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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:
The study was conducted between 11 June 2015 and 19 August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
liquid

Method

Target gene:
- S. typhimurium: Histidine
- E. coli: Tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
DOSE RANGE FINDING TEST 1
- TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 5000, 1250, 313, 78.1, 19.5, 4.88 µg/plate

DOSE RANGE FINDING TEST 2 (repeated for some strains due to cytotoxicity):
Without S9
- TA 1535, TA 1537, TA 100: 313, 156, 78.1, 39.1, 19.5, 9.77 µg/plate
With S9
- TA 1535, TA 1537: 313, 156, 78.1, 39.1, 19.5, 9.77 µg/plate

EXPERIMENT 1 (based on dose range finding tests)
Without S9
- TA 100, TA 1535, TA 1537: 313, 156, 78.1, 39.1, 19.5, 9.77 µg/plate
- TA 98: 5000, 2500, 1250, 625, 313 and 156 µg/plate
- WP2uvrA: 5000, 2500, 1250, 625, 313 µg/plate
With S9
- TA 100: 1250, 625, 313 and 156, 78.1, 39.1 µg/plate
- TA 1535 and TA 1537: 313, 156, 78.1, 39.1, 19.5, 9.77 µg/plate
- WP2uvrA and TA 98: 5000, 2500, 1250, 625, 313 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO up to 50 mg/mL and insoluble in water
Controls
Untreated negative controls:
yes
Remarks:
(untreated plates)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 2-aminoanthracene / acridine mutagen ICR-191 / AF-2: 2 -(2 -Furyl)-3- (5 -nitro-2 -furyl)acrylamide
Remarks:
A detailed overview of positive control substances per strain is included in "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation method
0.1 mL of test solution was incubated for 20 minutes at 37±0.5 °C and thereafter, placed on agar.

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in duplicate for each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation
Evaluation criteria:
For the test substance to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed when compared to the negative control. The responses must be dose-related and/or reproducible.
Statistics:
No statistical methods were used.

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
Remarks:
The number of revertant colonies was less than twice when compared to the negative control, in all strains, in the presence and absence of metabolic activation.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed.

RANGE-FINDING/SCREENING STUDIES:
- Test 1: cytotoxicity was detected at 313 µg/plate and above, with strains TA 100, TA1535 and TA 1537 in the absence of S9, and strains TA 1535 and TA 1537 in the presence of S9. At 5000 µg/plate cytotoxicity was seen with TA 98 without metabolic activation, and at 1250 µg/plate and above with strain TA 100. The number of revertant colonies was less than twice when compared to the negative control, in all strains, in the presence and absence of metabolic activation.
- Test 2: cytotoxicity was observed at 156 µg/plate and above, in strains TA 100, TA 1535 and TA 1537 in the absence of S9 mix, and in strains TA 1535 and TA 1537 with S9 mix. The number of revertant colonies was less than twice when compared to the negative control, in all strains, in the presence and absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:
The substance was found to be not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to the Japanese Guidelines for Screening Mutagenicity Testing of Chemicals (similar to OECD 471).
Executive summary:

The mutagenic activity of the substance was evaluated according to the Japanese Guidelines for Screening Mutagenicity Testing of Chemicals (similar to OECD 471) and according to GLP principles. The test was performed with the pre-incubation method assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in all strains. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98, TA100) and E.coli WP2, both in the absence and presence of S9-metabolic activation. Based on the results of this study, it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.