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Bacterial Mutation Assay



The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial ReverseMutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA(TSCA) OPPTS harmonised guidelines.


Salmonella typhimurium strains TA1535, TAl537, TA98, TAl00 and Escherichia coli strain WP2uvrA- were treated with the test material using the Ames plate incorporation method at a maximum of six dose levels, in triplicate, both with and without the addition of a rat liverhomogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate in the first experiment.The experiment was repeated on a separate day using an amended dose range of 15 to 5000 ug/plate, fresh cultures of the bacterial strains and fresh test material formulations. An additional dose level was included in the second experiment to allow for test material inducedtoxicity, ensuring that a minimum offour non-toxic dose levels were achieved. A third, confirmatory experiment was performed using tester strain TA1535 (without S9 only) in an effort to confirm both reproducibility and a dose-response relationship. A dose range of 300,500, 750, 1000 and 1500 ug/plate was employed using the plate incorporation method.


The vehicle (acetone) 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 thefrequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn of all the tester strains at 5000 ug/plate both with and without S9. A slight weakening in the integrity of the background lawn of TAl535 was noted at 1500 ug/plate (without S9 only). This response was slightly inconsistent however, as it was only observed in two out of three experiments. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/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 toxicologically 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. Statistically significant, and moderately reproducible increases in revertant colony frequency were observed in tester strain TA1535, without S9 only, at the upper sub-toxic dose levels of the test material in Experiments 1 and 2.Therefore, a third, confirmatory experiment was performed,using the plate incorporation method. The third experiment was designed to enhance the weak dose-response relationship observed in the previous experiments. Small but statistically significant increases in revertant colony frequency were observed at only one dose level (1500 ugplate). However, in all three experiments, the increases never exceeded 1.75 times the concurrent solvent control and two of the statistically significant responses (1500 ug/plate in Experiments 1 and 2)were accompanied by weakened bacterial background lawns. Furthermore,there was no clear evidence of a dose-response relationship, even after the inclusion ofa tightened dose range in Experiment 3. Therefore, the test material was considered not to be causing a mutagenic response.A statistically significant response was also noted in TA98, in the presence of S9 only, at 5000 pg/plate. This response was discounted however because it was non-reproducible and was accompanied by a weakened bacterial background lawn.


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


Mammalian Cell Chromosome Aberration Test



This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott etal,1990). The method used followed that described in the OECD Guidelinesfor Testing of Chemicals (1997) No.473 "Genetic Toxicology: Chromosome Aberration Test"and Method B1 of Commission Directive 2000/32/EC. The study design also meets therequirements of the UK Department of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals.


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, ie Experiment1, 4 hours in the presence of an induced rat liver homogenate metabolising system (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. A third experiment was also conducted using the same exposure conditions as Experiment 1 in the absence of S9 only.


All vehicle (solvent) 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 frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.The test material was toxic but did not induce any toxicologically significant, increases in the frequency of cells with aberrations, in any of four exposure conditions, using a dose range that included a dose level that induced approximately 50% mitotic inhibition.


The test material was considered to be non-clastogenic to human lymphocytes in vitro.


Micronucleus test in the mouse



The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed tocomply with the 1997 OECD Guidelines for Testing of Chemicals No.474"Micronucleus Test", Method B12 of the EC Commission Directive 2000/32/EC, the USA EPA, TSCA and FIFRA guidelines and the Japanese METL'MHLW guidelines for testing of new chemical substances.


The range-finding test was performed to find suitable dose levels of the test material, route of administration and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes; therefore the main study was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) 1500 mgkg with 750 and 375 mgkg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow was extracted, and smear preparations made and stained.

 Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single oral dose of dried arachis oil (7mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.


A statistically significant decrease in the PCE/NCE ratio was observed in the 48-hour 1500 mg/kg test material dose group when compared to the concurrent control group. This and the presence of clinical signs were taken to indicate that systemic absorption had occurred and exposure of the target tissue achieved.There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups.The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditionsof the test.


The test material was considered to be non-genotoxic under the conditions of the test.

Justification for selection of genetic toxicity endpoint
Two in vitro assays, an Ames test and a chromosome aberration test both showed negative results, both in the presence and absence of a metabolising activation system (S9). In addition, an in vivo micronucleus assay in the mouse also showed negative results.

Short description of key information:
Three key, OECD compliant GLP studies that include a bacterial gene mutation assay, an in vitro mammalian cells assay for chromosome aberrations and and in vivo micronucleus assay in the mouse.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for mutagenicity according to the Regulation (EC) No. 1272/2008..

Self classification:

Based on the available data, no additional classification for mutagenicity is proposed according to Regulation (EC) No. 1272/2008 (CLP).