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Additional information

There are reliable tests available investigating genetic toxicity of CAS No. 163520-33-0 in bacteria and mammalian cells in vitro and in mammalian cells in vivo.

 

Mutagenicity in bacteria was assessed in a study performed according to OECD guideline 471 and GLP (Müller, 1994). Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and E. coli strain WP2 uvr A were exposed to concentrations of 4, 20, 100, 500, 2500, 5000 µg/plate in the plate incorporation assay in the absence and presence of metabolic activation by rat liver S9-mix. No strain specific bacteriotoxic effect was observed in the tester strains. Precipitate was observed in the test plates of all strains at concentrations of 2500 µg/plate and above. For all tester strains the maximum number of revertants observed in the test plates was comparable to those of the negative controls with and without metabolic activation, whereas treatment with the positive control substances caused the expected statistically significant increase in revertants per test plate, demonstrating the validity of the test system. Thus, under the conditions of this test the test substance can be regarded as not mutagenic in bacteria.

 

The mutagenic potential of CAS No. 163520-33-0 in mammalian cells in vitro was assessed by a HPRT-assay according to OECD guideline 476 under GLP-conditions (Müller, 1997). In the pre-test Chinese hamster V79 cells were exposed for 4 hours to test substance concentrations of 100, 250, 500, 1000, 1500, 2000, 2500 and 2953.4 µg/mL (10 mM) in the absence and presence of metabolic activation by rat liver S9-mix. Dose-dependent toxic effects with and without metabolic activation were observed, but a high precipitation of the test substance at all doses tested without S9 mix and from 250 µg/mL onwards with S9 mix prevented evaluation of toxicity in the preliminary experiment. Therefore in the main assay the toxicity of the test substance was assessed by determining the plating efficiency of cells in the culture plates rather than by determining survival by measurement of crystal violet extinction. Based on the results of the preliminary experiment V79 cells were exposed to the test substance at concentrations of 15, 50, 150, 375, 750 and 1500 µg/mL without S9 mix and 15 (test 2), 50, 150, 375, 750, 1500 and 2953,4 µg/mL (10 mM, test 1) with S9 mix for 4 hours in two independent experiments. The relative plating efficiency decreased with dose, indicating toxicity. Precipitation was observed at 150 µg/mL (without S9 mix) and 375 µg/mL (with S9 mix) and above. No increases in mutant colony numbers were obtained in two independent experiments with the test substance in either the presence or absence of S9-mix. Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay. Therefore, the test substance is regarded as not mutagenic in mammalian cells, in vitro.

 

The clastogenic potential of CAS No. 163520-33-0 in vitro was assessed in a chromosomal aberration test in mammalian cells according to OECD guideline 473 and GLP (Müller, 1998). Following preliminary toxicity testing, Chinese hamster lung fibroblasts (V79) were exposed to 10, 25 or 40 µg/mL for 20 hours without S9-mix and to 100, 250 or 750 µg/mL for 3 hours with S9 mix. Duplicate cell cultures per dose level were used and cells sampled 20 or 28 (40 µg/mL -S9 only) hours after the start of treatment were examined for chromosome aberrations. Positive and vehicle (dimethylsulfoxide, DMSO) control cultures were included in each assay. At 750 µg/mL, a highly toxic and precipitating dose level, and the toxic 250 µg/mL concentration the test compound induced a significant increase in the number of chromosome aberrations in the presence of metabolic activation. No increases were seen in the absence of metabolic activation. Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix. Therefore the test substance was clastogenic at toxic and precipitating dose levels in the presence of metabolic activation in this in vitro test with Chinese hamster lung V79 cells.

