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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 May 1994 - 15 November 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

214-946-9

EC Name:

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

Cas Number:

1222-05-5

Molecular formula:

C18H26O

IUPAC Name:

4,6,6,7,8,8-hexamethyl-1,3,4,6,7,8-hexahydrocyclopenta[g]isochromene

Test material form:

liquid: viscous

Method

Target gene:

Histidine and tryptophan genes

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

-Preliminary test (TA100 and WP2 UvrA): 6.7-5000 µg/plate (with and without S9).
-First and second experiment (TA98, TA100, TA1535, TA1537, TA1538 and WP2 UvrA: 0,10,33,100,333,1000,5000 µg/plate (with and without S9).

The results of the dose range-finding study indicate that precipitate, but no appreciable toxicity was observed at highest dose. 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: HHCB can be dissolved in acetone

Controls

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Positive control substance:

2-nitrofluorene

other: 2-aminoanthracene (with S9 activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: reduction of background lawn.

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

METHOD OF TREATMENT/ EXPOSURE:
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. 0.5 mL 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 finding 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.

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

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to the recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to the recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to the recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to the recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to the recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: 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.

RANGE-FINDING/SCREENING STUDIES (if applicable): TA100 and WP2 UvrA: 6.7-5000 µg/plate (with and without S9). No cytotoxicity was observed up to the highest dose.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : All positive controls gave positive responses to the systems within acceptable ranges.
- Signs of toxicity : No cytotoxicity was observed up to the highest dose
- Mean number of revertant colonies per plate and standard deviation : 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.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see report
- Negative (solvent/vehicle) historical control data: see report

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD TG 471.

Executive summary:

Test material (>99% pure) in acetone was tested in the Ames test (OECD TG 471) in accordance with GLP in absence or presence of Aroclor-induced rat liver S9 at doses 0, 10, 33, 100, 333, 1000 and 5000 mg/plate using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA1538 and Escherichia Coli strain WP2 UVRA and appropriate positive controls in triplicate. The doses were based on a preliminary assay. Slight precipitation was seen at the three highest doses (≥333 µg/plate) but no cytotoxicity was observed. All positive controls gave positive responses to the systems within acceptable ranges. No significant increase in the number of revertant colonies was observed at any dose with any of the six strains with or without activation. Based on this the substance is not mutagenic in this test.