Methyl (R)-2-(4-hydroxyphenoxy)propionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: Under the conditions of this study, the test material is not mutagenic in the Ames test with and without metabolic activation.

Chromosome Aberration: Under the conditions of this study, the test material induced chromosome aberrations in cultured human lymphocytes with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Mouse Micronucleus: Molinier (1993): Under the conditions of the study, the test material did not induce cytogenetic damage to the bone marrow cells of mice when treated by the oral route at 2000 mg/kg in the micronucleus test.

Mouse Micronucleus: Haddouk (2002): Under the conditions of this study, the test material did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations with a 24-hour interval at the dose-levels of 500, 1000 or 2000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Ames test (Engelhardt, 1992)

The mutagenic potential of the test material was investigated in the bacterial reverse mutation assay in accordance with the standardised guidelines OECD 471 and EU Method B14 under GLP conditions. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

Four strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537 and TA 98) were exposed to the test material in DMSO at concentrations of 0, 20, 100, 500, 2500 and 5000 µg/plate in both the presence and absence of metabolic activation (S-9 mix). A standard plate test and a pre-incubation assay were carried out and following incubation the plates were assessed for cytotoxicity and an increase in the number of his+ revertants.

In the standard plate test both with and without S-9 mix there was no increase in the number of his+ revertants in any tester strain.

In the pre-incubation test both with and without S-9 mix there was no increase in the number of his+ revertants in any tester strain.

No bacteriotoxic effect (reduced his- background growth) was observed.

Under the conditions of this study, the test material is not mutagenic in the Ames test with and without metabolic activation.

Chromosome Aberration (Griffon, 2002)

The potential of the test material to induce chromosomal aberrations was investigated in accordance with the standardised guidelines OECD 473 and EU method B.10 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was tested in two independent experiments, both with and without a metabolic activation system (S9 mix). Dose-levels were selected on the basis of pH, osmolality and solubility. A wide-range of treatment-levels was used for the first experiment and dose-levels for scoring of chromosomal aberrations were selected on the basis of cytotoxicity indicated by reduction of mitotic index. For each culture, heparinised whole blood was added to culture medium containing a mitogen (phytohaemagglutinin) and incubated at 37 °C in a humidified atmosphere of 5 % CO2/ 95 % air, for 48 hours. The test material was dissolved in DMSO. Without S9 mix 3 μg/mL of mitomycin C was used as the positive control and with S9 mix 50 μg/mL of cyclophosphamide was used.

In the culture medium, the dose-level of 5000 μg/mL showed no precipitate. At this dose-level, the pH and the osmolality were equivalent to those of the vehicle control.

In experiments without S9 mix, the treatment-levels were: 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 μg/mL. In the experiments with S9 mix, the treatment-levels were: 78.125, 156.25, 312.5, 625, 1250, 2500, 3750 and 5000 μg/mL for the first experiment, and 156.25, 312.5, 625, 1250, 2500 and 3750 μg/mL for the second experiment

For the first experiment, lymphocyte cultures were exposed to the test or control materials, with or without S9 mix, for three hours then rinsed. Cells were harvested 20 hours after the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. One and a half hour before harvest, each culture was treated with a colcemid solution (10 μg/mL) to block cells at the metaphase-stage of mitosis. As this experiment gave clear positive results without S9 mix and equivocal results with S9 mix, an additional experiment with S9 mix, was performed.

For the second experiment, cells were exposed to the test or control materials for three 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. 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 the cells were fixed, spread on glass slides and stained.

In experiments without S9 mix, no precipitate was observed at the end of the treatment period at 5000 µg/mL. The test material was completely toxic at the highest dose-level. At the lower dose-levels, the test material was slightly to strongly toxic. The dose-levels selected for analysis of chromosomal aberrations were: 312.5, 625 and 1250 μg/mL. A clear and significant increase in the frequency of cells with chromosomal aberrations, when compared to vehicle control culture, was noted at 625 and 1250 μg/mL. As this first experiment was clearly positive, no other experiment without S9 mix was performed.

