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Genetic toxicity: in vitro

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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:
September 2002 - January 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from guideline study.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
not specified
Principles of method if other than guideline:
To evaluate the mutagenic potential of test substance in bacteria by AMES assay.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
histidine locus in the genome of Salmonella typhimurium and tryptophan locus of Escherichia coli
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagenic compounds. Mutation of either the uvrA gene (Escherichia coli) or the uvrB gene (Salmonella typhimurium) results in a deficient DNA excision repair system, which greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, Salmonella typhimurium tester strains containing this deletion also require the vitamin biotin for growth.

Salmonella typhimurium tester strains also contain the rfa wall mutation, which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e., benzo[a]pyrene) that would otherwise be excluded by a normal intact cell wall.

Tester strains TA98 and TAlOO also contain the pKMlOl plasmid, which further increases the sensitivity of these strains to some mutagens. The suggested mechanism by which this plasmid increases sensitivity to mutagens is by modification of an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strains TAlOO, TA1535, and WP2uvrA are reverted from auxotrophy to prototrophy by base substitution mutagens.
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 Homogenate (Aroclor) in S9Mix
Test concentrations with justification for top dose:
Salmonella tester strains (with S9 mix): 33.3, 100, 333, 1000, 3330, and 5000 ug per plate
Salmonella tester strains (without S9 mix): 3.33, 10.0, 33.3, 100, 333, 1000, 3330, and 5000 ug per plate
Escherichia coli tester strain (with and without S9 mix): 33.3, 100, 333, 1000, 3330, and 5000 ug per plate

Cytotoxicity was observed in the dose range finding study, and the highest dose level of test article used in the subsequent mutagenicity assay was a dose which gave a reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn.
Vehicle / solvent:
The test article was .observed to form a transparent, colorless solution at a concentration of 100 mg per mL in dimethylsulfoxide (DMSO). DMSO was selected as the vehicle. At 100 mg per mL, which was the most concentrated stock dilution prepared for the mutagenicity assay, the test article was observed to form a transparent, non-viscous, colorless solution. The test article remained a solution in all succeeding dilutions prepared for the mutagenicity assay.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene - TA100, TA1535, TA1537, WP2uvrA with S9 Mix; ICR-191 - TA1537 without S9 Mix
Details on test system and experimental conditions:
Tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, test article, tester strain, and S9 mix (when appropriate) were combined in molten agar, which was overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of test article, vehicle controls and positive controls were plated in triplicate.
Rationale for test conditions:
Experimental materials, methods and procedures are based on those described by Ames et al. (1975) and Green and Muriel (1976). The assay design is based on the OECD Guideline 471, updated and adopted 21 July 1997.
Evaluation criteria:
The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.
Lawns were scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted.

Revertant colonies were counted by automated colony counter or by hand.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
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:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose Range Finding Assay
Doses tested in the mutagenicity assay were selected based on results of the dose rangefinding assay conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per dose. Ten doses of test article, from 6.67 to 5000 ug per plate were tested.
Cytotoxicity was observed with tester strain TA100 at 333 ug per plate and above in the absence of S9 mix as evidenced by reduced background lawns and a decrease in the number of revertants per plate. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with tester strain WP2uvrA in the presence or absence of S9 mix.

Mutagenicity Assay
In the initial mutagenicity assay, first trial (B1), all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.
In the confirmatory mutagenicity assay, second trial (C1), contamination was observed on many of the assay plates and several of the plates were observed to have reduced or absent bacterial background lawns. Due to the multiple technical problems observed the data generated were not used in the evaluation of the test article (the results have not been included).
The confirmatory assay was repeated in third trial (D1). In the repeat confirmatory mutagenicity assay, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. In this trial, a 2.7-fold increase was observed with tester strain WP2uvrA in the presence of S9 mix, however, this increase was not clearly dose-responsive and did not meet the criteria for a positive evaluation. In order to clarify this response, the test article was retested with tester strain WP2uvrA at the same doses in the presence of S9 mix in
fourth trial (D2). Also, due to variability in the vehicle control counts for tester strain TA100 in the absence of S9 mix, the test article was retested with tester strain TAI100 at the same doses in the absence of S9 mix.
In the fourth trial, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with tester strain WP2uvrA in the presence of S9 mix or with tester strain T A100 in the absence of S9 mix.

All criteria for a valid study were met.

Applicant's summary and conclusion

Conclusions:
The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of Aroclor™ induced rat liver (S9).
Executive summary:

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, the test article did not cause a positive increase in the mean number of revertants per plate with any of the tester strains in either the presence or absence of Aroclor™ induced rat liver (S9).