Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-01-04 to 2018-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical state: powder/solid
- Appearance: white powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 397451
- Expiration date of the lot/batch: 2018-02-07
- Manufacturing date: 2018-29-08

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in dimethyl sulfoxide at a concentration of 50 mg/ml. Test item concentrations were used within 2 hours of preparation.

OTHER:
A correction factor of 1.00 for the purity/composition of the test item was applied in this study.

Method

Target gene:
The Histidine locus in S. typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Tryptophan locus in E. coli strains (WP2uvrA)
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9
Test concentrations with justification for top dose:
- Dose range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate with and without 5% S9-mix (TA100 and WP2uvrA) (top dose selected based on the solubility findings)
- Mutation experiment I: 52, 164, 512, 1600 and 5000 μg/plate with and without 5% S9-mix (TA98, TA1535 and TA1537) (top dose selected based on the dose range finding test results)
- Mutation experiment II: 492, 878, 1568, 2800 and 5000 μg/plate with and without 10% S9-mix (all tester strains) (top dose selected based on the dose range finding test and mutation experiment I results)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (dimethyl sulfoxide)
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water at 50 mg/ml. In DMSO, the test item formed a clear solution at 50 mg/ml (= 5000 μg/plate). Based on these solubility findings, DMSO was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation; 5 μg/plate (TA1535)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191
Remarks:
Without metabolic activation; 2.5 μg/plate (TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without metabolic activation; 10 μg/plate (TA98)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation; 650 μg/plate (TA100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without metabolic activation; 10 μg/plate (WP2uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation; 2.5 μg/plate (TA1535 at 5 and 10% S9; TA1537 at 5% S9), 5 μg/plate (TA153 7 at 10% S9), 1 μg/plate (TA98 at 5 and 10% S9; TA100 at 5% S9), 2 μg/plate (TA100 at 10% S9), 15 μ g/plate (WP2uvrA at 5 and 10% S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar:
- 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains,
- 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9-mix (in case of activation assays) or
- 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

DURATION
- Exposure duration: 48 ± 4 h
- Selection time: 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium histidine-dependent strains); Tryptophan (E. coli tryptophan-dependent strains)

NUMBER OF REPLICATIONS: All concentrations for all experiments were tested in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle 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 vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up 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.

In addition to the criteria for positive or negative repsonse, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.
Statistics:
No formal hypothesis testing was done.

Results and discussion

Test results
Species / strain:
other: TA1535, TA1537, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: see addional information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Water solubility: insoluble in water at 50 mg/mL
- Precipitation: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES:
The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and
presence of 5% (v/v) S9-mix. Normal bacterial background lawn and no precipitate were observed up to the highest dose. Based on these results, the dose ranges for mutation experiment I (TA98, TA1535 and TA1537) were selected

COMPARISON WITH HISTORICAL CONTROL DATA:
All bacterial strains showed negative responses over the entire dose range, i.e. no biologically relevant and/or significant dose-related increase in the number of revertants in any of the experiments.
The vehicle 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:
Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA1537 in the absence of S9-mix, ni teh second mutation experiment.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.