Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
28th September -18th November 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Annex V (Ames)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid

Method

Target gene:
The mutant strains of salmonella are incapable of synthesising histidine and are therefore dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic agent these bacteria may undergo a reverse mutation in histidine independent forms which are detected by their ability to grow on a histidine deficient medium.
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium Strains: TA1535, TA1537, TA102, TA98 and TA100
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/beta-naphthoflavone-induced rat liver S9.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate
Vehicle / solvent:
Solvent: Dimethyl sulphoxide
Details on test system and experimental conditions:
Preliminary toxicity test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were; 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100), 2 ml of molten, trace histidine supplemented, top agar, 0.1 ml of test material formulation., 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30ml/plate). Ten doses of the test material and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine supplemented, top agar was overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37ºC the plates were assessed for number of revertant colonies using a domino colony counter and examined for effects on the growth of the bacterial background lawn.
Mutation test- Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000µg/plate) were assayed in triplicate against each tester strain using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0ml of molten, trace histidine 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 test material both with and without S9-mix.
All the plates were incubated at 37ºC for approximately 478 hours and the frequency of the revertant colonies assessed using a Domino colony counter.
Mutation test- Experiment-2
The second experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50-5000 µg/plate)
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in reverent 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 only determining factor for positive response.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations: No biologically 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. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies.

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:

A GLP study was performed to meet the requirements of the OECD Guidelines for Testing for Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Directive 2000/32/EC and the USA, EPA (TSCA) OPPTS harmonised guidelines, in order to determine the mutagenic potential of Inhibitor AHM P500.

Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 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 the first experiment. 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. 

The test material caused no visible reduction in growth of the bacterial background lawn at any dose level, although substantial decreases in reverent colony frequency were noted to all of the strains at 5000µg/plate both in the presence and absence of S9. The test material was, therefore, tested up to the maximum recommended dose level of 5000µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9.

No biologically significant increases in the frequency of reverent colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation. A small increase in reverent colony frequency was observed in tester strain TA1535, without S9 only, at 500 and 1500µg/plate in the first experiment. The increases were not however, reproduced in experiment 2 and were considered to be of no biological relevance because there was no evidence of dose-response relationship or reproducibility. Furthermore, the relevant counts were generally within the in-house range for the tester strain and the fold increases were only 1.8 times the concurrent vehicle control.

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