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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19th July 2013 to 12nd August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
EC Number:
294-430-8
EC Name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
Cas Number:
91722-28-0
IUPAC Name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
Test material form:
liquid
Details on test material:
Name of test material (as cited in study report):Internal Olefin Sulfonate (IOS)

Method

Target gene:
S. typhimurium TA98; hisD 3052_GC rfa uvrB pKM101c
S. typhimurium TA100; hisG 46_GC rfa uvrB pKM101
S. typhimurium TA1535; hisG 46_GC rfa uvrB
S. typhimurium TA1537; hisC 3076_GC rfa uvrB
E. coli WP2uvrA; trpE65_AT rfa uvrA
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
For the tests without metabolic activation, 0.5 mL of 0.1 M Na-phosphate buffer (pH 7.4) and 0.1 mL of each fresh bacterial culture were added to each tube containing 0.1 mL of the test solution. For the tests with metabolic activation, 0.5 mL of the S9 mix was added to each tube instead of the 0.1 M Na-phosphate buffer. The mixture was pre-incubated in a water bath at 37℃ for 20 minutes while shaking horizontally, and then 2.0 mL of top agar were added to the mixture, and the contents of each tube were poured over the surface of the minimal glucose agar plate. For the sterility test, 0.1 mL of the test solution of the highest dose and 0.5 mL of the S9 mix were put into each tube, 2.0 mL of top agar were then added to the tube, and the contents of each tube were poured over the surface of the minimal glucose agar plate. These operations were conducted under lamps with ultraviolet absorbent filter.All plates were incubated at 37℃ for 48 hours, and the number of revertant colonies was counted. Afterwards, growth inhibition of the test strains was checked using a stereoscopic microscope.
As top agar, a soft agar solution (0.6% Agar and 0.5% NaCl) with 1/10 by volume of the solution of 0.5 mM biotin-0.5 mM L-histidine and the same ratio of 0.5 mM L-tryptophan were used for the S. typhimurium TA strains and the E. coli strain, respectively. One minimal glucose agar plate was used for each dose level in the preliminary test and three minimal glucose agar plates were used for each dose level in two main tests which were performed at the same doses.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water for injection (The Japanese Pharmacopoeia)
- Justification for choice of solvent/vehicle: From the result of the solubility test, the test substance was dissolved at 50 mg/mL in water, and neither exothermic reaction nor generation of gas was observed. Therefore water for injection was used as solvent for preparation.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl, 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation
DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
Evaluation criteria:
In the two main tests, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive. The results at each concentration were demonstrated with the mean and the standard deviation.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
It is concluded that Internal Olefin Sulfonate is not mutagenic in the bacterial reverse mutation assay carried out under the experimental conditions.
Executive summary:

In the two main tests, neither an increase in the number of revertant colonies (more than twice as many as that of the negative control) nor a dose-related response was observed at any doses in any strains of base-pair substitution type or frame-shift type, with or without metabolic activation. The revertant colonies of the positive controls showed an increase of more than twice that of the negative controls and they were within limit of controls (mean±3SD) in historical data, indicating that this study was performed correctly. From these results, mutagenicity of the test substance was judged negative.

The growth inhibition of the test strains by the test substance was observed at some doses. And the precipitate of the test substance on the plates was not observed either with or without metabolic activation. In the sterility test on the test solution and the S9 mix, no growth of bacteria was observed.