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In vitro assay

Gene mutation assay

The potential of the test item to induce reverse mutation was evaluated inSalmonella typhimurium (Sire, 2010a). The study was performed according to the international guidelines (OECD 471 and Commission Directive No. B13/14) and in compliance with the principles of Good Laboratory Practice.

A preliminary toxicity test was performed to define the dose-levels of the test item to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes). Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item was dissolved in water for injections. The selected treatment-levels were 312.5, 625, 1250, 2500 and 5000 µg/plate for all the strains in the first experiment and 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL for all the strains in the second experiment, both with and without S9 mix. No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels. Without S9 mix, no noteworthy toxicity was noted at any dose-levels in any tester strains. A slight increase in the number of revertants was noted in the TA 102 strain in the second experiment (1.8-fold the vehicle control value). Since this increase did not exceed the threshold of 2-fold the vehicle control value and was not observed in the first experiment, it was not considered as biologically relevant. The test item did not induce any other noteworthy increase in the number of revertants. With S9 mix, no noteworthy toxicity was noted at any dose-levels in any tester strains. Slight increases in the number of revertants were noted in the TA 102 strain in the first experiment (up to 1.9-fold the vehicle control value). Since these increases did not exceed the threshold of 2-fold the vehicle control value and were not observed in the second experiment, they were not considered as biologically relevant. Noteworthy increases in the number of revertants, exceeding the threshold of 2-fold the vehicle control value (up to 2.9-fold the vehicle control) were noted in the TA 98 strain in both experiments. The test item did not induce any other noteworthy increase in the number of revertants. The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Under the experimental conditions of this study, the test item showed a mutagenic activity in the bacterial reverse mutation test with the TA 98Salmonella typhimuriumstrain, in the presence of a metabolic activation system.

Chromosomal aberration assay

The potential of the test item to induce chromosome aberrations was evaluated in cultured human lymphocytes (Sire, 2010b). The study was performed according to the international guidelines (OECD 473 and Commission Directive No. B10) and in compliance with the Principles of Good Laboratory Practice. The test item was tested in two independent experiments, both with and without a liver metabolizing system (S9 mix), obtained from rats previously treated with Aroclor 1254. The highest dose-level for treatment in the first experiment was selected on the basis of pH, osmolality and solubility. For selection of the dose-levels for the second experiment, any toxicity indicated by the reduction of Mitotic Index (MI) in the first experiment was also taken into account. For each culture, heparinized whole blood was added to culture medium containing a mitogen (phytohemagglutinin) and incubated at, for 48 hours. In the first experiment, lymphocyte cultures were exposed to the test or control items (with or without S9 mix) for 3 hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. The second experiment was performed without S9 mix, cells were exposed continuously to the test or control items until harvest, and with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed. Cells were harvested 20 hours and 44 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later, respectively. As inconsistent results were obtained in the first and second experiments with S9 mix, a third experiment was undertaken in presence of S9 mix, under the following conditions: cells were exposed to the test or control items for 3 hours and then rinsed. Harvest time was 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa. All the slides were coded for scoring. The test item was dissolved in sterile water for injections. The positive controls were Mitomycin C ( without S9 mix) andCyclophosphamide (with S9 mix). In the culture medium, the dose-level of 5000 µg/mL showed no precipitate. At this dose-level, the pH and the osmolality values were equivalent to those of the vehicle control culture. With a treatment volume of 55 µL/5.5 mL culture medium, the treatment-levels were 39.06, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL for the first experiment, both with and without S9 mix, 78.13, 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL for the second experiment, both with and without S9 mix and 156.3, 312.5, 625, 1250, 1875, 2500 3750 and 5000 µg/mL for the third experiment with S9 mix. No precipitate was observed in the culture medium at the end of the treatment period. The frequency of cells with structural chromosome aberrations of the vehicle and positive controls was as specified in acceptance criteria. The study was therefore considered valid.

Experiments without S9 mix

Cytotoxicity

Following 3-hour treatment, a 40 to 60% decrease in the mitotic index was observed at all dose‑levels.

Following the 20-hour treatment, no noteworthy decrease in the mitotic index was noted at any dose-levels.

Following the 44-hour treatment, a 29 to 49% decrease in the mitotic index was induced at dose‑levels ≥ 2500 µg/mL.

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows.

           78.13, 156.3, 312.5, 1250, 2500 and 5000 µg/mL for the 3-hour treatment.

           1250, 2500 and 5000 µg/mL for the 20-hour treatment.

           5000 µg/mL for the 44-hour treatment.

Following the 3-hour treatment, a slight but statistically significant increase in the frequency of cells with structural chromosomal aberrations was noted at the dose-level of 2500 µg/mL (4.0%versus1.0% for the vehicle control, p<0.05). However, this increase was not dose-related, was mainly due to one out of the two treated cultures and remained within the corresponding historical data range (4%versus[0 to 5%] for the historical data), consequently, it was considered as biologically non-relevant.

