(1R)-1-[(4R,4aR,8aS)-2,6-bis(3,4-dimethylphenyl)tetrahydro[1,3]dioxino[5,4-d][1,3]dioxin-4-yl]ethane-1,2-diol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19th August 1992 to 2nd November 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. A guideline is not specified in the report but the study is similar to OECD guideline 471.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Principles of method if other than guideline:

The experimental materials, methods and procedures are based on those described by Ames et al (1975).

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Target gene:

S. typhimurium: histidine operon
E. coli: tryptophan operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538

Additional strain / cell type characteristics:

other: The tester strains contain rfa wall mutations and a deletion of the uvrB gene. Strains TA98 and TA100 also contain the R-factor plasmid, pKM101.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: The tester strain contains a uvrA DNA repair deficiency which enhances it's sensitivity to some mutagenic compounds.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Concentration range in the main test (with and without metabolic activation): 100, 250, 500, 1000. 2500 and 5000 µg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR-191 and 4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation).
The test article, the tester strain and the S9 mix (where appropriate) were combined in molten agar which was overlaid onto a minimal agar plate. Following incubation at 37 ± 2°C for 48 ± 8 hr, revertant colonies were counted.


DURATION
- Preincubation period: To ensure that cultures were harvested in late log phase, the length of incubation was determined by spectrophotometric monitoring of culture turbidity. Cultures were harvested once a predetermined turbidity was reached as determined by a percent transmittance (%T) reading on a spectrophotometer. This target turbidity ensures that cultures have reached a density of at least 0.5E+09 cells per mL and that the cultures have not overgrown. Overgrown (stationary) cultures may exhibit decreased sensitivity to some mutagens. Cultures were removed from incubation when the target %T was reached and were placed at 5 ± 3°C.
- Exposure duration: 48 ± 8 h
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 48 ± 8 h; 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.


NUMBER OF REPLICATIONS: All doses of test article, vehicle controls, negative controls and positive controls were plated in triplicate.


NUMBER OF CELLS EVALUATED: Not specified


DETERMINATION OF CYTOTOXICITY
The cytotoxicityof the test material was determined in a dose range finding study:
- Method: The dose rangefinding study was performed using tester strains TA100 and WP2uvrA- both in the presence and absence of S9 mix. Ten doses of test article were tested at one plate per dose. The test article was checked for cytotoxicity up to a maximum concentration of 5 mg per plate.
Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn.
No cytotoxicity was observed in the dose rangefinding study so the highest dose of test article used in the mutagenicity assay was the same as that tested in the rangefinding study.


OTHER EXAMINATIONS:
None performed

Evaluation criteria:

The condition of the bacterial background lawn was evaluated for evidence 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.
The number of revertant colonies per plate for the vehicle and negative controls and all plates containing test article were counted manually. The number of revertant colonies per plate for the positive controls were counted by an automated colony counter.
For all replicate platings, the mean revertants per plate and the standard deviation were calculated.

Statistics:

For all replicate platings, the mean revertants per plate and the standard deviation were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Test results 3

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In DMSO, the test article formed a cloudy white suspension at 50 mg per mL (5,000 µg per plate) which was the most concentrated stock dilution of test article prepared. The test article became a solution at 5 mg per mL (500 µg per plate) and remained in solution at all subsequent dilutions prepared for the mutagenicity assay.
- Other confounding effects: None specified


RANGE-FINDING/SCREENING STUDIES: Doses to be tested in the mutagenicity assay were selected based on the results of the dose rangefinding study conducted on the test article using tester strains TA100 and WP2uvrA- in both the presence and absence of S9 (one plate per dose). Ten doses of test article, from 6.67 to 5,000 µg per plate, were tested. No cytotoxicity was observed in either the presence or absence of S9 as evidenced by no observed decrease in the number of revertants per plate. The bacterial background lawn was evaluated as normal up to the 1,000 µg per plate dose in both the presence and absence of S9. The lawns on the plates above this dose could not be evaluated due to the presence of test article precipitate.

Remarks on result:

other: other: preliminary test

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

In the initial mutagenicity assay and the confirmatory assay, all data were acceptable and no positive increases in the number of revertants per plate were observed with any of the tester strains either in the presence or absence of 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, in both an initial and a confirmatory assay, the test article, 1,3:2,4 -Bis (3,4-dimethylbenzylidene) sorbitol, did not cause a positive increase in the numbers of revertants per plate of any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9).