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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:
2016-12-06 to 2016-12-14
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)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
liquid
Details on test material:
Physical state: liquid
Appearence: light brown liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I16FB3013
- Expiration date of the lot/batch: 2018-06-07 (retest date)
- Date of manufacture: 2016-06-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: not available
- 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

OTHER: A correction factor of 1.14 for the purity/composition of the test item was applied in this study.

Method

Target gene:
Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)
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 applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Aroclor 1254 induced rat liver metabolic activation system)
Test concentrations with justification for top dose:
Dose Range Finding Test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 µg/plate with and without 5% S9-mix (Top dose based on the solubility findings);
Mutation Assay: 0, 17, 52, 164, 512, 1000, 1600 µg/plate with and without 5% S9-mix (Top dose based on solubility finding and dose range finding test results)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- 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 was soluble at 50 mg/ml (= 5000 μg/plate). Based on these solubility findings, DMSO was selected as vehicle.
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 S9-mix; at 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 S9-mix; at 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 S9-mix; at 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 S9-mix; at 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 S9-mix; at 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 S9-mix; at 2.5 µg/plate (TA1535, TA1537), at 1 µg/plate (TA98, TA100), at 15 µg/plate (WP2uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: 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 (if incubation with a selection agent): 48 ± 4 h (simultaneous with exposure)

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains) or tryptophan (E. coli strains)

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

DETERMINATION OF CYTOTOXICITY
- Method: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
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.
Statistics:
No formal hypothesis testing was done.
In addition to the criteria stated above, 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.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
see details below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
see details below
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble at 50 mg/ml
- Precipitation: at 1600 and 5000 μg/plate (start of the incubation), no precipitation at the end of the incubation.

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 S9-mix. Based on the results of the dose range finding test, the following dose ranges were selected for the mutation experiment with the tester strains TA1535, TA1537 and TA98: 17, 52, 164, 512, 1000 and 1600 μg/plate in the absence, and 17, 52, 164, 512, 100, 1600 and 5000 μg/plate in the presence of S9-mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The 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.
- Negative (solvent/vehicle) historical control data: The vehicle 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:
- Measurement of cytotoxicity used: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies.
- Other observations when applicable: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or reduction of the bacterial background lawn, was observed in the tester strains TA100, TA1535, TA1537 and TA98 in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

Mutagenicity:
without S9: significant increase in TA1535, TA100 and TA1537, but biologically relevant in TA1535 and TA100 only; with S9: significant increase in TA1535, TA100, TA98 and TA1537, but biologically relevant in TA1535 and TA100 only

Any other information on results incl. tables

In the absence of S9-mix, the test item induced dose related increases in the number of revertant colonies. The increase observed in tester strain TA1535 was above the laboratory historical control data range, and was up to 21-fold the concurrent control. The increase observed in tester strain TA100 was above the laboratory historical control data range and was up to 8.6-fold the concurrent control. The increase observed in tester strain WP2uvrA was above the laboratory historical control data range and was up to 9.1-fold the concurrent control. The increase observed in tester strain TA1537 was more than three times the concurrent control (4.3-fold), but was within the laboratory historical control data range. Therefore, this increase is considered to be not biologically relevant.

In the presence of S9-mix, the test item induced dose related increases in the number of revertant colonies. The increase observed in tester strain TA1535 was above the laboratory historical control data range, and was up to 7.7-fold the concurrent control. The increase observed in tester strain TA100 was above the laboratory historical control data range and was up to 12-fold the concurrent control. The increase observed in tester strain WP2uvrA was above the laboratory historical control data range and was up to 7.0-fold the concurrent control. The increases observed in tester strain TA1537 and TA98 were more than three times the concurrent control (3.3 and 3.7-fold, respectively), but were within the laboratory historical control data range. Therefore, these increases are considered to be not biologically relevant.

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
positive with and without metabolic activaiton

Based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.