Appendix B: Toxicological Investigations Genotoxicity bacteria – Notified polymer (< 60%) OECD TG 471 Bacterial Reverse Mutation Test. EC Directive 2000/32/EC B.13/14 Mutagenicity Reverse Mutation Test – using Bacteria. Plate incorporation procedure (pre-experiment and test 1)/Pre incubation procedure (test 2) Species/Strain TA1535, TA1537, TA98, TA100 S. typhimurium: WP2uvrA E. coli: Metabolic Activation System S9 fraction from induced rat liver phenobarbital/β-naphthoflavone in Pre-experiment/test 1 with and without metabolic activation: 0, 3, 10, 33, Main Test 100, 333, 1000, 2500 and 5000 µg/plate Test 2 with and without metabolic activation: 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate Vehicle Deionised water Remarks - Method No deviations from the protocol. Metabolic Test Substance Concentration (µg/plate) Resulting in: Activation Cytotoxicity in Cytotoxicity in Precipitation Genotoxic Effect Test Main Test > 5,000 Absent Test 1 > 5,000 > 5,000 negative Test 2 > 5,000 > 5,000 negative > 5,000 Present Test 1 > 5,000 > 5,000 negative Test 2 > 5,000 > 5,000 negative Remarks - Results The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in both main experiments. No toxic effects, shown as a reduction in the number of revertants, occurred in the test groups with and without S9 mix. No substantial increase in revertant colony numbers of any of the five test strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the border of biological relevance. The positive controls produced satisfactory responses, thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. The test substance was not mutagenic to bacteria under the conditions of the test. RCC (2006)
Appendix B: Toxicological Investigations Genotoxicity – bacteria Notified polymer (< 60%) OECD TG 471 Bacterial Reverse Mutation Test. EC Directive 2000/32/EC B.13/14 Mutagenicity – Reverse Mutation Test using Bacteria. Plate incorporation procedure (pre-experiment and test 1)/Pre incubation procedure (test 2) Species/Strain S. typhimurium: TA1535, TA1537, TA98, TA100 E. coli: WP2uvrA Metabolic Activation System S9 fraction from phenobarbital/β-naphthoflavone induced rat liver in Main Test Pre-experiment/test 1 with and without metabolic activation: 0, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate Test 2 with and without metabolic activation: 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate Vehicle Deionised water Remarks - Method No deviations from the protocol. Metabolic Activation Test Substance Concentration (µg/plate) Resulting in: Cytotoxicity in Test Cytotoxicity in Main Test Precipitation Genotoxic Effect Absent > 5,000 Test 1 > 5,000 > 5,000 negative Test 2 > 5,000 > 5,000 negative Present > 5,000 Test 1 > 5,000 > 5,000 negative Test 2 > 5,000 > 5,000 negative Remarks - Results The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in both main experiments. No toxic effects, shown as a reduction in the number of revertants, occurred in the test groups with and without S9 mix. No substantial increase in revertant colony numbers of any of the five test strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of S9 mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the border of biological relevance. The positive controls produced satisfactory responses, thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. The test substance was not mutagenic to bacteria under the conditions of the test. RCC (2006)