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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

From the results of this study, it is concluded that the reaction mass is non-mutagenic to any of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified experimental conditions.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 16, 2018-October 12, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD 471, Bacterial Reverse Mutation Test, adopted by the Council on July 21, 1997.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
The Salmonella typhimurium strains used in this study were mutants derived from Salmonella typhimurium LT2. The strains used in the study were obtained from Molecular Toxicology Inc., 157 Industrial Park Dr. Boone, NC 28607, U.S.A.
Metabolic activation:
with and without
Metabolic activation system:
S9 Fraction and S9 Mix
Test concentrations with justification for top dose:
Tester strains were exposed to test concentrations of 78.125, 156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate of test item both in the absence and presence of metabolic activation (10 % v/v S9 mix).
Vehicle / solvent:
Dimethyl Sulfoxide (Stock, 50000 µg/mL)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
mitomycin C
other: 2-nitrofluorene for TA98
Details on test system and experimental conditions:
This bacterial reverse mutation test was performed to evaluate mutagenic potential of test itemI using Salmonella typhimurium tester strains TA1537, TA1535, TA98, TA100 and TA102 with and without metabolic activation system. The study was conducted in compliance with the OECD Principles of GLP (1998).
Rationale for test conditions:
The present study was conducted according to:
OECD, 1997: The Organisation for Economic Co operation and Development (OECD) Guidelines for the Testing of Chemicals, OECD 471, Bacterial Reverse Mutation Test, adopted by the Council on July 21, 1997. This assay measures the ability of the test item to induce reverse mutations at specific histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100 and TA102, which are known for their reliability and reproducibility in a short term mutagenicity assay and are also recommended by the OECD and other guidelines.
Evaluation criteria:
A result is considered positive if concentration-related increase over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
Strains TA1535, TA1537
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.
Strains TA98, TA100 and TA102
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.
Statistical analysis was used as an aid in the evaluation of dose response.
A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing.
Statistics:
Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose level > 1500 µg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose level > 1500 µg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose level > 1500 µg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose level > 1500 µg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose level > 1500 µg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Confirmatory Mutation Test
Normal background lawn pattern without reduction in revertant colonies was observed up to the tested concentration of 2500 µg/plate in the absence and presence of the metabolic activation system (10% v/v S9 mix), respectively, in the all tester strains. No increase in the number of revertant colonies was observed both in the absence and presence of the metabolic activation system (10% v/v S9 mix) in all the tester strains. Normal bacterial background lawn with 78-89% reduction in number of revertant colonies in absence of S9 and approximately 75-88% reduction in number of revertant colonies in presence of metabolic activation (10% v/v S9 mix) was observed at the tested concertation of 5000 µg/plate in all tester strains
Results revealed that there was no positive mutagenic effect in tester strains TA1537, TA1535, TA98, TA100 and TA102 at the tested concentrations of 78.125, 156.25, 312.5, 625, 1250, 2500, and 5000 µg/plate both in the absence and presence of metabolic activation system (10% v/v S9 mix), when compared with the concurrent negative control. Statistical analysis did not reveal any significant effect.
Conclusions:
From the results of this study, it is concluded that the test item is non-mutagenic to any of the five strains of Salmonella typhimurium viz., TA1537, TA1535, TA98, TA100 and TA102 when tested under the specified experimental conditions.
Executive summary:

This study was performed to evaluate mutagenic activity of the reaction mass by the bacterial reverse mutation test, using five histidine deficient mutant tester strains of Salmonella typhimurium (i.e., TA1537, TA1535, TA98, TA100 and TA102).

The substance was tested in two independent experiments, in the absence and presence of metabolic activation. Bacterial cultures were exposed totest itemat 8 concentrations (two plates/concentration) between 1.5 to 5000 µg/plate in the initial toxicity-mutation test. Normal background lawn pattern was observed up to the tested concentration of 5000 µg/plate in all tester strains (TA1537, TA1535, TA98, TA100 and TA102), no increase in the number of revertant colonies (no mutagenic effect) was observed both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in all the tester strains. To confirm the negative results obtained in the initial toxicity mutation test, confirmatory mutation test was conducted with an increased S9 concentration i.e., 10% v/v S9 mix andmodified concentration spacing. Bacterial cultures were exposed to test item at 7 concentrations (three plates/concentration) between 78.125 to 5000 µg/plate both in the absence and presence (10% v/v S9 mix) of metabolic activation. After 48 hours of incubation at 37 ± 1°C, the revertant colonies were scored.

Test item did not induce any significant increase in the number of revertant colonies, in both trials, with and without S9 mix, in any of the five tester strains. All the values for the negative control were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

All criteria for a valid study were met as described in the study plan.From results of this study, under the specified experimental conditions, the reaction mass is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification