested for mutagenicity in selected strains of S. typhimurium both in the presence and absence of in vitro activation by microsomal enzymes from rat liver (Ames test).

FAT 36038/C (a violet dyestuff) was tested in S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at eight dose levels: 0.2, 2, 20, 200, 500, 1000, 2000 and 4000 ml per Petri dish. The test substance was diluted in sterile water and every concentration was tested in triplicate.

Positive control was also used to perform the experiment. MNG, 9 -aminoacridine and daunomycine were used as positive control without S9. 2 -anthramine was used as positive control with S9.

Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, product FAT 36038/C was found to be mutagenic for S. typhimurium strain TA 1537 without metabolic activation.

 

Further, in another key study, FAT 36038/J was tested for chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system according to OECD Guideline 473.

A preliminary toxicity test was performed to establish the dose range for the chromosome aberration assay. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. In both phases, CHO cells were treated for 4 and 20 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 20 hours after treatment initiation.

Dimethyl formamide (DMF) was used as the vehicle. In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 μg/mL.

Cytotoxicity (≥ 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥ 200 μg/mL in the non-activated 4-hour exposure group, at dose levels 6, 20, 60, 200 and 2000 μg/mL in the S9-activated 4-hour exposure group, and at doses 0.6 and ≥ 200 μg/mL in the non-activated 20-hour exposure group.

Under the conditions of the assay described in this report, FAT 36038/J TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

In another key study, the test substance, FAT 36038/J TE, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).

Based on the results, FAT 36038/J TE was evaluated in the definitive mutagenicity assay at concentrations of 1.50, 3.00, 6.00, 7.00, 8.00, 9.00, 10.0, 11.0 and 11.7 μg/mL with and 1.25, 2.50, 5.00, 8.00 and 11.7 μg/mL without S9. No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH of the cultures. The average adjusted relative survival was 7.58 and 96.44 % at a concentration of 11.7 μg/mL with and without S9, respectively. However, the limit dose was not achieved due to a shift in precipitate profile, and the entire assay was retested with an adjustment in dose levels.

These results indicate FAT 36038/J was negative in the in vitro Mammlian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate cultures, under the conditons and according to the criteria of the test protocol.

Justification for classification or non-classification

FAT 36002 was found to be not mutagenic in a Salmonella reverse mutation asssay. However, read across substance FAT 36038 exerted a weak mutagenic action on strains S. typhimurium TA 98 and TA 1535. The metabolites of the test material were weakly mutagenic with strain TA 1537.

Read across substance, FAT 36038/J TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells as well as in the in vitro Mammlian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate cultures.

Hence, the test substance FAT 36038 as well as the target substance (FAT 36002) may be considered as non genotoxic and does not warrant classification as per the CLP (Regulation No. EC 1272/2008) criteria.