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In vitro bacterial mutagenicity:

The in vitro mutagenicity of benzyl chloride has been tested in in vitro bacterial systems mainly based on the Ames test. The three reported studies are all similar to the OECD guideline 471.

In a study from Brusick (1976), the authors tested the mutagenicity and recombinogenicity of benzylchloride (CAS n° 100-44-7) in the absence and presence of a metabolizing system with a methodology similar to the OECD guideline 471. The Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and the Saccharomyces cerevisiae strain D4 were used and the metabolizing system was obtained from rat liver homogenates. These strains were exposed to 0.1, 1, 5 and 10 µL/plate with and without metabolic activation for 48 to 72 h. Solvent controls and positive controls were performed.

The test showed that benzylchloride was mutagenic for Salmonella typhimurium strain TA 100 and recombinogenic for Saccharomyces cerevisiae strain D4. This respons was observed in absence and presence of the metabolic activation system, demonstrating that metabolic activation is not essential for mutagenicity or recombinogenicity. Furthermore, there was also an indication of a weak response at the high dose of the Salmonella typhimurium strain TA 1535. Effects were only seen at relatively high doses and dose-related responses at the lower end of the dose range were not observed. Hence, based on this information, further in vitro testing would be required with other systems to clarify if in vivo testing would be necessary.

In an other study from Flowers (1976), the authors tested the mutagenicity of benzylchloride (CAS n° 100-44-7) in the absence and presence of a mammalian metabolizing system with a methodology similar to the OECD guideline 471.Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were used. These strains were exposed to 0.001, 0.01, 0.1, 1, 5 and 10 µL/plate of the test substance (in DMSO) with and without mammalian metabolic activation. Solvent control and positive controls were performed. However, the rules for the interpretation of the results are not reported.

Hence, the authors concluded that benzylchloride was mutagenic for Salmonella typhimurium strain TA 100 at two dose levels (5 and 10 µL/plate) with metabolic activation. Questionable results were besides observed for strains TA 98 and TA 1538 at 5 µL/plate without metabolizing system. For all strains except TA 100, the authors noticed toxic effects at the highest tested concentration, 10 µL/plate.

Considering, the criteria in the OECD 471 for the interpretation of mutagenicity tests, the results should be interpretated as ambiguous since no clear dose-response relation was established. However, the overall results proves that benzylchloride shows obvious signs of mutagenicity in the tested Salmonella strains. Hence, further in vitro testing would be required to clarify the situation.

Finally, in an other study from Hyldig-Nielsen (1981), the authors tested the mutagenicity potential of benzyl chloride (CAS n° 100 -44 -7) in a Salmonella spot test according to the methodology of Ames et al. (1975). They imbibed discs of the test substance diluted in ethanol at different concentrations and applied the discs in petri dishes innoculated with the S. TA strains. They conducted two set of experiments. The first set included five common strains of Salmonella (TA 1535, TA 1537, TA 1538, TA 98, TA 100) and four concentrations as a solvent control and as

positive control with 2 -Amino-anthracene, tested all with and without metabolic activation. The second experiment was conducted with three TA strains (TA 1538, TA 98, TA 100) and five different concentrations, again with and without metabolic activation.

Results of the first sub-set of experiment are not reported as the applications of several discs per petry dish disturbed and avoided a clear reading of the results. However in the second experiment (with one disc per petry dish), they authors found a high increase of revertants in the highest tested concentration with and without metabolic activation.

They concluded that benzyl chloride is a potent mutagen in the Salmonella spot test. However, no explanation is given on their referential for interpretating these results. According to the OECD guideline 471, criteria to conclude on positive mutagenicity in in vitro bacterial reverse mutation assays are complex and varied but nonetheless, a significant increase in the number of revertants and a clear dose-response relation ship should established. At the present level of information, we may hypothesize that the increase in the number of revertants was their criteria. However, signs of toxicity were observed at the highest tested concentrations. Therefore, these results should be interpretated with caution and regarded as ambiguous. Hence, further in vitro testing would be required to clarify the situation.

The results of these experiments are similarly described and all based on the Ames test method. The differences between the Brusick study and the Flowers study, which are the more relevant and reliable, are not really big. Both exhibit deviations to the guideline. However, in the Flowers study (1976), no data is reported on the interpretation criteria of the mutagenicity and based on the OECD criteria, these results would be ambiguous. Thus, these elements were the basis for the choice of the Brusick study (1976) as a key study.

