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Key value for chemical safety assessment

Additional information

Genotoxicity in vitro:

In a reverse gene mutation assay (Ames test) in bacteria (Mortelmans, 1986), Salmonella typhimurium strains TA1535, 1537, 98 and 100 were exposed to methyl salicylate, at concentrations of 1.0 to 333.3 µg/plate with and without metabolic activation by S9 fraction from the livers of male rats and hamsters. Cytotoxicity was evident at the highest concentration. There was no evidence of induced mutant colonies over background either with or without metabolic activation in any tested strain. In a reverse gene mutation assay (Ishidate et al, 1984), Salmonella typhimurium strains TA 92, 94, 98, 100, 1535 and 1537 were exposed to MeS with and without metabolic activation by liver microsomal fraction, S9, from Fischer rats pretreated with polychlorinated biphenyls. Methyl salicylate was non-mutagenic under the assay conditions. It is concluded that MeS is not mutagenic in bacteria.

In a mammalian cell chromosome aberration assay (Ishidate et al., 1984),cell cultures were exposed to MeS at three concentrations up to 0.25 mg/ml for 24 or 48 hours without metabolic activation. The cytotoxic concentration was defined as the dose which induced 50% cell growth inhibition. Methyl salicylic acid did not induce a clastogenic effect. No positive controls were reported. In a mammalian cell cytogenetics assay for chromosome aberration (Stich et al., 1981), CHO cell cultures were exposed to the hydrolysis product SA at concentration of 25 mg/ml for 3 hours with and without metabolic activation. The concentration used was half the dose which induced mitotic inhibition, defined as one metaphase or less in 6000 CHO cells. In addition, CHO cells were exposed to 12 mg/ml of Salicylic acid with a transition metal (Cu2 +orMn2+at 10-4M)without a metabolic activation. Salicylic acid did not induce a clastogenic effect.

In a Rel. 1 mouse lymphoma L5178Y study (Wollny, 2008), SA was tested at 87.5, 175.0, 350.0, 1400.0 µg/ml. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours. Relevant cytotoxic effects were solely noted following 24 h treatment in the second experiment at 700 µg/ml and above. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration. SA was considered to be non-mutagenic in this mouse lymphoma assay.

 

Genotoxicity in vivo:

No in vivostudies have been carried out on MeS itself, however data are available from the read-across substance SA.

Two in vivochromosome aberrations on SA have been published by Giri et al. (1996). In the first study, Swiss albino male mice were treated with SA dissolved in DMSO (75 µl/mouse), in an i.p. administration at the doses of 50, 100 and 200 mg/kg. Four mice were used for each dose along with a negative control. Negative control mice received equal volume of only DMSO. Five mice were injected cyclophosphamide (25 mg/kg) in equal volume of DMSO (75 µl/mouse) and served as positive control. No significant increase in CA was observed for SA when compared with solvent control. A significant increase in MI was observed at the lowest dose for SA. Positive control chemical cyclophosphamide induced high chromosome aberrations over solvent control. In the second study, Swiss albino male mice were treated with SA suspended with 2% gum acacia in distilled water (0.3 ml/mouse), by gavage at the dose of 350 mg/kg. Five mice were used for treated group along with a negative control. Negative control mice were gavaged with 2% gum acacia in distilled water. No significant increase in CA was observed for SA when compared with solvent control. A significant increase in MI was observed for SA. Positive control chemical cyclophosphamide induced high chromosome aberrations over solvent control. Although each for these key studies had minor deviations from current guidelines, taken together they are considered as acceptable.

The publication by Giri et al (1996) also reports twoin vivosister chromatid exchange assays. In the first study, Swiss albino male mice were treated with SA in DMSO (75 µl/mouse) in a single i.p. injection at the dose of 25, 50 and 100 mg/kg. Negative control mice were injected with 75 µl DMSO while mitomycin C was used as a positive control at a dose of 1.5 mg/kg bw. No significant increase in SCE was observed for SA when compared with solvent control. Positive control chemical mitomycin C gave a very high frequency of SCE over those of solvent control. In the second study, Swiss albino male mice were treated with SA suspended in distilled water in 2 % gum acacia (0.3 ml/mouse), in a single oral administration at the dose of 350 mg/kg. Negative control mice were administered with gum acacia. No positive control was used. No significant increase in SCE was observed for SA when compared with solvent control. Taken together, these studies are acceptable.

Conclusion:

MeS did not show any mutagenicity in bacteria (S. typhimurium) or in mammalian cells and did not cause structural chromosome aberrations in CHO cells, fromin vitrostudies. Its hydrolysis product SA did not induce either chromosome aberrations or sister chromatid exchanges in i.p. or oral studiesin vivoin mice, indicating that SA is not genotoxic in the bone marrow cells of mice. Taken together, these results indicate that MeS is not genotoxic.

Short description of key information:

The genotoxic potential of MeS has been evaluated from in vitro studies on MeS itself and from in vitro and in vivo studies on the read-across substance salicylic acid, the initial metabolite by hydrolysis of MeS. All tests gave negative results, leading to the conclusion that MeS is not genotoxic. For in vitro gene mutation studies in bacteria, two publications (Rel. 2) describing Ames tests in various strains of S. typhimurium have been chosen as Key studies, Mertelmans (1986) and Ishidate (1984). For in vitro mammalian cell gene mutation, a study on SA (RCC, 2008) performed according to OECD guideline 476 (Rel 1) has been chosen as key study. For chromosomal aberration in mammalian cells in vitro, a publication (Rel. 2) by Ishidate (1984) has been chosen as key study, with another, on SA, by Stich et al. (1981) (Rel. 2) as supporting study. For genotoxicity in vivo, a publication by Giri et al. (1996) describing oral and intraperitoneal studies on chromosome aberrations and sister chromatid exchange in the bone marrow cells of mice on SA has been taken as key study.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Not genotoxic according to EU and GHS (UN/EU) criteria.

Based on absence of mutagenicity in bacterial or mammalian cells, absence of clastogenicity in chromosome aberration studiesin vitroand absence of induction of either chromosome aberrations or sister chromatid exchangesin vivoin mice, from studies on MeS and hydrolysis product SA. These results indicate that MeS is not genotoxic.