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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 July - 13 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
White solid

Method

Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102, TA 1535
Metabolic activation:
with and without
Metabolic activation system:
In this study, Aroclor 1254 induced rat liver S9 prepared in-house was used as the metabolic activation system.
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, and 1500 ug/plate. In the first experiment, the cytotoxicity was only observed at 1500 ug/plate to all tester strains without metaboli activation system, and at 1500 ug/plae to some tester strains with metabolic activation system. In the validation experiement the same results were obtained as in the first experiment.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
other: Dexon, 2-Aminofluorene, 1,8-Dihydroxyanthraquinone (Dorbane), 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The test system was exposed to the test article via the standard plate incorporation method and the pre-incubation method at six dose levels, in triplicate, with positive controls and solvent controls, both in the presence and absence of the cofactor-supplemented S9 mix. Based on the results of the preliminary test, six dose levels were selected.

PRELIMINARY TEST:
A preliminary test was performed and DMSO used as a solvent. The standard plate incororation method was performed at 3 dose levels with 4 intervals between test point, including 5000, 1250, and 312.5 ug/plate in 5 tester strains of TA 97a, TA 98, TA 100, TA 102, and TA 1535 with and without metabolic activation system.

THE FIRST EXPERIMENT:
Dose selection was based on the results from the preliminary test and the related requirements of the guidelines. In the first experiment 6 dose levels 1500, 500, 150, 50, 15, and 5 ug/plate were used.

DURATION:
Enrichment culture: One day prior to the plate-incorporation, the stored tester strains were thawed and 100 ul of bacterium suspension was cultivated in nutrient broth medium for approximately 16 hours at 36.1-37.0 degrees C.
Plate incorporation: After the top layer agar was solidified, the plates were inverted and incubated for approximately 48 hours at 36.2-37.7 degrees C.

SELECTION AGENT (mutation assays): histidine and biotin solution

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
Method: When being observed in the absense of S9 mix, three plates of each dose group of TA 98 were selected to watch by naked eye for the test item precipitation both before and after incubation. The number of revertant colonies in each plate was counted, and the background lawns were observed microscopically. When counting the plates of the positive controls, two diagonal sectors were chosen randomly and counted, then the result was multiplied by 4 to determine the number of revertant colonies. TA 1535, was divided into eight sectors on the back. In the presence of S9 mix all procedures were the same as in the absence of S9 mix ecept for PBS replaced with S9 mix.
Evaluation criteria:
CRITERIA OF A POSITIVE RESULT:
1.) When there is a concentration-related increased over the rage in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.
2.) When there is a reproducible increase at one or more concentrations in the mean number of revertant colonies in at least one strain with or without metabolic activation system, the test item should be evaluated as positive.

CRITERIA OF A NEGATIVE RESULT:
The test item should be evaluated as negative if none of the above criteria is met.

CRITERIA OF EQUIVOCAL RESULT:
Although most tests give a clear positive or negative result, some data generated prohibit making a definite judgement about the test item. Results of this type should be reported as equivocal.
Statistics:
For three plates at each dose and control group, the mean number and the standard deviation of the number of revertant colonies was determined. Additionally, the ratio of the mean number of revertant colonies between each group and the concurrent solvent control was calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA97a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Positive controls validity:
valid
Additional information on results:
Preliminary test results: Monitoring of TA 98 indicated that there was no precipitation at all doses and groups before and after the incubation, with and without metabolic activation system. When comparing the concurrent solvent controls, the dose-related cytotoxicity was observed at 5000 and 1250 ug/plate for all tester strains under two treatment conditions of the test. In the first experiment, the mean number of revertant colonies at each analyzable dose level in all tester strains was two times less than that of hte concurrent solvent controls in TA 97a, TA 98, TA 100, TA 102, and three times less than that of the concurrent solvent controls in TA 1535. Additionally, in the first experiment, the cytotoxicity was observed at 1500 ug/plate doses. The numner of revertant colonies were less and/or the background lawn was thinner than the concurrent solvent control.

The results of viable count in the two experiments showed that the density of the cultures for each tester strain was withinthe acceptable range of 0.9-9 x 10^9 CFU/ml. In both the first experiment and the validation experiment, the mean number of revertant colonies in the solvent controls and positive controls were within the range of the historical control data in the lab.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, results from both the first experiment and validation experiment were negative. Therefore, Difluorosulfonamide HCl is considered to be non-mutagenic in the bacterial reverse mutation assay using histidine deficient test strains of Salmonella typhimurium.