Exo-1-methyl-4-(1-methylethyl)-7-oxabicyclo[2.2.1]heptan-2-ol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Not the recommended combination of strains according to guideline tested, positive controls missing

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1S,2S)-1-methyl-4-(propan-2-yl)-7-oxabicyclo[2.2.1]heptan-2-ol

Cas Number:

87172-89-2

IUPAC Name:

(1S,2S)-1-methyl-4-(propan-2-yl)-7-oxabicyclo[2.2.1]heptan-2-ol

Test material form:

solid

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA1538, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague Dawley rats

Test concentrations with justification for top dose:

50, 167, 500, 1667 and 5000 µg/plate

Vehicle / solvent:

water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine (2-Aminoanthracene); with metabolic activation: all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: The plates were incubated for 48 - 72 hours in the dark at 37 °C

NUMBER OF REPLICATIONS: 3

PRELIMINARY TOXICITY SCREEN:
The preliminary toxicity screen for the Ames Assay performed without metabolic activation used two of the histidine auxotrophs of S. typhimurium, TA 1538 and TA 100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the S. typhimurium tester strains. The test compounds was prepared to a concentration of 50 mg/mL and five different levels tested for toxicity. Top agar, used as an overlay, was reconstituted into molten state and supplemented with 0.5 mM histidine - 0.5 mM biotin at a volume of 0.1 mL/mL of agar, and maintained at 45 °C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45 °C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 mL aliquotes of top agar solution, 0.1 mL of tester strain and 0.1 mL of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37 °C incubator. The plates were uncubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria.

Evaluation criteria:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies with at least one dose point inducing a mutant frequency value that is two-fold the solvent control value. Significance at the 95% confidence limit is determined by the program developed by Moore and Felton (1983). This program applies a linear regression analysis to the data points and any P value greater than 0.05 is considered significant. In addition, the greater the P value, the higher the probability that the data points fit a linear response. This dose-response relationship accasionally necessites slight modification of the original doses in a repeat assay. Alternatively, if the solvent control is within the 95% confidence limits of the test chemical procedures the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is definied as the absence of a reproducible increase in the number of histidine-independent colonies.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, 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

Any other information on results incl. tables

Table 1: Results of the Ames test with the test item

Test item	Mean spondaneous revertants/plate
	TA 1535	TA 1537	TA 1538	TA 98	TA 100
Without metabolic activation (-S9)
Solvent control; H2O	20±6	11±2	12±3	26±6	134±13
50	24±8	12±3	7±12	34±5	146±17
167	17±3	11±4	8±4	34±6	139±34
500	21±6	13±4	11±2	28±3	148±14
1667	18±1	10±2	8±3	25±6	149±17
5000	22±4	12±4	12±4	27±3	118±16
Positive controls
Sodium azide	443±30*				516±50*
9-Aminoacridine		520±100*
2-Nitrofluorene			319±26*	273±14*
With metabolic activation (+S9)
Solvent control; H2O	17±2	11±2	17±1	35±1	112±31
50	18±1	14±1	15±1	40±3	122±110
167	23±7	19±6	18±6	29±7	133±4
500	13±5	16±3	17±5	49±114	117±12
1667	15±2	10±4	17±3	34±6	113±10
5000	18±6	16±3	18±5	39±4	120±23

Applicant's summary and conclusion

Conclusions:

Under the conditions of this Ames test die test substance did not induce gene mutation in strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of Salmonella typhimurium with and without metabolic activation.