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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative results in the following in-vitro studies:

- bacterical reverse mutation assay

- in vitro mammalian chromosome aberration test (on structural analogue substance)

- mammalian cell gene mutation assay (on structural analgoe substance)

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

Genetic toxicity in vivo

Description of key information

Negative result in:

- micronucleus assay (chromosome aberration) (on structural analgoe substance)

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

Additional information

mammalian cell gene mutation assay:

In a GLP compliant in vitro mammalian gene mutation study, Chinese Hamster V79 cells were exposed to the test material (cyclohexyl salicylate) up to cytotoxic concentrations without metabolic activation, and with metabolic activation, up to the limit of solubility. The ability of the test material to induce mutations at the HPRT locus in Chinese Hamster V79 cells was investigated in two independent experiments, both performed with and without metabolic activation. The test material was dissolved in ethanol and tested in the following concentrations:

Experiment I:

Without S9 mix: 0.75, 2.5, 5.0 7.5 and 8.5 µg/mL

With S9 mix: 3.0, 6.0 10.0, 30.0 and 60.0 µg/mL

Experiment II:

Without S9 mix: 1.0, 2.5, 5.0, 7.5 and 10.0 µg/mL

With S9 mix: 6.0, 10.0, 30.0 and 60.0 µg/mL

Doses were selected based on a pre-test for toxicity. Without S9 mix, the highest concentration demonstrated strong toxicity. With S9 mix, no toxicity was observed up to the limit of solubility (60 µg/mL). No relevant increase in mutant colony numbers were obtained in either experiment with or without metabolic activation. The positive negative, and vehicle controls were all demonstrated to be valid under the conditions of the test. Under the conditions of the test, the test material did not induce gene mutations at the HRPT locus in V79 cells.

in vitro mammalian chromosome aberration test:

In a GLP compliant in vitro mammalian chromosome aberration test, Chinese Hamster V79 cells were exposed to the test material (cyclohexyl salicylate) up to cytotoxic concentrations with and without metabolic activation. The ability of the test material to induce structural chromosomal aberrations in Chinese Hamster V79 cells was investigated. Cell cultures were exposed to the test material with or without metabolic activation. The test material was dissolved in DMSO and tested in the following concentrations:

- Without metabolic activation (S9 mix)

7 hour harvest time: 8 and 40 µg/mL

20 hour harvest time: 1, 4 and 8 µg/mL

28 hour harvest time: 6 µg/mL

- With metabolic activation (S9 mix)

7 hour harvest time: 40 µg/mL

20 hour harvest time: 5, 20 and 60 µg/mL

28 hour harvest time: 40 µg/mL

Doses were selected based on a pre-test for toxicity. When evaluating plating efficiency, strong cytotoxicity was observed at 6.2 µg/mL without S9 mix, and at 50 µg/mL with S9 mix. In the mitotic index assay, cytotoxicity was observed at the highest concentrations applied 28 hours after treatment without metabolic activation and at 7 and 28 hours after treatment with metabolic activation. A significant reduction of the mitotic index was observed at the highest non-lethal concentration 7 hours after treatment with and without metabolic activation. In the definitive assay, biologically and statistically relevant chromosomal aberrations were not observed neither with nor without metabolic activation. Under the conditions of the test, the test material did not induce structural chromosomal aberrations in V79 cells.

bacterical reverse mutation assay:

The mutagenic potential of the test substance was assessed according to OECD guideline 471. Salmonella typhimuriumstrains TA1535, TA1537, TA98, TAl 00 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at 7 dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 5 to 5000µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both 4 non-toxic dose levels and the toxic limit of the test item.

In the first experiment (plate incorporation method), the test item induced a substantial reduction in the revertant colony frequency of all of the Salmonella strains dosed in the presence of S9-mix from 500µg/plate (TA 1537) and 1500 µg/plate (TA 100, TAI 535 and TA98). No toxicity was noted to any of the Salmonella strains dosed in the absence of S9-mix and Escherichia coli strain WP2uvrA dosed in both the absence and presence of S9-mix. In Experiment 2 (pre-incubation method), the test item again induced a substantial reduction in revertant colony frequency for all of the Salmonella strains dosed in the presence of S9-mix from 1500 µg/plate. However, in this experiment weakened bacterial background lawns were also noted for all of the Salmonella strains dosed in the absence of S9-mix at 5000µg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA dosed in both the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000µg/plate. A test item precipitate (light and oily in apearance) was noted at 5000µg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. In Experiment 2, small, statistically significant increases in revertant colony frequency were observed in the absence of S9-mix at 15 µg/plate (TA100) and 500 µg/plate (TA98). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at each statistically significant dose level were within the in-house historical untreated/vehicle control range for the tester strains and the maximum fold increase was only 1.36 times the concurrent vehicle control.

The test item was considered to be non-mutagenic under the conditions of this test.

micronucleus assay (chromosome aberration):

The cytogenicity of the test material in vivo was investigated in a GLP compliant study. The test material (cyclohexyl salicylate) was administered by oral gavage in arachis oil at doses up to 3000 mg/kg in male and female mice. The animals were sacrificed at 24, 48 and 72 hours, and smears were prepared from the bone marrow. Under the conditions of the test, the test material did not induce a statistically or a time dependent increase in micronucleated polychromatic erythrocytes in male and female mice when tested up to concentrations of 3000 mg/kg.

Read-across justification:

Due to the limited availability of genotoxicity data on (z)-3-hexenyl salicylate, the mutagenic and cytogenic potential of (z)-3-hexenyl salicylate was assessed by reading-across to cyclohexyl salicylate. The reported results with cyclohexyl salicylate were considered representative of the effects of (z)-3-hexenyl salicylate. The substances are structurally very similar, the only difference being, (z)-3-hexenyl salicylate contains an acyclic hexenyl chain and cyclohexyl salicylate contains a cyclic hexyl group. Both molecules have very similar physical-chemical properties and are anticipated to undergo the same metabolic pathway. The results obtained with cyclohexyl salicylate are therefore expected to be representative of (z)-3-hexenyl salicylate.

Justification for selection of genetic toxicity endpoint

Four studies were available to address the genetic toxicity of the substance. As all 4 studies examined different endpoints, it was not possible to select one representative key study. All studies were performed in line with current accepted guidelines and under GLP conditions and reported to a high quality. All studies were assigned a reliability score of 1 in accordance with the criteria for assessing data quality as defined in Klimisch (1997).

Justification for classification or non-classification

According to the EU Regulation EC 1272/2008, the substance does not meet the criteria for classification for germ cell mutagenicity nor mutagenicity, respectively.