1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylindeno[5,6-c]pyran

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

study initiation at May 17th 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study is conducted according to OECD TG 471, without deviations that influence the quality of the results, in compliance with GLP.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report Date:

1994

Reference 2

Reference Type:

publication

Title:

Genotoxicity Tests with 6-Acetyl-1,1,2,4,4,7- hexamethyltetraline and|1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma|--benzopyran.

Author:

Api, A.M., and San, R.H.C.

Year:

1999

Bibliographic source:

Mutation Research, 446: 67-81.

Materials and methods

Test guideline

Qualifier:

according to

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): HHCB
- Physical state: clear, colorless viscous liquid
- Analytical purity: >99%
- Storage condition of test material: 2 - 8 degrees celcius, protected from exposure to light. Headspace was purged with nitrogen.

Method

Target gene:

TA98, TA100, TA1535, TA1537, TA1538
WP2 UVRA

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538; Escherichia coli WP2 uvrA with and without S-9 activation.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

10 and 5000 ug/plate

The maximum dose tested was 5000 µg per plate. This dose was delivered to the test system as a clear solution in acetone using a plating aliquot of 50 µl. The results of the dose range-finding study indicate that precipitate but no appreciable toxicity was observed. Therefore, the maximum dose that was plated in the mutagenicity assay was 5000 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: no data

Controls

Untreated negative controls:

yes

Remarks:

acetone

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (with S9 activation); 2-nitrofluorene (without S9 activation)

Details on test system and experimental conditions:

Ames test
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: 12 hours in shaker/incubator
- Exposure duration: 48 - 72 hours

Frozen Permanent Stocks were prepared by growing fresh overnight cultures, adding DMSO (0.09 ml/ml of culture) and freezing away approximately 1.5 ml aliquots in glass vials. Frozen Permanent Stocks were stored at <_-70°C. Master plates were prepared by streaking each Salmonella tester strain from a frozen permanent onto minimal medium supplemented with histidine (260 µM), biotin (3 µM) and, for strains containing the R-factor, ampicillin (25 µg/ml). Master plates for E. coli were prepared by streaking onto nutrient bottom agar. Master plates were incubated at 37±2°C for 24 to 48 hours and stored at 4±2°C.

Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing ~50 ml of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37±2°C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

On the day of their use in the mutagenicity assay, all tester strain cultures were checked for the correct genotype. The presence of the rfa wall mutation and the deletion in the uvrB gene were confirmed for Salmonella by demonstration of sensitivity to crystal violet and ultraviolet light, respectively. The presence of the pKM101 plasmid was confirmed for Salmonella tester strains TA98 and TA100 by demonstration of resistance to ampicillin. The deletion in the uvrA gene was confirmed for E. coli by demonstration of sensitivity to ultraviolet light. Spontaneous reversion frequencies in the vehicle controls that are characteristic of the respective strains were demonstrated by plating 100 µl aliquots of the culture along with the appropriate vehicle on selective media.

Test article dilutions were prepared immediately before use. One-half (0.5) milliliter of S9 or Sham mix, 100 µl of tester strain and 50 µl of vehicle or test article were added to 2.0 ml of molten selective top agar at 45±2°C. After vortexing, the mixture was overlaid onto the surface of 25 ml of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 4±2°C until colony counting could be conducted. The condition of the bacterial background lawn was evaluated for evidence of test article toxicity and precipitate by using a dissecting microscope. This toxicity and precipitate were scored relative to the vehicle control plate, using the criteria and codes that appear in Figure 3. Revertant colonies for a given tester strain and
activation condition, except for the positive controls, were counted either entirely by automated colony counter or entirely by hand unless 1) the assay is the dose range f i nding assay, 2) the plate meets the criteria for toxicity defined in the protocol or 3) the plate exhibits definitive mutagenic activity and precipitate accounts for <25 % of the machine-determined revertant count. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually except as noted above.




OTHER:

Evaluation criteria:

For a test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:

Strains TA1535, TA1537 and TA1538
Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value.

Strains TA98, TA100 and WP2 uvrA
Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate was calculated.

Results and discussion

Test results

Species / strain:

bacteria, other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No cytotoxicity at the highest dose

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Slight precipitation is seen at the three highest doses (≥333 µg/plate). All dose levels of HHCB, acetone (negative control) and positive controls were plated in triplicate. All positive controls gave positive responses to the systems within acceptable ranges. No significant increase in the number of revertant colonies was observed for HHCB at any dose with any of the six strains with or without activation.

Remarks on result:

other: other: Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538; Escherichia coli WP2 uvrA with and without S-9 activation.

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

No significant increase in the number of revertant colonies
was observed at doses of 10 - 5000 ug/plate.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

All positive controls gave positive responses to the systems within acceptable ranges. No significant increase in the number of revertant colonies was observed for HHCB at any dose with any of the six strains with or without activation

Executive summary:

HHCB (>99% pure) in acetone was tested in the Ames test in absence or presence of Aroclor-induced rat liver S9 at a dose ranging from 10 to 5000 mg/plate according to OECD guideline 471 using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and Escherichia Coli strain WP2 UVRA and appropriate positive controls. Based on preliminary range-finding studies, doses of 10 (not tested in confirmation assay), 33, 100, 333, 1000, 3333 (only tested in confirmation assay) or 5000 µg HHCB/plate were used. Slight precipitation was seen at the three highest doses (≥333 µg/plate). All dose levels of HHCB, acetone (negative control) and positive controls were plated in triplicate. All positive controls gave positive responses to the systems within acceptable ranges. No significant increase in the number of revertant colonies was observed for HHCB at any dose with any of the six strains with or without activation.