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EC number: 700-579-6 | CAS number: -
- S9 mix 10%
+ S9 mix 10%
+ S9 mix 10% and pre-incubation
R = (Number of revertants colonies wiith the test material) / (Number of revertant colonies without the test material)
The search for any mutagenic activity of Sepisol Fast Blue 85219, was studied by means of the Ames test (Salmonella his- and E.coli Trp-/microsome system) in compliance with the OECD Guideline 471 using the maximum concentration recommended by OECD Guideline, i.e. 5000 μg/plate for the toxicity assay. Four lower dilutions chosen according to a geometrical (half-log) ratio were also tested. In the main assay, the maximum dose was chosen in accordance with the recommendations of OECD Guideline, i.e. 5000μg/plate.
Preparation of test material solution
The test material (Sepisol Fast Blue 85219) is diluted in ethanol so as to prepare a stock solution of 100 mg/L.
Preliminary assay: Cytotoxicity
Five concentrations have been tested (50; 150; 500; 1500 and 5000 µg/plate). 0.1 mL of a bacterial suspension from a culture containing 1 to 5 x 109bacteria/mL and 0.1 mL of the test item at the relevant concentration were successively added to 2 mL of top agar to which 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar and then incubated for 24 or 48 hours at 37 °C (n=3). Colonies are counted. A negative sample with pure solvent is run the same way.
- Without metabolic activation:
The following technic was performed for each one of the Salmonella strains used in the test: 0.1 mL of a bacterial suspension containing 1 to 5 x 109bacteria/mL and 0.1 mL of the test item at the relevant concentration were successively added to 2 mL of top agar to which 10 % of 0.5 mM biotin histidine solution, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar.
For E.Coli the following technic was applied: 0.1 mL of a bacterial suspension containing 1 to 5 x 109bacteria/mL and 0.1 mL of the test item at the relevant concentration were successively added to 2 mL of top agar to which 5 % (v/v) of Nutrient broth n°2 and an extra 5µL of a L-Tryptophan solution at 2 mg/mL, maintained in a state of superfusion at 45°C, has been added. The content of each tube was agitated, then spread out in a Petri plate containing 20 mL of minimum agar.
Three plates were used per treatment. The plates were then incubated at 37°C for 48 h approximately. Finally, colonies of revertants were counted for each plate.
- With metabolic activation :
Two techniques have been used. The first method (plate incorporation) was the same as the one described above except that immediately before spreading in the plates, 0.5 mL of the S9 mix metabolic activation system was added in soft agar. The other method: Pre-incubation, in which the mixture of bacteria, the test material, and the S9 fractioin is first incubated at 37 °C during at least 20 minutes before to be added to the top agar.
For the first essay, incorporation plate method have been used. The pre-incubation method have beee used for the second assay with S9 mix.
Solvent controls, positive controls and assay for the control of media sterility were performed like in the mutagenicity assay without metabolic activation.
Cytotoxic rate of 50,5%, 23,5 % and 13,5% were observed with respectively 5000, 1500 and 500 µg/plate of test material. Those rates stand below the tolerated threshold of 75%. No cytotoxic effect observed for the following doses: 150 et 50 µg/plate.
A significative decrease of the number of revertants colonies is observed for the dose 5000 µg/plate.This finding must be correlated with the high toxicity measured at this concentration.
The mutagenic activity of the test item was assessed by means of the Ames’s test in the four Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and in the E. Coli WP2 (uvr A-) (pKM 101) tested either in presence or in absence of metabolic activation, in two independent assays. No mutagenic activity was found either with or without metabolic activation in any of the 5 strains. Values fall within the range of historical values observed at the facility.
Under the experimental conditions described, the test material Sepisol Fast Blue 85219 did not show any mutagenic activity regarding 4 strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and Escherichia Coli WP2 (uvr A-) (pKM 101) with and without metabolic actvation.
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