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In vitro

- Gene mutation in bacteria

A bacterial gene mutation assay (Ames test) was conducted with ethiprole in compliance with OECD guideline 471 and under GLP conditions (Ballantyne, 1998). In a preliminary cytotoxicity test with S. typhimurium TA 100 at concentrations ranging from 8 to 5000 µg/plate, evidence of cytotoxicity and precipitation of the test substance were observed at the maximum test concentration and only in in the presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). However, this concentration was included in the main study as being the best estimate of the precipitation and toxicity limit. The main study included two series of experiments, both performed in the absence and in the presence of S9-mix. In the first experiment, the direct plate incorporation procedure was conducted with Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537 as well as with Escherichia coli WP2 uvrA using the same concentrations as in the preliminary test. In the second experiment, concentrations ranging from 312.5 to 5000 µg/plate were used in the same strains of both species, with the exception that a preincubation period was applied. Furthermore, an additional concentration of 156.25 µg/plate was tested in the presence of the metabolic activation system. In the first experiment (plate incorporation), cytotoxic effects were only observed in S. typhimurium TA 98 and TA 1537 and at the highest concentration used (with or without S9-mix). In the second experiment (with preincubation), cytotoxic effects only occurred in the S. typhimurium strains at concentrations ≥ 625 µg/plate (with S9-mix) and concentrations ≥ 2500 µg/plate (without S9-mix). Precipitation of the test substance was observed in the presence or absence of S9-mix in both experiments (5000 µg/plate in experiment 1 and ≥ 625 µg/plate in experiment 2). The positive and negative controls included showed the expected results in each experiment, but only one positive control substance was tested in the presence of metabolic activation and not in all strains. However, each batch of the metabolic activation system was checked for sterility, purity and functionality by the manufacturer. Therefore, these deviations were not considered to prejudice the validity of this study. The test substance did not induce mutations in the bacterial mutation tests in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E.coli (WP2 uvrA).

- Gene mutation in mammalian cells

The genotoxic potential of ethiprole was further assessed using a gene mutation assay in cultured mammalian cells (mouse lymphoma L5178Y cells) according to OECD guideline 476 and complying with GLP (Fellows, 1999). A preliminary cytotoxicity study in this cell line was performed with concentrations ranging from 31.25 to 1000 µg/mL. After a 4-day incubation period with the test substance, marked cytotoxicity was observed at ≥ 250 or ≥ 500 µg/mL in the absence or presence of metabolic activation (Aroclor 1254-induced rat liver S9-mix). Precipitation of the test substance was observed at the highest concentration tested (1000 µg/mL), thus a top concentration of 500 µg/mL was selected for the main study experiments. Two independent assays with test concentrations ranging from 15.625 to 500 µg/mL were conducted with and without metabolic activation. In these experiments, cells were exposed to the test material for 3 h. Two days after treatment, viability and 5-trifluorothymidine (TFT) resistance were determined. In the first assay, marked cytotoxicity was observed at the highest concentration (500 µg/mL), resulting in a relative survival of 18 and 10% in the absence or presence of S9-mix, respectively. An additional concentration of 375 µg/mL was included in the second assay in the presence of the S9-mix. In this experiment, similar results for cytotoxicity were obtained compared to the first experiment, yielding 19% and 17% relative growth at 500 µg/mL in the absence and presence of S9-mix, respectively. The negative (solvent) controls included in these experiments were within the expected ranges for mutant frequencies and the positive controls induced clear increases in mutation. In both assays, a weak linear trend was observed in the presence of S9-mix (p < 0.05). This linear trend was solely due to a very small, but not statistically significant increase in mutant frequency at the highest concentration tested (500 µg/mL). No increase in mutant frequency was observed at any other concentration tested. Reanalysis of the data under exclusion of the highest concentration tested confirmed that no linear trend was seen in both assays. Therefore, it was concluded that under the conditions used in the study, the test material was not mutagenic at the TK-locus of mouse lymphoma L5178Y cells in the absence and presence of metabolic activation.