 

The genetic toxicity of CAS No. 163520-33-0 in vivo was addressed in a Mammalian Erythrocyte Micronucleus test in the mouse, performed according to OECD guideline 474 under GLP conditions (Müller, 1996). Groups of 5 male and 5 female NMRI mice were used at each time point. Based on the results of a preliminary toxicity study, each was given a single dose by gavage of either 20, 100 or 200 mg/kg body weight, suspended in sesame oil. Bone marrow smears were obtained from 5 males and 5 females from each of the test groups and negative control groups at the three sampling times 12, 24, and 48 hours after dosing. Smears from the positive control group were obtained 24 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes and 1000 normochromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examining at least 1000 erythrocytes from each animal. Clinical signs of toxicity were observed in the preliminary toxicity study and in one male receiving 200 mg/kg bw, demonstrating relevant systemic exposure of the animals to the test substance in the absence of mortality. Treatment with the test substance did not increase the number of polychromatic and normochromatic erythrocytes containing micronuclei. The ratio of polychromatic to normochromatic erythrocytes in both male and female animals also remained unaffected by treatment. Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei in both males and females, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes was not significantly affected. These results gave no indication of a clastogenic effect of the test substance in vivo in NMRI mice under the conditions of this test.

 

In addition the potential of CAS No. 163520-33-0 to induce unscheduled DNA synthesis (indicative of DNA damage and subsequent repair) in vivo was also addressed in rat hepatocytes following acute oral exposure, performed according to the draft version of OECD guideline 486 under GLP conditions (Proudlock, 1997). The test substance was assessed for the induction of DNA repair in hepatocytes at dosages of 600 and 2000 mg/kg bw (limit dose for acute oral toxicity testing). A concurrent negative control group was treated with the vehicle (aqueous 1% (w/v) methyl cellulose) and positive control groups were treated with dimethylnitrosamine at 4 mg/kg bw (2 hours expression) or 2-acetylaminofluorene at 50 mg/kg (14 hours expression). Hepatocytes were isolated by enzymatic dissociation at 2 or 14 hours after exposure of the animals to the test substance. Four animals were assessed at each experimental point with the exception that only two animals from the positive control group were assessed at each expression time. The isolated hepatocytes were allowed to attach to glass coverslips and were cultured in vitro with (methyl-³H) thymidine at 10 µCi/mL for 4 hours to 'radiolabel’ replicating DNA. The hepatocytes were 'chased' for 24 hours with unlabelled thymidine then they were fixed and processed for autoradiography. DNA repair was assessed by comparing the labelling levels of hepatocyte nuclei from treated animals with control values and with the accompanying cytoplasmic labelling levels (usually a total of 150 cells from 3 different slides per animal were examined). The test substance did not cause any significant increases in either the gross nuclear grain count or the net nuclear grain count at any dose level at either sampling time. Positive control group animals showed a large and highly significant increase in the net nuclear grain count which was accompanied by a large increase in the gross nuclear grain count.

In conclusion, the test substance did not induce gene mutations in bacteria and mammalian cells in vitro. The clastogenic effect of the test substance seen in the presence of S9 mix in vitro at toxic and precipitating dose levels in Chinese hamster fibroblasts was not evident in vivo in mice. In addition no unscheduled DNA synthesis, as indicator of DNA damage, was observed after in vivo treatment with the test substance in rat hepatocytes. Thus, the test substance did not prove to be genotoxic in vivo.


Justification for selection of genetic toxicity endpoint
Positive results were obtained in an in vitro chromosome aberration test in the presence of a metabolic activation system in Chinese hamster V79 cells at toxic precipitating dose levels. These results could not be confirmed in an in vivo mouse erythrocyte micronucleus test where no indication of a clastogenic effect was found. As negative results were obtained in all other in vitro and in vivo tests the test substance is not genotoxic based on the available information and no study was selected.

Short description of key information:
OECD 471, Ames, plate incorporation, ±S9: not mutagenic in bacteria
OECD 473, Chromosomal Aberration, V79 cells, ±S9: clastogenic in mammalian cells in vitro with metabolic activation
OECD 476, HPRT, V79 cells; ±S9: not mutagenic in mammalian cells in vitro
OECD 474, Micronucleus test, mouse: not clastogenic in mammalian cells in vivo
OECD 486, Unscheduled DNA synthesis in hepatocytes: no unscheduled DNA synthesis in vivo

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.