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Gene mutation studies in bacteria: The ability of Lyral to cause gene mutations in prokaryotic cells was investigated in two studies, comparable to OECD guidelines 471. The most recent one, conducted with GLP (BioReliance, 1999) was selected as a key study. The S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2 uvr A were treated with 0 (vehicle control), 75, 200, 600, 1800 and 5000 μg/plate of the test substance in DMSO in the presence and absence of metabolic activation, using a plate incorporation method. The doses were selected based on the results of a preliminary range-finding study. Cytotoxicity was observed at the highest concentration levels in strains TA98 and TA1537. No statistically significant increase in the number of revertants was noted in any cases, indicating that the substance gave a negative result in the Ames test.

In an older study of Takasago International Corporation (1984), conducted without GLP, S. typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and E. coli strain WP2 uvr A were exposed to test concentrations of 0 (vehicle control), 10, 50, 100, 500, 1000 and 5000 μg/plate of the test substance in DMSO using the preincubation method, with and without metabolic activation. Cytotoxicity was observed in all strains at the highest concentration level. No statistically significant increase in the number of revertants was noted in any strain either in the absence or presence of metabolic activation, indicating that the substance is negative in the Ames test.

Chromosome aberrations in mammalian cells in vitro: The ability of Lyral to induce chromosome aberrations in vitro was investigated in a GLP-compliant study, performed according to a protocol similar to OECD Guideline 473, in Chinese hamster ovary (CHO) cells (Bioreliance 2000). The cells were treated with concentration levels of 200, 400, and 600 µg/ml and 100, 200, and 400 µg/ml of the test substance in DMSO for 4 and 20 hour exposure periods in the absence of metabolic activation. In the S-9 activated study, dose levels of 200, 800, and 900 µg/mL were evaluated for a 4 hour exposure period with a 20 hour cell harvest time. Dose levels were selected based on the results of a preliminary dose-range finding study. Statistically significant increases in structural chromosome aberrations (primarily chromatid-type breaks/exchanges) were observed in the non-activated and S-9 activated 4 hour exposure groups relative to the solvent control group: at 400μg/mL in the non-activated 4 hour exposure group and at the dose levels of 200, 800 and 900μg/mL in the S-9 activated exposure group. However, the statistically significant increase in the percentage of structurally aberrant cells at 400μg/mL in the non-activated 4 hour exposure group (7.0%) was only 1% outside of the range of the percentage of structurally aberrant cells observed with the historical solvent control (0.0-6.0%). Therefore, the statistically significant increase in structurally aberrant cells in the non-activated 4 hour exposure group was considered not biologically significant. No statistically significant increases in numerical chromosome aberrations were observed in the non-activated or S-9-activated 4-hour exposure groups relative to the solvent control group, regardless of dose level. A statistically significant increase in structural chromosome aberrations was observed in the non-activated 20 hour continuous exposure group relative to the solvent control group, at 400μg/ml. However, the statistically significant increase in the percentage of structurally aberrant cells at 400μg/ml (3.5%) was within the range of the percentage of structurally aberrant cells observed with the historical solvent control (0.0-6.0%). Therefore, the statistically significant increase in structurally aberrant cells in the non-activated 20 hour exposure group was considered not biologically significant. No statistically significant increases in numerical chromosome aberrations were observed in the non-activated 20 hour continuous exposure group relative to the solvent control group, regardless of dose level.

The statistically significant increase in structural chromosome aberrations of 7.5, 11, 24% at the 200, 800 and 900 μg/mL dose levels, respectively in the S-9 activated exposure group could partially be explained by increased cytotoxicity. At the 800 and 900 µg/mL dose levels, a 46 and 52% cell growth inhibition, respectively, was observed. However, no cell growth inhibition was observed at the 200 µg/mL dose level. Therefore, based on the findings of the study, Lyral was concluded to be positive for the induction of structural chromosome aberrations in the S9 activated test system, negative for the induction of structural aberrations in the non-activated test system, and negative for the induction of numerical chromosome aberrations in both the non-activated and S9 activated test systems in Chinese hamster ovary (CHO) cells.

Micronucleus test in vivo: A GLP-compliant micronucleus test with mice, performed according to a protocol similar to OECD Guideline 474, was conducted by BioReliance (2000b). Groups of male and female ICR mice (vehicle control: 10/sex/dose, low and mid dose groups and positive control group: 5/sex/dose, high dose group: 15/sex/dose) were administered the test substance at dose levels of 0 (corn oil), 225, 450 and 900 mg/kg bw by a single intraperitoneal administration. Animals of low and mid-dose groups and positive control animals were sacrificed 24 hours post-administration, high dose and vehicle control groups were sacrificed 24 and 48 hours post-administration, and bone marrow slides were prepared. Mortality was observed in 1/15 females at 900 mg/kg bw. Clinical signs included lethargy and piloerection in males and females at 225, 450 and 900 mg/kg bw and irregular breathing in males and females at 900 mg/kg bw. Slight to moderate reductions (up to 31%) in the ratio of polychromic erythrocytes to total erythrocytes were observed in the test article-treated groups relative to the respective controls. These reductions suggest bioavailability of the substance to the bone marrow target. No significant increase in micronucleated polychromatic erythrocytes in the test article-treated group relative to the respective vehicle control group was observed in male or female mice at 24 or 48 hr after dosing (p > 0.05). The substance was thus concluded to give negative results in this micronucleus test in vivo.

Conclusion on genotoxicity of Lyral: In summary, for Lyral negative Ames tests (gene mutations), a positive in vitro chromosome aberration test, and a negative in vivo chromosome aberration test were available. According to chapter R.7A, pp. 374-402 and in accordance with Table R.7.7-5 of the REACH Guidance chapter R.7A, no further testing on genotoxicity is required in this case. Furthermore, according to Table R.7.7 -5, the conclusion to be drawn on genotoxicity is "not genotoxic". Thus, based on the data available for Lyral, it is valid to conclude that Lyral is negative for genotoxicity.


Short description of key information:
The substance gave negative results in two Ames tests with S. typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and E. coli strain WP2 uvr A, with and without metabolic activation, at concentration levels up to 5000 μg/plate. Cytotoxicity was observed at the highest concentration level. The substance was concluded to be positive for the induction of structural chromosome aberrations in CHO cells in the presence of S9 metabolic activation, and negative for the induction of numerical chromosome aberrations in both the non-activated and S9 activated test systems. In the in vivo micronucleus assay with male and female mice no statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed at dose levels up to and including 900 mg/kg bw, following a single intraperitoneal administration. Based on these results, the substance is considered to be not genotoxic in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative results in three genotoxicity studies, two in bacteria (Ames), and one in an in vivo micronucleus study in mice, classification for genotoxicity is not warranted according to EU Directive 67/548 (DSD) and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.