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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Remarks:
source of read across
Adequacy of study:
key study
Study period:
From 02nd to 25th October, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Source: University of California at Berkeley.
- Storage: at -196 °C in a Stateboume liquid nitrogen freezer, model SXR 34.
- Check: prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Source: British Industrial Biological Research Association.
- Storage: at -196 °C in a Stateboume liquid nitrogen freezer, model SXR 34.
- Check: prior to the master strains being used, characterisation checks were carried out to confirm the amino-acid requirement, presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.
Metabolic activation:
with and without
Metabolic activation system:
Microsomal enzyime fraction (S9)
Test concentrations with justification for top dose:
MAIN STUDY - EXPERIMENT II: 0, 50, 150, 500, 1500 and 5000 µg/plate
RANGE-FINDING - EXPERIMENT I: 0, 15, 50, 150, 500, 1500 and 5000 µg/plate
PRELIMINATY TOXICITY TEST: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
The test material was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer and sonication for 10 minutes at 40 °C on the day of each experiment.
Controls
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Arninoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation). Top agar was prepared using 0.6 % Difco Bacto agar and 0.5 % sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar. Vogel-Bonner Minimal agar plates were purchased from Fred Baker Scientific.
Overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

NUMBER OF REPLICATIONS: three

RANGE-FINDING - EXPERIMENT I
Six concentrations of the test material were assayed in triplicate against each tester strain, using the direct plate incorporation method. An additional dose level was employed in the range finding study to allow for the toxicity of the test material, ensuring there was a minimum of four non-toxic doses.
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. 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 of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at and above 1500 µg/plate because of intense colouration.

MAIN STUDY - EXPERIMENT II
The second experiment was performed using methodology as described for the range-finding study, using fresh bacterial cultures, test material and control solutions. The test material dose range was modified slightly, based on the results of the range-finding study, and was 50 to 5000 µg/plate.

PRELIMINARY TOXICITY STUDY
In order to select appropriate dose levels for use in the main study, 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 study was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA), 0.1 ml of test material formulation, 0.5 ml of S9-mix or phosphate buffer and 2 ml of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material and a vehicle control (sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan 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 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at and above 1500 pg/plate because of intense colouration.

MICROSOMAL ENZYME FRACTION
S9 was prepared in-house from the livers of male Sprague-Dawley rats weighing ~ 250g. These had each orally received three consecutive daily doses of phenobarbitone/B-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on Day 4. Before use, each batch of S9 was assayed for its ability to metabolize the indirect mutagens 2-Aminoanthracene and Benzo(a)pyrene. The S9 was stored at -196 °C.

S9-Mix and Agar
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.
S9 5.0 ml, 1.65 M KCI/0.4 M MgCl2 1.0 ml, 0.1 M Glucose-6-phosphate 2.5 ml, 0.1 M NADPH 2.0 ml, 0.1 M NADH 2.0 ml, 0.2 M Sodium phosphate buffer (pH 7.4) 25.0 ml and Sterile distilled water 12.5 ml.
A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine or tryptophan supplemented, top agar was overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

ACCEPTANCE CRITERIA
The reverse mutation assay may be considered valid if the following criteria are met:
- all tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls;
- the appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pkM101 plasmid R-factor etc;
- all tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per ml;
- each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix;
- there should be a minimum of four non-toxic test material dose levels;
- there should not be an excessive loss of plates due to contamination.
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met: the test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression(5)) significant increase in the revertant count in at least one strain of bacteria.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

MUTATION STUDY
A purple colouration was observed at 500 µg/plate becoming intense from 1500 µg/plate. This observation did not prevent the scoring of revertant colonies. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. 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.

The vehicle (sterile distilled water) 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.

PRELIMINARY TOXICITY STUDY
The test material was toxic to tester strain TA100 at 5000 µg/plate and was non-toxic to WP2uvrA. The test material formulation and S9-mix used in this experiment were both shown to be effectively sterile.

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-mutagenic under the tested conditions.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with solutions of the test material using the Ames plate incorporation method at up to six dose levels, in triplicate, both with and without metabolic activation. The method meets the requirements of the OECD Guidelines 471, Method B13/14 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

The dose range was determined in a preliminary toxicity assay and was 15 to 5000 µg/plate in the range-finding study. The experiment was repeated on a separate day using a modified dose range, based on results obtained from the range-finding study, and was 50 to 5000 µg/plate. An additional dose level was employed in the range finding study to allow for the toxicity of the test material, ensuring there was a minimum of four non-toxic doses.

The vehicle (sterile distilled water) 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 either a visible reduction in the growth of the bacterial background lawn and/or a significant reduction in the frequency of revertant colonies at 5000 µg/plate to all of the Salmonella tester strains both with and without metabolic activation. No toxicity was noted to E.coli strain WP2uvrA-. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A purple colouration was observed at 500 µg/plate becoming intense from 1500 µg/plate. This observation did not prevent the scoring of revertant colonies. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. 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 tested conditions.