Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09October2001 to 26July2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
See individual study reports for purity information and CofA where applicable
Specific details on test material used for the study:
Batch No. 013673A1

Method

Target gene:
The bacterial reverse mutation assay detects point mutations, both frameshifts and/or base pair substitutions. The strains of Salmonella typhimurium and Escherichia coli used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his-) or tryptophan (trp-) dependant cells, are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan) only those cells which revert to histidine (his+) or tryptophan (trp+) independence are able to form colonies. The trace amount of histidine or tryptophan in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The his+ or trp+ revertant are readily discernible as colonies against the limited background growth of the his- or trp- cells. By utilizing several different tester strains, both base pair substitution mutations and frameshift mutations can be detected. The bacterial reverse mutation assay has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
other: Salmonella typhimurium histidine auxotrophs: uvrB gene (repair); rfa wall mutation (LPS); Escherichia coli tryptophan auxotroph WP2uvrA; uvrB gene (repair);
Metabolic activation:
with and without
Metabolic activation system:
Standard (Aroclor 1254-induced male Sprague-Dawley rat liver) and reductive (uninduced male Golden Syrian hamster liver with FMN) metabolic activation systems (S9s).
Test concentrations with justification for top dose:
A dose range finding study was performed using tester strains TA100 and WP2MvrA, and ten doses of test article ranging from 6.67 to 5000 µg/plate, in the absence of S9 and in the presence of standard S9 (one plate per dose). Inhibited growth (characterized by a decreased revertant frequency or thinning/absence of the background lawn) was observed in tester strain TA100 at doses >333 (µg/plate with S9 and >33.3 µg/plate without S9, and in tester strain WP2uvrA at doses >333 µg/plate with and without S9. In addition, the test article was found to be freely soluble at all doses evaluated with and without S9.

Based upon these results, the test article was evaluated in the initial mutagenicity assay in Salmonella tester strains at doses of 33.3, 100, 333,1000, 2000 and 3330 µg/plate in the presence of S9 (both types), and 10.0, 33.3, 100, 333, 1000 and 2000 ug/plate in the absence of S9. MIP Orange 3100 also was evaluated in tester strain WP2uvrA at doses of 33.3, 100, 333, 1000, 2000 and 3330 µg/plate in the presence of S9 (both types), and 3.33, 10.0, 33.3,100, 333 and 500 µg/plate in the absence of S9.
Vehicle / solvent:
Water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
congo red
other: 2-aminoanthracene; ICR-191
Details on test system and experimental conditions:
Frequency and Route of Administration. The tester strains were exposed to the test article via the preincubation modification of the Ames Test originally described by Yahagi et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In addition, the inclusion of a reductive metabolic activation system, as described by Prival and Mitchell (1982), allows detection of the mutagenicity of certain free aromatic amines released by the reduction of azo compounds.

In the preincubation methodology, the S9 mix (or phosphate buffer, where appropriate), the tester strain, and the test article were preincubated prior to the addition of molten agar. The agar and the preincubation reaction mixture were mixed and then overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of the test article, the vehicle controls and the positive controls were plated in triplicate.

Plating Procedures
These procedures were used in the dose range-finding study and the mutagenicity assays. Each plate was labelled with a code which identified the test article, test phase, tester strain, activation condition and dose level. The S9 mixes and dilutions of the test article were prepared immediately prior to their use.


When S9 mix was required, 500 µL of the appropriate mix was added to 13 x 100 mm glass culture tubes which had been pre-heated to 30 or 37 °C ± 2°C (for reductive and standard S9 mixes, respectively). To these tubes were added 100 µL of tester strain and 200 µL of test or control article. If S9 mix was not required, 500 µL of O.IM phosphate buffer was substituted for the standard S9 mix. After the required components had been added, the mixture was vortexed. Cultures treated with standard S9 or without S9 were incubated for 20 ± 2 minutes at 37 ± 2°C, while those treated with reductive S9 were incubated for 30 ± 2 minutes at 30 ± 2°C.

Following the 20- or 30-minute pre-incubation, 2 mL of molten selective top agar was then added to each tube, and the mixture was vortexed and overlaid onto 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay had solidified, the plates were inverted and incubated for 52 ± 4 hr at 37 + 2°C. Positive control articles were plated using a 50 µL plating aliquot.


Scoring the Plates
Plates which were not evaluated immediately following the incubation period were held at 5 ± 3°C until such time that colony counting and bacterial background lawn evaluation could take place.

Bacterial Background Lawn Evaluation. The condition of the bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate (except as noted below; see Protocol Deviation). 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 (O), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) also were noted.

Counting Revertant Colonies. Revertant colonies were counted by automated colony counter or by hand.
Rationale for test conditions:
The bacterial reverse mutation assay detects point mutations, both frameshifts and/or base pair substitutions. The strains of Salmonella typhimurium and Escherichia coli used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his-) or tryptophan (trp-) dependant cells, are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan) only those cells which revert to histidine (his+) or tryptophan (trp+) independence are able to form colonies. The trace amount of histidine or tryptophan in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The his+ or trp+ revertant are readily discernible as colonies against the limited background growth of the his- or trp- cells. By utilizing several different tester strains, both base pair substitution mutations and frameshift mutations can be detected. The bacterial reverse mutation assay has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes.
Evaluation criteria:
Bacterial Background Lawn Evaluation. The condition of the bacterial background lawn was evaluated macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate (except as noted below; see Protocol Deviation). 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 (O), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) also were noted.

Counting Revertant Colonies. 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
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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:
negative
Cytotoxicity / choice of top concentrations:
not determined
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:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay: Preincubation Method with a Confirmatory Assay indicate that, under the conditions of this study, the test item was positive in tester strain TA98 in the presence of reductive S9. The test item was uniformly negative in all other tester strain/S9 combinations.
Executive summary:

The results of the Salmonella-Escherichia coli/Mammalian-Microsome Reverse Mutation Assay: Preincubation Method with a Confirmatory Assay indicate that, under the conditions of this study, the test item was positive in tester strain TA98 in the presence of reductive S9. The test item was uniformly negative in all other tester strain/S9 combinations.