Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
06 September 2007 to 12 October 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted similar or equivalent to OECD 471 Guidelines as well as the Japanese Authorities and US EPA (TSCA) OPPTS harmonised guidelines. There were no deviations and the study met the validation criteria. The study was conducted on a substance different to the registered substance but with a robust read-across justification.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Principles of method if other than guideline:
The method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW, and MAFF
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: pale yellow liquid
Details on test material:
The justification for read-across is attached in Section 13 of the IUCLID.

- Name of test material (as cited in study report): EX 802502
- Physical state: Pale yellow liquid
- Storage condition of test material: room temprature in the dark

Method

Target gene:
The mutant strains of Salmonella with a deletion through the excision repair gene (uvrB- Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa), are incapable of synthesising histidine, and may undergo a reverse mutation to histidine independent forms.
A mutant strain of Escherichia coli (WP2uvrA-, a deletion in an excision repair gene uvrA) requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta-naphthoflavone induced S9
Test concentrations with justification for top dose:
0, 50, 150, 500, 1500 and 5000 ug/plate
Vehicle / solvent:
acetone
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2 ug/plate for WP2uvrA-, 3 ug/plate for TA100, 5 ug/plate for TA1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 ug/plate for TA1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 ug/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Benzo(a)pyrene
Remarks:
5 ug/plate for TA98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
1ug/plate for TA100, 2 ug/plate for TA1535 and TA1537, and 10 ug/plate for WP2uvrA-
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates per dose level with and without metabolic activation

DETERMINATION OF CYTOTOXICITY
- Method: growth of the background lawn of bacteria

OTHER:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer.
Evaluation criteria:
Acceptance Criteria:

- An appropriate number of spontaneous revertant colonies are observed per plate for the tester strain cultures in the vehicle and with the untreated controls.
- Confirmation of tester strain characteristics such as rfa cell-wall mutation and pKM101 plasmid R-factor, etc.
- Tester strain cultures should contain 1 to 9.9 x 10^9 bacteria per ml
- The mean for each positive control value should be a minimum of 2x the respective vehicle control for each strain; this will demonstrate an intrinsic sensitivity of the tester strains to mutagenic exposure and the S9-mix
- Must be at least four non-toxic test material dose levels
- No evidence of (excessive) contamination

Statistics:
Methods as recommended by the UKEMS 1989.

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
- Precipitation: Precipitation was observed at the upper dose level but this did not affect the scoring of the revertant colonies.

COMPARISON WITH HISTORICAL CONTROL DATA: All control values were within the expected ranges.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:
Test Guidance This method is similar or equivalent to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, and meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", and the USA, EPA (TSCA) OPPTS harmonised guidelines. Method The following bacterial strains were used: Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-. Each strain was treated with solutions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without metabolic activation. The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The same dose levels were used for experiment 1 and experiment 2 which were performed on separate days. Results The vehicle (acetone) 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. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. A precipitate was observed at 5000 μg/plate, however, this did not prevent the scoring of revertant colonies. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose of the test material, either with or without metabolic activation. Conclusion In conclusion, the test material was considered to be non-mutagenic under the conditions of this test. This study was performed on a different substance to the registered substance, however, a robust read-across justification for the read-across has been made and the results of this study are considered to be relevant to the registered substance.