Altogether, these results bring evidences on the mutagenicity potential of benzyl chloride in in vitro bacterial system. Hence based on all available information, benzyl chloride should be considered as a mutagen in in vitro bacterial system.

So far, based on all results, further testing as required in the annex VIII should be required. However, according to the column 2 of the specific rules for adaptation from column 1 of the REACH regulation n° 1907/2006, an in vitro study on cytogenicity in mammalian cells or an in vitro micronucleus study of the annex VIII requirement does not usually need to be conducted if the substance is known to be carcinogenic category 1 or 2 or mutagenic category 1, 2 or 3. In this case, benzyl chloride is classified in the CLP regulation n° 1272/2008 EC as a carcinogen category 1B, hence no study on cytogenicity in mammalian cells or an in vitro micronucleus is required.

In vivo Mammalian cytogenicity:

The in vivo mutagenicity of benzyl chloride has been tested in in vivo Mammalians systems mainly based on the in vivo micronucleus test. Two reported studies are similar to the OECD guideline 474 and one test is based on the sperm head abnormality test.

In a study from Sison (1990), the authors tested the mutagenicity of benzyl chloride in vivo according to a methodology similar to the OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test) on Swiss-Webster male and female mice. Eight mice per dose were injected intraperitoneally less than 8 mL/kg bw of benzyl chloride solutions freshly diluted in DMSO twice. The concentrations corresponded to 1/8 LD50, 1/4 LD50 and 1/2 LD50 previously established in a screening test. The injections of benzyl chloride solutions took place 30 and 6 hours prior the preparation of the bone marrow. At the end of the exposure period, mice were sacrificed and bone marrow fluids were collected, stained and the number of micronucleated polychromatic erythrocytes on per thousand were counted to establish cytogenetic damages. Results for the benzyl chloride were then compared with other tested substances.

In the test conditions, the authors established that benzyl chloride induced significantly micronucleated polychromatic erythrocytes compared with solvent control. However, this increase was not estimated dose dependant and the induction rate was lower than the 3-fold threshold generally recommended. Benzyl chloride should therefore be considered as an ambiguous in vivo potential mutagen and would require further testing to establish its mutagenicity on somatic cells.

In an other study from Danford (1982), the authors tested the mutagenicity of benzyl chloride also in vivo in a micronucleus test following a methodology similar to the OECD guideline 474. Three dose were tested, 75, 150 and 300 mg.kg bw corresponding to 1/8 LD50, 1/4 LD50 and 1/2 LD50 established in screening test prior this experiment. Four mice per dose were injected either, one dose and killed 24 hours later, or one dose and a second identical dose 24 hours later then sacrificed 6 hours later. In each experiment, the bone marrow was extracted from the femurs, stained and scored for micronuclei in one thousand mature polychromatic erythrocytes.

No increase in micronuclei in polychromatic erythrocytes was observed at any dose. Therefore, benzyl chloride was not be considered as a potent mutagen in vivo in Tuck To mice.

In an other study from Scott (1982), the authors tested the mutagenicity of benzyl chloride (CAS n° 100 -44 -7) in a sperm head abnormality test on (CBAxBALB/C) F1 male mice.Three similar experiments were conducted. Each time, mice received five equal consecutive doses of benzyl chloride formulated in 0.5 % Tween 80, corresponding in one experiment to 125, 250 and 500 mg/kg bw/day subcutaneanously and in two other experiments to 50, 100, 200 and 400 mg/kg bw/day intraperitoneally. Thirty five days later, all surviving mice were killed and scored for sperm head abnormilties.

Considering that the criteria for a positive response are that the increase in abnormal sperm heads shall be at least double the negative control and be significant at least at the P>0.05 and besides, that reproducibility and a dose-response are required to confirm a positive response, benzyl chloride did not induce a positive response in the sperm head abnormality test.

Therefore, at this level of information, benzyl chloride was not considered as a potent mutagen for germ cells of mice. However as the specific target genes of this test are unknown, this study gives only support to the other results.

All three studies are sufficiently documented and gives relatively consistent results. The studies based on the micronucleus test are the most relevant thanks to their recognized testing method. Besides, altogehter these results prove that benzyl chloride does not induce significant genetic damages in vivo. At this level of information, further testing in vivo mutagenicity on somatic cells does not seem to be required and either recommanded.

Short description of key information:

- In vitro bacterial mutagenicity: positive

- In vivo Mammalian mutagenicity: negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification