(2α,4aα,8β)-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalen-8(5H)-one

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:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication.

Data source

Reference

Reference Type:

publication

Title:

Salmonella/microsome assay was performed for the given test chemical.

Author:

Gomes-Carneiro et al.

Year:

1998

Bibliographic source:

Mutation Research

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cineole

EC Number:

207-431-5

EC Name:

Cineole

Cas Number:

470-82-6

Molecular formula:

C10H18O

IUPAC Name:

1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane

Details on test material:

- Name of test material: Cineol (eucalyptol)
- IUPAC name: 1,3,3-Trimethyl-2-oxabicyclo[2.2.2]octane
- Molecular formula: C10H18O
- Molecular weight: 154.2512 g/mol

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA102, TA100, TA98 and TA97a

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Lyophilized rat liver S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

0, 250, 500, 750, 1000, 1250, 1500, 2000, 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The chemical was soluble in Ethanol

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

benzo(a)pyrene

mitomycin C

other: NPD (1 µg/plate). 2AF (10 µg/plate), 2AA (0.5 or 1 µg/plate)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration - triplicate
- Number of independent experiments - each experiment was repeated at least once

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): approximately 1.2x10^9 bacteria per milliliter
- Test substance added - in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No data
- Exposure duration/duration of treatment: 72 h
- Harvest time after the end of treatment (sampling/recovery times): No data

Evaluation criteria:

A dose related increase in the number of revertants was noted whether it be twofold over background or not

Statistics:

means ±SD

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA102, TA100, TA98 and TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable): A preliminary test carried out with TA100 strain without and with addition of S9 mixture. The upper limit of the dose interval tested was either the highest non-toxic dose or the lowest toxic dose determined in this preliminary assay.

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table - Mutagenicity testing of the test chemical in the Salmonella/microsome assay TA100, TA98, TA97a and TA102 tester strains

Monoterpene	Dose (µg/plate)	Number of revertants (Mean±SD)
		TA 100		TA 98		TA 97a		TA 102
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Test chemical	2500	-	-	-	-	-	142*	-	-
	2000	98±6*	157±12	-	51±18	146±8	180±4	-	514±129*
	1500	149±26	180±12	37±2	57±13	170±13	140±11	617±140	722±21
	1250	164±10	185±14	34±5	58±4	168±6	177±7	605±83	706±4
	1000	183±13	179±13	38±1	60±6	166±12	190±40	692±31	777±31
	750	196±10	177±26	47±6	59±8	174±19	180±10	642±23	698±30
	500	199±16	190±19	41±7	59±2	163±26	210±9	753±41	758±26
	250	193±12	-	43±2	-	168±17	-	686±19	794±32
	0	176±14	196±18	48±5	54±6	160±22	185±22	660±91	776±36
	PC	898±51	1003±66	204±34	210±34	998±52	1048±92	4102±317	1952±86

(–) Dose not tested.

(*) Toxicity apparent as an alteration of the background lawn.

(/+) Mutant counts of individual plates.

Values are the means±SD of three plates of one (out of two) representative experiment.

Applicant's summary and conclusion

Conclusions:

Test chemical did not induce mutation in the Salmonella typhimurium strains TA102, TA100, TA98 and TA97a both in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Salmonella reverse mutation assay was performed to determine the mutagenic nature of the given test chemical on a tester strains of Salmonella typhimurium TA102, TA100, TA98 and TA97 with and without metabolic activation system extracted from Lyophilized rat liver induced by Aroclor 1254 at dose concentrations of 0, 250, 500, 750, 1000, 1250, 1500, 2000, 2500 µg/plate. The Salmonella mutagenicity test was performed by the plate incorporation method. Briefly, 2 ml of top-agar was mixed with 100µl of an overnight grown culture of S. typhimurium, 100µl of the test substance (diluted in ethanol analytical grade, Merck, KGaA), the negative control, or the positive control (PC) and 500µl of the phosphate buffer or the S9 mixture. Ethanol served as the negative (solvent) controls, while the positive control substances were: SA (0.5µg/plate), NPD (1µg/plate), MC (0.5µg/plate), 4-NQNO (1µg/plate), 2AF (10µg/plate), 2AA (0.5 or 1µg/plate), and B-(α)-P (50µg/plate). SA and MC were dissolved in distilled water and dimethyl sulfoxide (DMSO) was used as vehicle for the other positive controls. Plates were incubated at 37°C for 72 h in the dark and then scored for revertant his+ bacteria colonies. Every determination was made in triplicate and each experiment was repeated at least once in order to check the reproducibility of the results. A preliminary test carried out with TA100 strain without and with addition of S9 mixture. The upper limit of the dose interval tested was either the highest non-toxic dose or the lowest toxic dose determined in this preliminary assay. The given test chemical was non toxic at highest doses of 2000–2500µg/plate. No mutagenic effect was observed with the four bacterial tester strains, in the absence as well as in the presence of extrinsic metabolic activation. Hence, the given test chemical is not likely to classify as a gene mutant in vitro.