- Chromosome aberrations

Ethiprole was assayed in an in vitro mammalian chromosome aberration test conducted in accordance with GLP and similar to OECD guideline 473 (Marshall, 1998). In two independent experiments, human lymphocytes from two healthy donors were treated with the test substance at concentrations up to 800 µg/mL, with and without metabolic activation (S9-mix from Aroclor 1254-induced rat liver). In the first experiment, continuous treatment for 20 h was performed in the absence of metabolic activation, whereas treatment in the presence of S9-mix included the incubation with the test substance for 3 h followed by a 17 h-recovery period. Concentrations for chromosome analysis were selected based on the cytotoxicity data after these treatments. At the highest concentration tested, the mitotic index was reduced by 31% (with S9-mix) and 42% (without S9-mix) compared to that of the solvent control. In contrast, the mitotic index in the absence of metabolic activation was reduced by 61and 73% at concentrations of 253.1 and 337.5 µg/mL, respectively. These results demonstrated no clear dose-response relationship for cytotoxic effects in the absence of S9-mix. The author suggested that the absence of a clear dose-response relationship was due to an accumulation of cell in mitosis at higher concentrations. However, this assumption was not substantiated by respective data. Moreover, test substance precipitation was observed at concentrations ≥ 337.5 µg/mL (with or without S9-mix). Thus, the decrease in cytotoxicity at higher concentrations may have been a result of result of low test substance availability in the culture medium. According to OECD guideline 473, the highest exposure concentrations for chromosome analyses should be those, which reduce the mitotic index by at least 50%. Since the highest concentrations applied for chromosome analysis in the absence of S9-mix resulted in less than 50% reduction of mitotic index at 450, 600 and 800 µg/mL, the test concentration of 253.1 µg/mL (corresponding to 61% mitotic inhibition) was included in this experiment. In the presence of S9-mix, no effects on the mitotic index were observed up to 337.5 µg/mL. Thus, concentrations ≥ 450 µg/mL (corresponding to 22-38% mitotic inhibition) were selected for chromosome analysis in the presence of metabolic activation.

The second experiment was performed with an additional group of cells treated for 44 h in the absence of S9-mix. An additional test group was also included in the presence of S9-mix (3 h treatment and a 41 h-recovery period). Similar patterns for the reduction in mitotic inhibition were observed in the absence of S9-mix compared to the first experiment, showing no dose-response relationship at higher concentrations. Moreover, little or no reduction in the mitotic index was observed at the highest concentration used in all treatments. Test substance precipitation occurred at ≥ 253.1 µg/mL (with S9-mix) and ≥ 337.5 µg/mL (without S9-mix). Based on the results of mitotic inhibition after 20 h incubation with test substance and S9-mix, chromosomes were analysed at concentrations ≥ 450 µg/mL. An additional concentration of 253.1 µg/mL (corresponding to 66% mitotic inhibition) was included in the absence of S9-mix. The effects on chromosomes at the delayed sampling time were only investigated at a single concentration of 800 µg/mL (with and without S9-mix), since no or only minor effects on the mitotic index were seen at the lower concentrations. In the first experiment, the test substance did not induce a statistically significant increase in the number of cells with chromosomal aberrations at any of the concentration analysed. In the second experiment, a small, but statistically significant (p ≤ 0.05) increase in cells with aberrations at the highest concentration (800 µg/mL) following treatment in the absence of S9-mix for 20 h was observed. Since no dose-response relationship was seen and the numbers of aberrant cells fell within the historical range of the solvent control, this result was not considered to be biologically significant. The positive control substances yielded the expected results. Under the conditions of this chromosome aberration assay, it was concluded that the test substance did not show clastogenic activity in cultured human lymphocytes.

