Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

IN VITRO BACTERIAL REVERSE MUTATION ASSAY

Negative in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA with and without metabolic activation, OECD 471, EU Method B.13/14, US EPA OPPTS 870.5100 and the Japanese guidelines published by METI, MHLW and MAFF

IN VITRO MAMMALIAN CHROMOSOME ABERRATION

Negative in human lymphocytes with and without metabolic activation, OECD 473, EU Method B.10, EPA OPPTS 870.5375, 40 CFR 799.9537 TSCA and the Japanese guidelines published by METI, MHLW and MAFF

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Two bacterial reverse mutation assays are available; one was conducted on commercially purchased material and the other was conducted using material manufactured by the registrant to confirm the parity of the two materials. On this basis, the study performed using the material manufactured by the registrant was selected as key.

In the key bacterial reverse mutation assay conducted with the material produced by the registrant, the potential of the test material to induce genetic toxicity was investigated in a study conducted in accordance with the standardised guidelines OECD 471, EU Method B.13/14, US EPA OPPTS 870.5100 and the Japanese guidelines published by METI, MHLW and MAFF under GLP conditions.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test material in acetone using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the range-finding test was predetermined and was 1.5 to 5000 µg/plate. The main experiment was conducted on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended following the results of the range-finding test and was 5 to 5000 µg/plate; seven test material dose levels were selected in order to achieve both four non-toxic dose levels and the potential toxic limit of the test material following the change in test methodology.

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Range-finding experiment (plate incorporation method) results showed that the test material induced toxicity as weakened bacterial background lawns to all of the Salmonella strains at 5000 µg/plate in both the presence and absence of metabolic activation. No toxicity was noted to Escherichia coli strain WP2uvrA. These results were not indicative of toxicity sufficiently severe to prevent the test material being tested up to the maximum recommended dose level in the main test.

 Main experiment results (pre-incubation method) showed that the test material induced toxicity as weakened bacterial background lawns to all of the Salmonella strains in the absence of S9-mix at and above 1500 µg/plate and to all of the Salmonella strains and WP2uvrA in both the presence and absence of S9-mix at 5000 µg/plate. A slight test material precipitate (particulate in appearance) was observed under an inverted microscope at 5000 µg/plate, though this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

The test material was therefore considered to be non-mutagenic under the conditions of this test.

In the supporting bacterial reverse mutation assay conducted with the commercially purchased material, the mutagenicity of the test material was assessed in a bacterial reverse mutation assay (Ames test) in accordance with standardised guidelines OECD guideline 471 and EU Method B.13/14 under conditions of GLP.

During the study, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using the plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for the study was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Under the conditions of the study, the vehicle control plates gave counts of revertant colonies generally within the normal range. All positive controls used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Therefore, the test material was concluded to be non-mutagenic under the conditions of the test.

An in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells was conducted in accordance with the standardised guidelines OECD 473, EU Method B.10, EPA OPPTS 870.5375, 40 CFR 799.9537 TSCA and the Japanese guidelines published by METI, MHLW and MAFF under GLP conditions.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study; in Experiment 1, 4 hours in the presence of metabolic activation (S9) at a 2 % final concentration with cell harvest after a 20-hour expression period, and a 4 hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hour exposure with addition of S9 was repeated (using a 1 % final S9 concentration) whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

The dose levels used in the main experiments were selected using data from the preliminary toxicity test and were as follows: 0, 43.5, 87, 174, 348, 696 and 1392 µg/mL. The vehicle used was DMSO.

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequencies of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50 % mitotic inhibition, in either the absence or presence of a liver enzyme metabolising system.

Under the conditions of this study, the test material is considered to be non-clastogenic to human lymphocytes in vitro.

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.

In accordance with the criteria for classification as defined in Annex VI, Directive 67/548/EEC (DSD), the substance does not require classification with respect to genetic toxicity.