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Toxicological information

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:
17 March 2004 to 05 April 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Annex V (Ames)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS harmonised guidelines
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 2 December 2002 Date of Signature: 13 February 2003
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): TIPX
- Substance type: Colourless liquid.
- Physical state: Liquid.
- Lot/batch No.: 042028.
- Storage condition of test material: Approximately 4ºC in the dark.

Method

Target gene:
Histidine operon (his) for Salmonella.
Tryptophan operon (trp) for E.Coli.
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell lines (if applicable):
Not applicable.
Additional strain characteristics:
other: Including a deletion through the excision repair gene (uvrB-) which renders the capability of DNA exision repair and deep rough mutation (rfa)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/beta-naphthoflavone induced, rat-liver S9.
Species / strain:
E. coli WP2 uvr A
Details on mammalian cell lines (if applicable):
Not applicable.
Additional strain characteristics:
other: Including a deletion through the excision repair gene (uvrA-)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/beta-naphthoflavone induced, rat-liver S9.
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Main Test (Experiments 1 and 2): 0, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle:
Dimethyl sulphoxide
Controlsopen allclose all
Negative controls:
yes
Remarks:
(concurrent untreated control )
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
(concurrent untreated control )
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix
Negative controls:
yes
Remarks:
(concurrent untreated control )
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix
Negative controls:
yes
Remarks:
(concurrent untreated control )
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix
Negative controls:
yes
Remarks:
(concurrent untreated control )
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix
Details on test system and conditions:
Concentration of the test substance resulting in precipitation: 5000 µg/plate

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h.

NUMBER OF REPLICATIONS: Triplicate.

DETERMINATION OF CYTOTOXICITY: Plates were assessed for effects on the growth of the bacterial background lawn.

OTHER EXAMINATIONS
- Other:
Solubility: Test material precipitation was examined on the plates.
Sterlility: (Preliminary study only) The aliquot of 0.1 ml of maximum concentration of the test material (5000 µg/plate) and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material.
Evaluation criteria:
A test material may be considered positive in the test system if the following criteria are met: the test material should have induced a reproducible, dose-related and statistically significant increase in the relevant count in at least one strain of bacteria.
Statistics:
Dunnett’s method of linear regression.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: E. coli WP2 uvr A- and S. typhimurium TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
The results of the main test is in tables 2 - 5 as attached.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A slight, oily precipitate was observed under the microscope only at 5000 ug/plate, this did not prevent the scoring of revertant colobies.

PRELIMINARY TOXICITY TEST: The test material was non-toxic to E. coli WP2 uvr A- and S. typhimurium TA100. See Table 1 as attached. The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level.

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:

Introduction.The method conforms 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 Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using the Ames plate incorporation method 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 was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in Experiment 1as the Main test. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material 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.

The test material caused no visible reduction in the growth of the bacterial background lawns at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 ug/plate. A slight, oily precipitate was observed under the microscope only at 5000 ug/plate, this did not prevent the scoring of revertant colobies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.The test material was considered to be non-mutagenic under the conditions of this test.