In vivo

The in vivo clastogenicity of ethiprole was investigated in a GLP-conform Mammalian Erythrocyte Micronucleus Test in male and female CD-1 mice according to OECD guideline 474 (Burmann, 1999). Based on a preliminary range-finding toxicity test, the test substance diluted in 0.5% methyl cellulose was administered as a single dose to groups of five animals per sex at dose levels of 0, 500, 1000 and 2000 mg/kg bw via oral gavage. At 1000 mg/kg bw, mortalities were observed in 1/5 females 24 h after treatment and in 1/5 males 48 h after treatment. Further mortalities were observed in 1/5 females 24 h after treatment and in 2/5 females 48 h after treatment. After a post-exposure period of 24, 48 and 72 h, femoral bone marrow was taken from animals of each treatment and control groups. To determine the frequency of micro-nucleated erythrocytes, 1000 erythrocytes per animal were scored. No increases in the frequency of micronuclei in polychromatic erythrocytes of the femoral bone marrow of CD 1 mice exposed to dose levels up to 2000 mg/kg bw compared to vehicle control values were observed. The mean ratios of polychromatic to normochromatic erythrocytes, being indicative of possible bone marrow toxicity, were similar to the values for vehicle control groups at all sampling times and dose levels and also fell within normal ranges of historical control data. The positive control substance (40 mg/kg bw cyclophosphamide in saline) significantly increased the number of polychromatic erythrocytes with micronuclei in male and female animals, thus verifying the sensitivity of the assay. Under the conditions of this Mammalian Erythrocyte Micronucleus Test, the test substance was not clastogenic in male and female CD1 mice up to dose levels of 2000 mg/kg bw.

A further GLP-conform in vivo genotoxicity test is available, in which the ability of ethiprole to induce to unscheduled DNA synthesis (UDS) in isolated hepatocytes of treated rats was investigated according to OECD guideline 486 (Howe, 2001). Based on a preliminary range-finding toxicity test, groups of 4 male Han Wistar rats were treated with the test substance diluted in 0.5% methyl cellulose at dose levels of 800 and 2000 mg/kg bw via oral gavage. A similar group of animals received the vehicle alone and served as vehicle control group. Furthermore, two positive controls groups, each consisting of 4 animals, were administered a single oral dose of 75 mg/kg 2-acetamidofluorene (2-AAF) suspended in corn oil (12-14 hour experiment) or 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour experiment) via gavage. The main study was performed in two independent experiments, comprising two different sampling times (12-14 h and 2-4 h) of hepatocytes after single administration of the test substance. At each sampling time, hepatocytes were isolated from the livers of three of four animals of each treatment and control group by collagenase perfusion and then radioactively labelled with [3H] thymidine. The incorporation of [3H] thymidine into newly synthesised DNA, being indicative of DNA damage and subsequent repair, was analysed using autoradiography. Slides were examined microscopically after development of the photographic emulsion and staining, and the net nuclear grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for each of two of the three slides, for each animal and dose group. The vehicle control animals showed a group mean NNG value of less than zero with only 1% cells in repair. Group mean NNG values of positive control animals were increased by 2-AAF and DMN treatment to more than 19.5 and more than 50% cells found to be in repair. The vehicle and positive control NNG values were within the respective historical control data, and thus verified the validity and sensitivity of the assay. Treatment with 800 or 2000 mg/kg bw of the test substance did not produce a group mean NNG value greater than -2.0 nor were any more than 1% cells found in repair at either dose in isolated hepatocytes approximately 12-14 or 2-4 hours after dosing. No clinical signs of toxicity were noted, except for a slight reduction in body weights at 2000 mg/kg bw/day 12-14 h after test substance administration. Based on these results, test substance did not induce UDS in isolated hepatocytes of male Han Wistar rats treated with single doses up to 2000 mg/kg bw.


Short description of key information:
In vitro:
Negative Ames tests with S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, and E. coli WP2 uvr A, with and without metabolic activation.
Negative results in a mammalian cell gene mutation test using mouse lymphoma L5178Y cells, with and without metabolic activation.
Negative results in a chromosome aberration tests using cultured peripheral human lymphocytes, with and without metabolic activation.

In vivo:
Negative results in an erythrocyte micronucleus test in mice.
Negative results in an unscheduled DNA synthesis test in rat.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The available data on genetic toxicity of ethiprole 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.