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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phase of this study was performed between 19 July 2012 and 22 September 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
other: Solid

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.coli
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:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
test concentrations
To achieve the maximum recommended test concentration (5000 μg/plate), formulated concentrations were adjusted to allow for the stated solvent content (28.5% propane-1,2-diol) of the test item. However for reporting purposes the concentrations are given as test item as a whole.

Preliminary Toxicity Test: 0, 0.2, 0.7, 2, 7, 20, 70, 210, 690, 2070 and 6890 µg/plate

main test:
Experiment one: 20, 70, 210, 700, 2090 and 6970 µg/plate
Experiment two: 7, 20, 70, 210, 700, 2100 and 6990µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl Sulphoxide
- Justification for choice of solvent/vehicle: The test material was fully soluble in Dimethyl Sulphoxide at 50 mg/ml in solubility checks performed in-house
- Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) 3 μg/plate
Remarks:
Without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 5 ug/plate
Remarks:
Without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-Aminoacridine (9AA): 80 μg/plate
Remarks:
Without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline-1-oxide (4NQO): 0.2 μg/plate
Remarks:
Without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG): 2 μg/plate
Remarks:
Without S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA): 1 μg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA): 2 μg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA): 2 μg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA): 10 μg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Benzo(a)pyrene (BP): 5 μg/plate
Remarks:
With S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and 20 minutes pre-incubation at 37 deg C in Experiment 2.

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: Approximately 42 h
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.
Evaluation criteria:
Acceptance Criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
4. Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivi ty of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the onldetermining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation
Statistical analysis of data as determined by UKEMS Dunnett's t test

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
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:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Dimethyl sulphoxide solubility: The test item was fully soluble in Dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house.
- Precipitation: test item precipitation was observed on the plates at 6990 μg/plate, this observation did not prevent the scoring of revertant colonies. Precipitation was observed in the presence or absence of S9-mix at this dose.

RANGE-FINDING/SCREENING STUDIES:
In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns and/ or substantial reductions in the frequency of revertant colonies of all of the Salmonella strains in both the absence and presence of S9-mix at 6970 μg/plate. No toxicity in terms of weakened bacterial background lawns or substantial decreases in revertant colony frequency were noted for Escherichia coli strain WP2uvrA. In the main test (pre-incubation method) the test item induced a much greater toxic response with weakened bacterial background lawns noted to all of the bacterial strains, initially from 700 μg/plate in the absence of S9-mix and 2100 μg/plate in the presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level. A test item precipitate (particulate in appearance) was noted at 6990 μg/plate, this observation did not prevent the scoring of revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

See attachment for results

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test item for this study was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the, EPA (TSCA) OPPTS harmonised guidelines.

Method

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item, using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 20 to 6890 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test item formulations.

Results.

The vehicle (Dimethyl Sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns and/or substantial reductions in the frequency of revertant colonies of all of the Salmonella strains in both the absence and presence of S9-mix at 6970 μg/plate. No toxicity in terms of weakened bacterial background lawns or substantial decreases in revertant colony frequency were noted for Escherichia coli strain WP2uvrA. In the main test (pre-incubation method) the test item induced a much greater toxic response with weakened bacterial background lawns noted to all of the bacterial strains, initially from 700 μg/plate in the absence of S9-mix and 2100 μg/plate in the presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level. A test item precipitate (particulate in appearance) was noted at 6990 μg/plate, this observation did not prevent the scoring of revertant colonies.

Conclusion.

The test item for this study was considered to be non-mutagenic under the conditions of this test.