OECD SIDS 5. TOXICITY
ID: 79-50-5 DATE: 18.01.2006
Critical study for SIDS endpoint
5.5 Genetic Toxicity 'in Vitro'
Type: System of testing:
Ames test Salmonella typhimurium, strains TA97, TA98, TA100, TA102, TA1535
(control), 50, 158.1, 500, 1581 and 5000 µg/plate
Cytotoxic Concentration: >5000 µg/plate
with and without
Method: Year: GLP:
OECD Guide-line 471 1999 yes
as prescribed by 1.1 - 1.4
Strains Salmonella typhimurium strains TA1535, TA97, TA98, TA100 and TA102 were obtained from BN Ames. Nutrient broth cultures of each strain, supplemented with 9% DMSO, were stored in liquid nitrogen. Strain identities and characteristics were periodically checked by recommended procedures (full details in report). For use in tests, cultures of the strains were grown overnight at 37°C in a shaking water bath in a nutrient broth liquid medium (described in full detail including sources of chemicals in the report). The growth of overnight cultures was controlled by measuring the optical density on a photometer at 650 nm. Each bacterial strain was diluted 10E-6 in 0.85% NaCl, 100 µl of the last dilution step was plated on a nutrient broth complete medium (details in report). Two replicate plates were incubated at 37°C, upside down, for 2 days. The number of colonies was registered and the number of cells plated on Vogel-Bronner minimal medium (full details in report) was calculated. The sensitivity of the S. typhimurium strains was verified using the following positive controls: sodium azide with TA1535 and TA100, ICR191 with TA97, 2-nitrofluorene with TA98 and Mitomycin C with TA102. Moreover, 2-aminoanthracene was used with all strains with and without metabolic activation to examine the activity of the S9 mix; S9 from Molecular Toxicology, Boone NC, USA (all chemicals and S9 fully detailed in report). A toxicity prescreen with plate incorporation and TA100 in duplicate was negative up to 5000 µg/plate and led to the selection of the tested doses as listed. Standard Ames procedure Test tubes containing 2 ml of 0.7% agar medium were autoclaved and kept in a prewarmed bath at 42-45°C; the following solutions were added in order. 0.2 ml of histidine/biotin mixture corresponding to 21 µg L-histidine and 24.4 µg biotin; 0.1 ml of test compound at different concentrations or of the solvent or 0.05 ml of the different reference substances;
ml of overnight cultures of the bacterial strain;
ml of S9 mix or, for tubes without metabolic activation,
ml sodium-phosphate-buffered saline at 0.2M, pH7.4.