No noteworthy increase in the frequency of cells with structural chromosomal aberrations was noted following the 20-hour treatment.

A slight but statistically significant increase in the frequency of cells with structural chromosomal aberrations was noted following the 44-hour treatment at the dose-level of 5000 µg/mL (6.0%versus1.0% for the vehicle control; p<0.05). This increase is only due to one out of the two treated cultures (culture 2). Moreover, the culture 2 showed a decrease in the mitotic index of 68% at the dose-level of 5000 µg/mL, indicating an important cytotoxicity. Consequently, the slight increase in the frequency of cells with structural chromosomal aberrations observed following the 44-hour treatment was not considered as biologically relevant and was attributed to secondary effect related to the cytotoxicity.

Experiments with S9 mix

Cytotoxicity

At the 20-hour harvest time in the first experiment, a slight decrease in the mitotic index (up to 42%) was noted without any clear evidence of a dose-response relationship.

At the 20-hour harvest time in the second experiment, no noteworthy decrease in the mitotic index was noted at any dose-levels.

At the 44-hour harvest time (second experiment), no noteworthy decrease in the mitotic index was noted at any dose-levels.

At the 20-hour harvest time in the third experiment, a slight decrease in the mitotic index was noted at 1875 µg/mL only (27% decrease).

Metaphase analysis

The dose-levels selected for metaphase analysis were as follows.

           1250, 2500 and 5000 µg/mL, for the 20-hour harvest time in the first and second experiments.

           5000 µg/mL, for the 44-hour harvest time (second experiment).

           2500, 3750 and 5000 µg/mL for the 20-hour harvest time in the third experiment.

A significant increase in the frequency of cells with structural chromosomal aberrations was noted at 5000 µg/mL in the first experiment (5.5%versus0.5% for the vehicle control; p<0.01).

Since this increase was reproduced neither in the second experiment using the same experimental conditions (20-hour harvest time) and using a 44-hour harvest time, nor in the third experiment using a closer range of dose-levels, it was not considered as biologically relevant.

The test item did not induce chromosome aberrations in cultured human lymphocytes.

In vivo assay

Micronucleus assay

The potential of the test item to induce structural or numerical damage was evaluated in bone marrow cells of mice (Sire, 2010c). The study was performed according to the international guidelines (OECD 474 and Commission Directive No. B12) and in compliance with the Principles of Good Laboratory Practices. A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice were given oral administrations of the test item at dose-levels of 500, 1000 and 2000 mg/kg/day, over a 2-day period. One group of five males and five females received the vehicle (water for injections) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg/day. The animals of the test item treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE). Neither mortality, nor clinical signs were observed in the animals of either sex given 500, 1000 or 2000 mg/kg/day, throughout the observation period. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data. Cyclophosphamide induced a statistically significant increase in the frequency of MPE (p<0.01), indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid. The mean values of MPE as well as the PE/NE ratio in the groups treated with the test item, were not found different from those of the vehicle group. The test item did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.

UDS assay

The test item was examined for its potential to induce unscheduled DNA synthesis (UDS) in primary rat hepatocytes after a single exposure of male Fisher rats by oral route (Simar, 2010). The study was conducted in compliance with OECD guideline # 486. From oral acute and repeated dose toxicity studies, the maximum tolerated dose (MTD) was estimated at 2000 mg/kg bw. The highest dose used for the UDS test was the MTD, i. e. 2000 mg/kg. The lower doses was 50% of the highest dose, i. e. 1000 mg/kg. Two to four hours and 12-16hoursafter exposure, animals were sacrificed for isolation of hepatocytes. The DNA-repair activities were examined by autoradiography in monolayer cultures of hepatocytes, incubated in the presence of [methyl-3H]thymidine. The hepatocarcinogens dimethylhydrazine (10 mg/kg, 2-4 hours expression time) and 2-acetamidofluorene (25 mg/kg, 12-16 hours expression time) were used as a positive control. Hepatocytes isolated from animals treated with the vehicle (distilled water) served as negative controls. At both expression times, the test item did not induce DNA-repair activities in hepatocytes. The positive control substances induced the expected increase in DNA-repair activities. It was concluded that the test item did not induce DNA-repair in rat hepatocytes.


Short description of key information:
The substance induced a mutagenic activity in the bacterial reverse mutation test with the TA 98 Salmonella typhimurium strain, in the presence of a metabolic activation system, did not induce chromosome aberrations in cultured human lymphocytes, did not induce chromosome aberrations in cultured human lymphocytes, did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations at the dose-levels of 500, 1000 and 2000 mg/kg/day and did not induce DNA-repair in an in vivo/in vitro Unscheduled DNA Synthesis (UDS) test in hepatocytes from Fischer male rats, treated once orally with 2000 and 1000 mg/kg. On a weight of evidence basis, the substance is considered as not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification is warranted for germ cells mutagenicity under EU Dangerous Substances Directive 67/548/EEC or CLP EU Regulation 1272/2008.