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In vitro

Imidazole was tested in the standard Ames test and in the preincubation Ames test conducted under GLP and according to the OECD TG 471. The substance was tested with Salmonella typhimurium TA 1535, TA 100, TA 1537, and TA 98 both in the presence and absence of metabolic activation in concentrations up to 5000 μg/plate. No mutagenic or bacteriotoxic effect was noted (BASF SE, 1992). According to OECD TG 471, an additional strain of either E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 needs to be investigated. In a second Ames test, the S. typhimurium strain TA102 was investigated. In this study by Forster et al. (1992), imidazole and its metabolites hydantoin, hydantoic acid, and N-acetyl-imidazole were also negative in a standard-plate Ames-test equivalent to the OECD TG 471 with S. typhimurium TA 97, TA 98, TA 100, and TA 102 in the presence and absence of metabolic activation. Test substance concentrations were up to and including 10,000 μg/plate without reaching cytotoxicity. In a third Ames test, Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were exposed to concentrations of 0, 62.5, 125, 250, 500, and 1000 µg/plate were tested in triplicate, in the presence and absence of S9 -mix (BASF SE, 1979). The number of revertant colonies in all cases was less than twice the corresponding solvent control values, when tested up to the maximum level of 1000 µg/plate. Imidazole and its metabolites showed no mutagenicity in bacterial in-vitro test systems in the presence or absence of metabolic activation.

In the same study by Forster et al. (1992) it was determined that imidazole did not induce Unscheduled DNA Synthesis (UDS) in rat primary hepatocytes. The test method used was equivalent to the OECD TG 482. The test substance concentrations (0.25, 0.5, 1, 2, 4 mg/mL) reached the cytotoxic concentration range. Cell survival was 50% at 1 mg/mL (Forster et al., 1992). However, a negative UDS is not sufficient information to conclude on the induction of gene mutation by a substance.

Besides this UDS to investigate the potential mutagenicity of the test substance, a mammalian gene mutation test according to OECD 476 and GLP in V79 Chinese hamster cells (HPRT locus) was conducted with imidazole (Harlan, 2010). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum concentration was 700.0 µg/mL, corresponding to a molar concentration of about 10 mM of the test item. 7,12-dimethylbenz(a)anthracene (DMBA) and Ethylmethane sulfonate (EMS) were used as positive controls in experiment with and without metabolic activation, respectively. Positive as well as negative controls gave expected results.

The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of experiment I with metabolic activation at 700 µg/mL and in the second culture without metabolic activation at 43.8 and 350 µg/mL. The increase observed in the second culture without metabolic activation was not dose dependent as indicated by the lacking statistical significance and not reproduced in the parallel culture under identical conditions. Although there was a statistically significant increase of the mutation frequency in the presence of metabolic activation, the effect was not reproducible in the parallel culture or in both cultures of the second experiment with metabolic activation.

No other relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximal concentration. Therefore, Imidazole was considered to be non-mutagenic in this HPRT assay.

In order to confirm the assessment of the HPRT assay, a new guideline conforming mouse lymphoma

assay was performed as this test system is considered more sensitive than the HPRT assay. The test substance was tested for its ability to induce gene mutations at the thymidine kinase (TK) locus in L5178Y TK+/- mouse lymphoma cells in vitro with the microwell method (BASF SE 2015). Two independent experiments were carried out with and without the addition of liver S9-mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). An initial range-finding cytotoxicity test for the determination of the experimental doses was performed. The first experiment was conducted with and without S9-mix (4-hour exposure period) with concentrations of 0, 87.5, 175.0, 350.0, and 700.0 μg/mL test substance. The second experiment was conducted without S9-mix (24 -hour exposure period) with concentrations of 0, 87.5, 175.0, 350.0, and 700.0 μg/mL test substance and with S9-mix (4 -hour exposure period) and test substance concentrations of 0, 100.0, 200.0, 400.0, and 700.0 μg/mL. Subsequently, cells were cultured for an expression period of about 48 hours and then cultured in selection medium for another approx. 10 days. Finally, the number of large and small colonies was determined. The negative controls gave mutant frequencies within the range expected for the L5178Y TK+/- mouse lymphoma cell line. Both positive controls, methyl methansulfonate and cyclophosphamide, led to the expected increase in the frequencies of forward mutations. No cytotoxicity indicated by either reduced relative cloning efficiency or reduced relative total growth of below 20% of control was observed in both experiments. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9-mix and/or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions described, the test substance did not induce forward mutations in vitro in the mouse lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic activation.

In vivo

Imidazole hydrochloride was tested in a micronucleus test in accordance with the OECD TG 474 under GLP conditions in mice, dosed once by gavage with 500, 1000, and 2000 mg/kg bw/d. The salt imidazole hydrochloride dissociates into protonated imidazole and chloride in the stomach following oral gavage and did not induce micronuclei at any dose or any harvesting time, which were set at 16, 24, and 48 hrs after dosing. The animals showed signs of toxicity at 500 mg/kg bw and above. The number of polychromatic and normochromatic erythrocytes was not statistical significantly different from the control, therefore it may be concluded, that imidazole was not toxic to the bone marrow. However, imidazole was detected in the plasma of Wistar rats dosed orally (gavage) with approximately 17 mg/kg bw of imidazole, indicating imidazole is absorbed (Pagella et al 1983). This suggests that the bone marrow will have been exposed in this micronucles test. Imidazol was found to be not clastogenic or aneugenic in this test (BASF SE, 1993).

Short description of key information:
No mutagenicity was observed in the Ames tests performed. No clastogenic or aneugenic effects were found in the in vivo mouse micronucleus test. No unscheduled DNA synthesis was induced in primary rat hepatocytes, and no mutagenicity was observed in the HPRT gene of V79 Chinese hamster cells and in a mouse lymphoma assay in the absence and presence of metabolic activation. Based on all available data it can be concluded that imidazole is not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Imidazole was not mutagenic in the Ames test, did not induce unscheduled DNA synthesis in primary rat hepatocytes, and imidazole was negative in the in vivo micronucleus test in mice. However inconclusive results were obtained in a HPRT assay. To exclude a potential mutagenic effect of the test substance, an additional mouse lymphoma assay was performed which showed no mutagenic effects. Therefore, classification according to Regulation 1272/2008/EC is not warranted.

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