Appendix B: Toxicological Investigations Acute toxicity – oral Notified chemical OECD TG 423 Acute Oral Toxicity – Acute Toxic Class Method. Species/Strain Rat/Wistar Vehicle Propylene glycol Remarks - Method No significant protocol deviations were noted. The test substance contains > 10% impurity and no correction was made for the purity. The substance was mixed with the vehicle, heated in a water bath at a maximum temperature of 96 C for 1 h to obtain homogeneity and dosed at ≤ 40 C. Group Number and Sex of Animals Dose mg/kg bw Mortality 1 3F 2000 0/3 2 3F 2000 0/3 LD50 > 2000 mg/kg bw Signs of Toxicity Clinical signs including lethargy, hunched posture and/or piloerection were noted for all animals on Days 1 and/or 2. Effects in Organs No abnormalities were noted at macroscopic post mortem examination. Remarks - Results All animals showed expected body weight development. The notified chemical is of low toxicity via the oral route. WIL (2015a) Acute toxicity – dermal Notified chemical OECD TG 402 Acute Dermal Toxicity – Limit Test. Species/Strain Rat/Wistar Vehicle Propylene glycol Type of dressing Occlusive Remarks - Method No significant protocol deviations were noted. The test substance contains > 10% impurities and no correction was made for the purity. The substance was mixed with the vehicle, heated to a maximum of 93 C for 1 h to obtain visual homogeneity and dosed at < 40 C. Group Number and Sex of Animals Dose mg/kg bw Mortality 1 5M 2000 0/5 2 5F 2000 0/5 LD50 > 2000 mg/kg bw Signs of Toxicity - Local Erythema, fissuring, scales and/or scabs were noted on the treated skin area and/or left flank of all animals. In addition, necrosis of the treated skin area and/or left flank was noted on Days 4-8 for all female animals, with one of them having a wound on the left flank on Days 7 and 8 and scar formation on Days 14 and 15. Signs of Toxicity - Systemic None reported Effects in Organs No abnormalities were noted at macroscopic post mortem examination. Remarks - Results All animals showed expected body weight development.
the validity of the test system. The notified chemical was not mutagenic to bacteria under the conditions of the test. WIL (2014d) Genotoxicity – in vitro Notified chemical OECD TG 473 In vitro Mammalian Chromosome Aberration Test. Species/Strain Human peripheral lymphocytes Cell Type/Cell Line Lymphocytes Metabolic Activation System S9 mix prepared from phenobarbital/β-naphthoflavone induced rat liver Vehicle Dimethyl sulfoxide (DMSO) Remarks - Method The test substance contains > 10% impurities and no correction was made for the purity/composition. A dose range-finding study was carried out at 5.4-512 μg/mL (3 h exposure) and 1.7-1600 μg/mL (24 h or 48 h exposure). The dose selection for the main experiments was based on the solubility and results from the preliminary study. Vehicle and positive controls (mitomycin C and cyclophosphamide) were run concurrently with the notified chemical. Metabolic Activation Test Substance Concentration (μg/mL) Exposure Period Harvest Time Absent Test 1 52*, 164*, 512* 3 h 24 h Test 2 50, 75*, 100*, 125*, 150, 175, 200 24 h 24 h Test 3 50*, 75*, 100*, 125, 150, 175, 200 48 h 48 h Present Test 1 52*, 164*, 512* 3 h 24 h *Cultures selected for metaphase analysis. Metabolic Activation Test Substance Concentration (µg/mL) Resulting in: Cytotoxicity in Preliminary Test Cytotoxicity in Main Test Precipitation Genotoxic Effect Absent Test 1 > 512 > 512 ≥ 512 negative Test 2 ≥ 164 ≥ 125 ≥ 200 negative Test 3 ≥ 164 ≥ 100 ≥ 200 negative Present Test 1 > 512 > 512 ≥ 512 negative Remarks - Results In the tests, no effect of the test substance on the number of polyploidy cells and cells with endoreduplicated chromosomes were noted in the presence or absence of metabolic activation. The notified chemical was not clastogenic to human lymphocytes treated in vitro under the conditions of the test. WIL (2014e)