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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 March 2003 - 28 April 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC Commission Directive 2000/32/EC Annex mutation test in bacteria. 4D-B.13/14. Mutagenicity - Reverse. No. L136/57.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Joint Directives of JEPA, JMHW and JMITI. (31 October 1997) Kanpoan No. 287, Eisei No. 127 and Kikyoku No. 2 (31 October 1997).
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMHW Genotoxicity Testing Guideline, PAB Notification No. 1604 (1 November 1999).
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Official Notice of J MOL (8 February 1999).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
DL-serine
EC Number:
206-130-6
EC Name:
DL-serine
Cas Number:
302-84-1
Molecular formula:
C3H7NO3
IUPAC Name:
DL-serine
Test material form:
other: powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 20017
- Expiration date of the lot/batch: 5 July 2003
- Purity: 98.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in the dark.

Method

Target gene:
Histidine, ampicillin, 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 uvr A pKM 101
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 µg/plate.
The highest concentration of the tets substance tested was 50 mg/ml in the chosen solvent, which provided a final concentration of 5000 µg/plate. This is the standard limit concentration recommended in the regulatory guidelines this assay follows.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water (purified in-house by reverse osmosis) containing 0.15% agar
- Justification for choice of solvent/vehicle: the test substance was insoluble in water, in dimethyl sulphoxide (DMSO) and in acetone.
Controlsopen allclose all
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
First test (range-finding):
Aliquots of 0.1 ml of the test dilution, positive control or negative control were placed in glass vessels.
All plates were incubated at 37°C for approximately 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted.
Second test:
The pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes.
The maximum concentration chosen was 5000 µg/plate, but only five concentrations were used.
Rationale for test conditions:
For a test to be considered valid the mean of the solvent/vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent solvent/vehicle controls.
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent solvent/vehicle controls, with some evidence of a positive dose-response relationship, it will be considered to exhibit mutagenic activity in this test system. No statistical analysis will be performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers.
Statistics:
The mean number and standard deviation of revertant colonies are calculated for all groups. The means for all treatment groups are compared with those obtained for the solvent/vehicle control groups.
The statistical procedures used will be those described by Mahon et al (1989) and will usually be Dunnett's test followed, if appropriate, by trend analysis. Biological significance should always be considered along with statistical significance.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
other:
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
It is concluded that, under the test conditions employed, the test material showed no evidence of mutagenic activity in this bacterial system.
Executive summary:

The mutagenic potential of the test material to histidine dependent auxotrophic mutants of Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli strain WP2uvrA/pKM101 was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japanese Genotoxicity Testing Guideline No. 1604.

Two independent mutation tests were performed in the presence and absence of metabolic activation. The first (range-finding) test was a standard plate incorporation assay, the second involved a pre-incubation stage.

Concentrations of test material up to 5000 µg/plate were tested. No signs of toxicity were observed towards the tester strains in either mutation test.

No evidence of mutagenic activity was seen at any concentration of test material in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions employed, the test material showed no evidence of mutagenic activity in this bacterial system.