AMES TEST - Aim, Materials & Methods - Greenquat BT
AIM
The purpose of the test is to evaluate the mutagenicity of finished products/raw materials aimed to be used on the skin or on the mucosae, following the OECD Guideline 471 standard.
MATERIALS AND METHODS
Assay system:
Mutant Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 were used.
The bacerial strains were kept frozen at -80°C into cryovials.
One day before the use, the strains were thawed out and transferred into 100 ml of Cultural Medium (Nutrient Broth, Oxoid), and were incubated at 37°C ± 1°C for 15-16 hours to obtain fresh cultures in exponential growth so to expose at mutagenic effects an high number of cells. During the test procedures, bacterial strains were kept on ice to avoid viability reduction.
Reference substances:
The following substances were used as reference mutagens:
– Sodium Azide, (MP Biomedicals, batch. M5284);
– Daunomycin, (MP, batch. SR03054);
– ICR 191 Acridine, (Sigma, batch. SLCH0813);
– Mitomycin C (MMC), (Santa Cruz, batch. C0218);
– 2-Aminoantracene, (Sigma, batch. STBG0630V).
Culture media and reagents:
– Nutrient Broth, (Oxoid, batch. 56059);
– Minimal Glucose Agar plates, (BD, batch. 56739);
– Nutrient Agar plates, (Trinova Biochem, batch. 56798);
– Top agar supplemented with Biotin and Histidine (Trinova Biochem, batch 56716);
– Lyophilized Rat Liver S9, (Moltox, batch. 4409);
– Regensys A, (Moltox, batch 21117RA);
– Regensys B, ((Moltox, batch 08668RB);
– Crystal Violet, (Trinova Biochem);
– ST Quads plates (Trinova Biochem, batch. 57054).
Enzymatic system for metabolism activation:
The enzymatic system for metabolism activation (S9 mix) was prepared at 5% concentration, adding S9 (an hepatic homogenate obtained from the liver of adult male rats which had previously been induced with “aroclor 1254” soybean oil solution) to Regensys A and Regensys B containing respectively phosphate-buffered salt solution and Glucose -6- phosphate and NADP for the activation.
Equipment and substances:
Standard microbiology laboratory equipment and in particular:
Pipettes (Gilson);
Petri Dishes (Trinova Biochem);
Test tubes (VWR);
Laminar-flow hood (Faster);
Vortex Stirrer (Froilabo);
Incubator (Froilabo).
Experimental design:
The product to be tested was diluted in water and the following concentrations were tested: 5 – 2.5 – 1.25 – 0.625 – 0.313 %.
Positive controls were prepared at these concentrations:
Performance of the assay:
For the plate test without metabolic activation, 0.1 ml of assay sample, 0.1 ml of the bacterial cell suspensions and 2 ml of molten top agar (containing Biotin and Histidine) was pipetted in a glass tube, then briefly vortexed and poured into minimal glucose agar plates. Negative controls, solvent controls and positive controls were also prepared. The plates were then incubated at 37 ±1°C for at least 48 hours. After the incubation, the reverted colonies of the assay sample at different concentrations, as well as those of the negative controls (with and without solvent) ad positive controls, were counted in each plate. Two replicates were performed for each sample.
For the plate test with metabolic activation, 0.1 ml of assay sample, 0.1 ml of the bacterial cell suspensions and 0.5 ml of the enzymatic system for metabolism activation was added to aliquoted top agar (containing Biotin and Histidine) in a glass tube, then briefly vortexed and poured into minimal glucose agar plates. Negative controls, solvent controls and positive controls were also prepared. The plates were then incubated at 37 ±1°C for at least 48 hours. After the incubation, the reverted colonies of the assay sample at different concentrations, as well as those of the negative controls (with and without solvent) ad positive controls, were counted in each plate. Two replicates were performed for each sample.
