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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

This study was performedbythe bacterial reverse mutation test, using five histidine deficient mutant tester strains ofSalmonella typhimurium(i.e., TA1537, TA1535, TA98, TA100 and TA102).

Test substancewas 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 0.05 to 160 µg/plate in the initial toxicity-mutation test. Normal background lawn pattern was observed up to the tested concentration of 160 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 totest itemat 6 concentrations (three plates/concentration) between 5 to 160 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 itemdid 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 the results of this study, under the specified experimental conditions,test itemisconcluded to be non-mutagenic in the Bacterial Reverse Mutation Test usingSalmonella typhimurium.

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:
Study Initiation : November 15, 2017 and Study Completion date : April 16, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 Fraction and S9 Mix
Test concentrations with justification for top dose:
5, 10, 20, 40, 80 and 160 µg/plate both in the absence and presence of metabolic activation system (10% v/v S9 mix)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours of incubation at 37 ± 1°C
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):

SPINDLE INHIBITOR (cytogenetic assays):

STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS:

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED:

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:

DETERMINATION OF CYTOTOXICITY
- cloning efficiency

- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:
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.
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:
bacteria, other: TA1537, TA1535, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.
Conclusions:
test item is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.
Executive summary:

This study was performed by the bacterial reverse mutation test, using five histidine deficient mutant tester strains ofSalmonella typhimurium(i.e., TA1537, TA1535, TA98, TA100 and TA102).

Test 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 0.05 to 160 µg/plate in the initial toxicity-mutation test. Normal background lawn pattern was observed up to the tested concentration of 160 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 totest itemat 6 concentrations (three plates/concentration) between 5 to 160 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 itemdid 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 the results of this study, under the specified experimental conditions,test item 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

This study was performedbythe bacterial reverse mutation test, using five histidine deficient mutant tester strains ofSalmonella typhimurium(i.e., TA1537, TA1535, TA98, TA100 and TA102).

Test substancewas 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 0.05 to 160 µg/plate in the initial toxicity-mutation test. Normal background lawn pattern was observed up to the tested concentration of 160 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 totest itemat 6 concentrations (three plates/concentration) between 5 to 160 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 itemdid 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 the results of this study, under the specified experimental conditions,test item is concluded to be non-mutagenic in the Bacterial Reverse Mutation Test usingSalmonella typhimurium.