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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2000-16-04
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
description: white powder
Chemical name: Undecylenoyl phenilalanine
Purity > 98.7%
Batch: 02 144 00005
Manufactured: 29/05/2002
Expiry: 19/05/2003
Received: 03 september 2002
Storage: 4°C in the dark

Method

Target gene:
Salmonella typhimurium: histidine
Escerichia coli: tryptophan
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
Additional strain / cell type characteristics:
other: WP2uvrA-
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
The dose range for the range finding study was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate for E. coli strain WP2uvrA- and 5 to 5000 ug/plate for all the remaining tester strains
Vehicle / solvent:
Solvent: DMSO
Details on test system and experimental conditions:
0.1mL 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. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of the test mateial both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

The main study was performed using methodology as previously described, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as the range finding study (50 to 5000 ug/plate for WP2uvrA- and 5 to 5000 ug/plate for the remaining tester strains.
Evaluation criteria:
the test material should have induced a reproductible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method

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:
cytotoxicity
Remarks:
(> 1500 µg/plate)
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
An oily precipitate was observed at 5000 ug/plate, 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, with any dose of the test material, either woth or without metabolic activation.
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 with metabolic activation
negative without metabolic activation

The test material was considered to be non mutagenic under the conditions of this test.
Executive summary:

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 n°471 "bacterial reverse mutation test", method B13/14 of comission directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA100 and Escherichia coli strain WP2uvrA- were treated with solutions of the test material using the AMES plate incorporation method up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% rat liver S9 in stadndard co-factors). The dose range for the range finding study was determined in a preliminary toxicity assay and was 50 to 5000 ug/plate for E. coli strain WP2uvrA- and 5 to 5000 ug/plate for all the remaining tester strains. The experiment was repeated on a separate day using the same dose range as the range finding study, fresh cultures of the bacterial strains and fresh test material formulations. Additional dose levels were used in both experiments to allow the toxicity of the test material, ansuring there were a minimum of four non toxic doses.

Results

The vehicle ( dimethylsulfoxide) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9 mix were validated.

The test material caused a visible reduction in growth of the bacterial background lawn to all of the Salmonella tester strains both with and without S9 -mix, initially at 1500 ug/plate. No toxicity was noted to E. coli strain WP2uvrA-. The test material was, therefore, tested using an extended dose range up to the maximum recommended dose level of 5000 ug/plate. An oily precipitate was observed at 5000 ug/plate, this did not prevent the scoring or revertant colonies.

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 nonsidered to be non mutagenic under the conditions of this test.