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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
Adequacy of study:
key study
Study period:
26 August 2003 to 5 November 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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)
Deviations:
no
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:
- Sponsor’s Identification: EK2003-0090
- CIN: 10084241
- Lot No.: DEV34241
- KAN: 632021-1
- Date Received: 11 August 2003
- Physical Description: Light-orange powder
- Storage Conditions: Room temperature

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-) dependent 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+ revertants are readily discernable as colonies against the limited background growth of the his- or trp- cells. By using 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 / 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):
Tester Strain Genotypes:
TA98: hisD3052, uvrB, rfa and pKM101
TA100: hisG46, uvrB, rfa and pKM101
TA1535: hisG46, uvrB and rfa
TA1537: hisC3076, uvrB and rfa
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Tester Strain Genotype:
WP2uvrA: trp, uvrA and pKM101
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
10.0, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In solubility testing, the test article was observed to be insoluble in deionized water at approximately 100 mg per mL.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene and ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION
In agar (plate incorporation)

DURATION
- Exposure duration: After required components had been added, the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar contained in a 15 x 100 mm petri dish. After the overlay solidified, plates were inverted and incubated for 52 ± 4 hours at 37 ± 2 °C.
- Fixation time (start of exposure up to fixation or harvest of cells): To ensure that cultures were harvested in late log phase, length of incubation was determined by spectrophotometric monitoring of culture density. Cultures were harvested once a predetermined density was reached which ensured that cultures had reached a density of at least 0.5 x 10ˆ9 cells per mL and had not overgrown. Overgrown (stationary) cultures may exhibit decreased sensitivity to some mutagens. Cultures were removed from incubation when target density was reached and were held at >0 to 10 °C until used in the assay.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate andor by thinning or disappearance of the bacterial background lawn.
Evaluation criteria:
ASSAY EVALUATION CRITERIA
Once criteria for a valid assay had been met, responses observed in the assay were evaluated.

Tester Strains TA98, TA100, and WP2uvrA(pKM101)
For a test article to be considered positive, it must have produced at least a 2-fold increase in mean revertants per plate of at least one of these tester strains over the mean revertants per plate of its respective vehicle control.
This increase in mean number of revertants per plate must have been accompanied by a dose response to increasing concentrations of test article.

Tester Strains TA1535 and TA1537.
For a test article to be considered positive, it must have produced at least a 3-fold increase in mean revertants per plate of at least one of these tester strains over the mean revertants per plate of its respective vehicle control. This increase in mean number of revertants per plate must have been accompanied by a dose response to increasing concentrations of test article.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
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(pKM101) in the presence and absence of S9 (one plate per dose). Ten doses of test article, from 6.67 to 5000 µg per plate, were tested in Trial A1. No cytotoxicity was observed with either tester strain in the presence or absence of S9 mix, as evidenced by no dose-related decreases in the number of revertants per plate. Bacterial background lawns were observed to be normal up to 1000 µg per plate in the presence of S9 mix and up to 5000 µg per plate in the absence of S9 mix. Test article precipitate was observed on the plates at 667 µg per plate and above in the absence of S9 mix. In the presence of S9 mix, test article precipitate was observed at 3330 µg per plate and above with TA100 and at 1000 µg per plate and above with WP2uvrA(pKM101). The bacterial background lawns were obscured by precipitate at 3330 µg per plate and above in the presence of S9 mix.

Any other information on results incl. tables

Test Article Handling

The test article was stored at room temperature. In solubility testing, the test article was observed to be insoluble in deionized water at approximately 100 mg per mL. In dimethylsulfoxide (DMSO) the test article was observed to form a transparent, brown, non-viscous, solution at 100 mg per mL . For this reason DMSO (Acros Organics, Lot No. A017777101 for Trial 25446-Al; EM Science, Lot No. 42233 for Trial 25446-B1 and Lot No. 42317 for trials 25446-Cl and 25446-Dl) was chosen as the vehicle. In the initial mutagenicity assay (Trial 25446-Bl), the test article was observed to form a very viscous, heterogeneous suspension at 100 mg per mL. For this reason, the test article was diluted with additional DMSO to achieve a top stock concentration of 50.0 mg per mL. At a concentration of 50.0 mg per mL, the test article was observed to form a transparent, dark-orange, non-viscous solution. The test article was observed to remain in solution at all succeeding dilutions prepared for the mutagenicity assay.

Mutagenicity Assay

The mutagenicity assay results are presented in the attached Tables.

Doses tested in the mutagenicity assay with all tester strains were 10.0, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate in the presence and absence of S9 mix.

In the initial mutagenicity assay, Trial B1, all data were acceptable and no positive increases in the mean number of revertants per plate were observed with any tester strain in either the presence or absence of S9 mix. A dose-related reduction in revertants per plate was observed with tester strain WP2uvrA(pKM101) in the absence of S9 mix. No other dose-related reductions in revertant count were observed. Test article precipitate was observed on the plates in the presence of S9 mix at 3330 µg per plate and above and in the absence of S9 mix at 1000 to 3330 µg per plate and above. The test article precipitate obscured the bacterial background lawns at 3330 µg per plate and above in both the presence and absence of S9 mix.

In the confirmatory mutagenicity assay, Trial C1, the mean vehicle control value for tester strain TA98 in the absence of S9 mix was not within the acceptable range for this strain as specified in the protocol. For this reason, the data generated with TA98 in the absence of S9 mix in Trial C1 were not used in the evaluation of the test article. The test article was re-tested

with tester strain TA98 in the absence of S9 mix in Trial D1. All other data generated in Trial C1 were acceptable and no positive increases in the mean number of revertants per plate were observed with any tester strain in either the presence or absence of S9 mix. A dose-related reduction in revertants per plate was observed with tester strain WP2uvrA(pKM101) in the absence of S9 mix. No other dose-related reductions in revertant count were observed. Test article precipitate was observed on the plates in the presence of S9 mix at 1000 to 3330 µg per plate and above and in the absence of S9 mix at 333 to 3330 µg per plate and above. The test article precipitate obscured the bacterial background lawns at 3330 pg per plate and above in both the presence and absence of S9 mix.

In the repeat mutagenicity assay, Trial D1, all data were acceptable and no positive increases were observed with tester strain TA98 in the absence of S9 mix. No cytotoxicity was observed as evidenced by no dose-related decreases in the mean number of revertants per plate.

Test article precipitate was observed on the plates (obscuring the bacterial background lawns) at 3330 µg per plate and above.

All criteria for a valid study were met.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

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

In a GLP compliant study conducted in line with OECD Guideline 471, butanoic acid, 4-[[4-[7-chloro-6-(1,1-dimethylethyl)-3H-pyrazolo[1,5-b][1,2,4]triazol-2-yl]phenyl]amino]-4-oxo, tetradecyl ester was evaluated for the ability to induce reverse mutations either in the presence or absence of mammalian microsomal enzymes at the histidine locus in the genome of several strains of Salmonella typhimunium and at the tryptophan locus of Escherichia coli tester strain WP2uvrA(pKM101).

Doses tested in the mutagenicity assay were selected based on the results of a dose range finding assay using tester strains TA100 and WP2uvrA(pKM101) and ten doses of test article ranging from 6.67 to 5000 µg per plate, one plate per dose, both in the presence and absence of S9 mix.

Tester strains used in the mutagenicity assay were Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA(pKM101). The assay was conducted in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. Doses tested with all tester strains in the initial mutagenicity assay were 10.0, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate in both the presence and absence of S9 mix. Results of the initial mutagenicity assay were confirmed in an independent experiment.

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