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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study has been conducted to OECD guidelines and to GLP, and therefore meets the criteria for Klimisch code 1. However as this study is used in a read-across approach Klimisch 2 is assigned.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1988

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
Details on test material:
The test substance was an analogue of CAS 70024-69-0 described as C20-24 alkaryl calcium salt derivative

Method

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
Metabolic activation:
with and without
Metabolic activation system:
araclor
Test concentrations with justification for top dose:
0.1, 0.33, 1.0, 3.33 and 10 mg/plate
Vehicle / solvent:
pluronic F127 25 % w/w in ethanol
Controls
Negative solvent / vehicle controls:
yes
Remarks:
Pluronic F127 25% w/w in ethanol
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
other: 2-aminoanthracene and ICR-191
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3/dose group/strain/treatment set

Evaluation criteria:
Number of revertant colonies.
Statistics:
Mean revertant colony count and standard deviation were determined for each dose point.

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle 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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: No reduction in the number of revertants/plate was observed in the range-finding study with strains TA100 and WP2uvrA.
ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed in the dose range-finding study with strains TA100 and WP2uvrA with or without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

No cytotoxic response was seen in the dose range finding study.

The positive control exhibited at least a 3 fold increase in revertant colonies.

Results: The test substance was not genotoxic in this assay with or without metabolic activation.

Remarks

No cytotoxicity was observed in the dose range-finding study with tester strains TA100 and WP2uvrA with or without metabolic activation as evidenced by normal background lawn and no reduction in the number of revertants/plate. The S9 optimization study was performed using TA98 and TA100 with the highest non-cytotoxic dose of test article, (10,000 ug/plate) and concentrations of S9 mix of 25-400 µl. In the absence of any effect 25 µl S9 mix/plate was used in the mutagenicity study. The test material formed a stable emulsion with the vehicle and the dilutions were well dispersed in the top agar. However after incubation test material was visible at all dose levels in the top layer. The test material was not cytotoxic to any tester strain. In the repeat study statistically significant increases in revertant colonies were observed in TA1535 without metabolic activation and in WP2uvrA with metabolic activation. However since these findings were not found during the first experiment they were not considered biologically significant. The positive control for each respective test strain exhibited at least a 3-fold increase (with or without S9) over the mean value of the vehicle control for a given strain, confirming the expected positive control response. Dosing solution analysis confirmed that high dose concentration was acceptable.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was not mutagenic to Salmonella and E. Coli strains with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA98, TA100, TA1535 and TA 1537 of S. typhimurium and E. coli WP2uvrA were exposed to an analogue of C20-C24 alkaryl calcium salt derivative (Analogue of CAS 70024 -69 -0), at concentrations of 100, 330, 1000, 3330 and 10000 µg/plate in the presence and absence of mammalian metabolic activation (US-EPA, 1989, Ames Test according to OECD 471). A Dose Range-finding Study was conducted using tester strains TA98 and TA100, and dose levels of test material ranging from 0.003 to 10 mg/plate were used. No cytotoxicity was observed in the dose range-finding study with tester strains TA100 and WP2uvrA with or without metabolic activation as evidenced by normal background lawn and no reduction in the number of revertants/plate. The S9 optimization study was performed using TA98 and TA100 with the highest non-cytotoxic dose of test article, (10,000 µg/plate) and concentrations of S9 mix of 25400 µl. In the absence of any effect 25 µl S9 mix/plate was used in the mutagenicity study. In the main study there were two treatment sets for each tester strain, with (+S9) and without (-S9) metabolic activation. Each of the tester strains was dosed with five concentrations of test substance, vehicle controls, and a positive control. Three plates/dose group/strain/treatment set were evaluated. The results of the initial assay were confirmed in a second independent experiment. 100 µl of test material, positive control or vehicle control were added to each plate along with 100 µl of tester strain, S9 mix (if needed) and 2.0 ml of top agar. This was overlaid onto the surface of 25 ml minimal bottom agar in a petri dish. Plates were incubated for 48 hours at 37oC. The condition of the bacterial background lawn was evaluated for cytotoxicity and test article precipitate. The test material formed a stable emulsion with the vehicle and the dilutions were well dispersed in the top agar. However after incubation test material was visible at all dose levels in the top layer. The test material was not cytotoxic to any tester strain. In the repeat study statistically significant increases in revertant colonies were observed in TA1535 without metabolic activation and in WP2uvrA with metabolic activation. However since these findings were not found during the first experiment they were not considered biologically significant. The positive control for each respective test strain exhibited at least a 3-fold increase (with or without S9) over the mean value of the vehicle control for a given strain, confirming the expected positive control response. Dosing solution analysis confirmed that high dose concentration was acceptable. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.