Appendix B: Toxicological Investigations Acute toxicity oral – RUCO-GUARD AFB6 CONC (LTD/1837) OECD TG 423 Acute Oral Toxicity Acute Toxic Class Method. – EC Council Regulation No 440/2008 B.1 tris Acute Oral Toxicity Acute – Toxic Class Method. Species/Strain Rat/Wistar (RccHan™:WIST) Vehicle Arachis oil BP Remarks - Method No protocol deviations Group Number and Sex Dose Mortality of Animals mg/kg bw 1 3 F 2,000 0/3 2 3 F 2,000 0/3 LD50 > 2,000 mg/kg bw Signs of Toxicity No signs of systemic toxicity were noted. Effects in Organs No abnormalities were noted at necropsy. Remarks - Results All animals showed expected bodyweight gain. The notified polymer is of low toxicity via the oral route. Harlan (2013a) Genotoxicity bacteria – RUCO-GUARD AFB6 CONC (LTD/1837) OECD TG 471 Bacterial Reverse Mutation Test. EC Directive 440/2008 B.13/14 Mutagenicity Reverse Mutation Test – using Bacteria. Test 1: Plate incorporation procedure/Test 2: Pre incubation procedure Species/Strain TA1535, TA1537, TA98, TA100 S. typhimurium: WP2uvrA E. coli: Metabolic Activation System S9 fraction from induced rat liver Phenobarbitone/β-Naphthoflavone Concentration Range in Test 1: with and without metabolic activation: 0, 3, 10, 33, 100, 333, Main Test 1,000, 2,500 and 5,000 µg/plate Test 2: with and without metabolic activation: 0, 10, 33, 100, 333, 1,000, 2,500 and 5,000 µg/plate Vehicle DMSO (at ratio 1:1) Remarks - Method No protocol deviations Metabolic Test Substance Concentration (µg/plate) Resulting in: Activation Cytotoxicity in Main Test Precipitation Genotoxic Effect Absent Test 1 > 5,000 1,000 negative ≥ Test 2 > 5,000 1,000 negative ≥ Present Test 1 > 5,000 1,000 negative ≥ Test 2 > 5,000 2,500 negative ≥ Remarks - Results No visible reduction in the growth of the bacterial background lawn was caused by the test substance at any dose level. Precipitation of the test
Appendix B: Toxicological Investigations Acute toxicity – oral RUCO-GUARD AFB6 CONC (LTD/1837) OECD TG 423 Acute Oral Toxicity – Acute Toxic Class Method. EC Council Regulation No 440/2008 B.1 tris Acute Oral Toxicity – Acute Toxic Class Method. Species/Strain Rat/Wistar (RccHan™:WIST) Vehicle Arachis oil BP Remarks - Method No protocol deviations Group Number and Sex of Animals Dose mg/kg bw Mortality 1 3 F 2,000 0/3 2 3 F 2,000 0/3 LD50 > 2,000 mg/kg bw Signs of Toxicity No signs of systemic toxicity were noted. Effects in Organs No abnormalities were noted at necropsy. Remarks - Results All animals showed expected bodyweight gain. The notified polymer is of low toxicity via the oral route. Harlan (2013a) Genotoxicity – bacteria RUCO-GUARD AFB6 CONC (LTD/1837) OECD TG 471 Bacterial Reverse Mutation Test. EC Directive 440/2008 B.13/14 Mutagenicity – Reverse Mutation Test using Bacteria. Test 1: Plate incorporation procedure/Test 2: Pre incubation procedure Species/Strain S. typhimurium: TA1535, TA1537, TA98, TA100 E. coli: WP2uvrA Metabolic Activation System S9 fraction from Phenobarbitone/β-Naphthoflavone induced rat liver Concentration Range in Main Test Test 1: with and without metabolic activation: 0, 3, 10, 33, 100, 333, 1,000, 2,500 and 5,000 µg/plate Test 2: with and without metabolic activation: 0, 10, 33, 100, 333, 1,000, 2,500 and 5,000 µg/plate Vehicle DMSO (at ratio 1:1) Remarks - Method No protocol deviations Metabolic Activation Test Substance Concentration (µg/plate) Resulting in: Cytotoxicity in Main Test Precipitation Genotoxic Effect Absent Test 1 > 5,000 ≥ 1,000 negative Test 2 > 5,000 ≥ 1,000 negative Present Test 1 > 5,000 ≥ 1,000 negative Test 2 > 5,000 ≥ 2,500 negative Remarks - Results No visible reduction in the growth of the bacterial background lawn was caused by the test substance at any dose level. Precipitation of the test