In experiments with S9 mix, no precipitate was observed at the end of the treatment period at 5000 μg/mL. In the first experiment, the test material was markedly to strongly toxic at dose-levels ≥2500 µg/mL. In the second experiment, a slight to moderate toxicity was induced at all dose-levels, but without clear evidence of a dose-relationship. Analysis of metaphases was performed at the following dose-levels: 1250, 2500 and 3750 μg/mL for the 20 hour harvest time in both experiments, and 2500 μg/mL for the 44-hour harvest time in the second experiment.

In the first experiment, a significant increase (p<0.05) in the frequency of cells with chromosomal aberrations was noted at 1250 μg/mL but not at the higher dose-levels. In the second experiment, at the 20-hour harvest time, a dose-related but not significant increase in the frequency of cells with chromosomal aberrations when compared to vehicle control cultures was noted at 2500 and 3750 μg/mL but the frequency of cells with chromosomal aberrations was out of the vehicle historical data and the non-significance was attributed to the high vehicle control frequency observed at the 20 hour-harvest time. At the 44-hour harvest time, a significant increase in the frequency of cells with chromosomal aberrations was noted at 2500 μg/mL.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls remained consistent with acceptance criteria. The study was therefore considered valid.

Under the conditions of this study, the test material induced chromosome aberrations in cultured human lymphocytes with and without metabolic activation.

In vivo

Mouse Micronucleus (Molinier, 1993)

The potential for the test material to induce cytogenetic damage to the bone marrow cells of mice was investigated in accordance with the standardised guidelines OECD 474 and EU method B12 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Following a preliminary study to define the dose to be used, the animals of the treated groups received a single administration of the test material at the limit dose of 2000 mg/kg by oral gavage. Two groups each comprising 5 males and 5 females (and one additional group composed of 3 males and 3 females) were dosed. The vehicle was 1 % aqueous methylcellulose and 2 vehicle groups were also treated. The animals of the positive control group (1 group, comprising 5 males and 5 females) received a single administration of cyclophosphamide at 50 mg/kg.

The animals of the treated and vehicle control groups were sacrificed 24 and 48 hours after treatment and the animals of the positive control group were sacrificed 24 hours after treatment. Bone marrow smears were then prepared.

For each animal, the micronuclei were counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

There were no clinical signs of toxicity after treatment with the test material. At the two sampling times, the number of MPE and the PE/NE ratio in mice exposed to the test material did not differ statistically from the concurrent vehicle control values. As no mortality occurred in the study, the supplementary animals were not needed and no smears were prepared for these animals.

In the two vehicle control groups, the mean values of micronucleated polychromatic erythrocytes (MPE) were within the range of the historical data. Cyclophosphamide induced a highly significant increase (p<0.001) in MPE, indicating the sensitivity of the test system under the experimental conditions.

Under the conditions of the study, the test material did not induce cytogenetic damage to the bone marrow cells of mice when treated by the oral route at 2000 mg/kg in the micronucleus test.

Mouse Micronucleus (Haddouk, 2002)

The potential of the test material to induce damage to the chromosomes or the mitotic apparatus in bone marrow cells of mice was investigated in accordance with the standardised guidelines OECD 474 and EU method B12 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Following a preliminary toxicity test, the top dose-level for the cytogenetic test was 2000 mg/kg/day. In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice received two oral treatments of the test material at dose-levels of 500, 1000 or 2000 mg/kg/day, at a 24-hour interval. One group of five males and five females received the vehicle (0.5 % methylcellulose) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test material (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.

The animals of the 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).

For both males and females, the mean values of MPE as well as the PF/NE ratio in the groups treated with the test material were equivalent to those of the vehicle control group. The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data. Cyclophosphamide induced a highly significant increase (p<0.001) in the frequency of MPE, indicating the sensitivity of the test system under the experimental conditions. The study was therefore considered valid.

Under the conditions of this study, the test material did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations with a 24-hour interval at the dose-levels of 500, 1000 or 2000 mg/kg/day.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.