Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 April 2016 - 28 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 31, 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced with Aroclor 1254 (500 mg/kg bw/day)
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced with Aroclor 1254 (500 mg/kg bw/day)
Test concentrations with justification for top dose:
- Dose range finding test:
TA 100 and WP2uvrA:
Absence and presence of S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate.

- First experiment: direct plate assay
TA1535, TA1537 and TA98:
Absence of S9-mix: 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate.
Presence of S9-mix: 1.7, 5.4, 17, 52, 164 and 512 μg/plate.

- Second experiment: Pre-incubation assay
TA1535, TA1537, TA98 and TA100:
Absence of S9-mix: 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate.
Presence of S9-mix: 1.7, 5.4, 17, 52, 164 and 512 μg/plate.
WP2uvrA:
Absence and presence of S9-mix: 17, 52, 164, 512, 1600 and 5000 μg/plate
Vehicle:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be miscible in DMSO.
Controls
Negative controls:
no
Solvent controls:
yes
Positive controls:
other:
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION:
- Exposure duration: incubation 37.0 ± 1.0°C for 48 ± 4 hours

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

NUMBER OF CELLS EVALUATED:
- 10^8 cells were evaluated

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:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Dose range finding: Precipitation was observed at the start of the incubation period at and above 1600 μg/plate. No precipitation was observed at the end of the incubation period.
Direct plate experiment: Precipitation was observed at the start of the incubation period at concentrations of 52 and 164 μg/plate plate in the absence of S9-mix and at 164 and 512 μg/plate in the presence of S9-mix. No precipitation was observed at the end of the incubation period.
Pre-incubatiuon experiment: Precipitation was observed at the start of the incubation period at and above 1600 μg/plate. No precipitation was observed at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: The test material was toxic at and above 52 μg/plate (without S9) and at and above 164 μg/plate (with S9) to strain TA100 and at 5000 μg/plate to WP2 uvrA.

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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material was toxic to all of the Salmonella strains at the highest dose level (164 μg/plate without S9 and 512 μg/plate with S9) in the direct plate assay. In the pre-incubation experiment the test material was toxic to all of the Salmonella strains at and above 17 μg/plate without S9 and at and above 52 μg/plate with S9, except for TA98 where cytotoxicity was observed at and above 164 μg/plate with S9. The test material was toxic to E.coli strain WP2 uvrA at and above 164 μg/plate without S9 and not toxic with S9.

Applicant's summary and conclusion

Conclusions:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 guideline and GLP principles.
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed in two independent experiments. The direct plate experiment was performed up to and including 5000 μg/plate in TA100 and WP2uvrA, and up to and including 164 μg/plate and 512 μg/plate in TA1535, TA1537 and TA98 in the absence and presence of S9 -mix, respectively. In the pre-incubation experiment the same dose ranges were used except for TA100 which was tested up to and including 164 μg/plate and 512 μg/plate, in the absence and presence of S9-mix, respectively. The dose levels were selected based on observed cytotoxicity in the dose range finding test (TA100 >= 164 μg/plate without S9 and 512 μg/plate with S9, WP2uvrA =5000 μg/plate). 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 and TA100) and in the number of revertant (Tryp+) colonies in E.coli WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiment. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay.