Eucalyptus dives, ext.

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:

1999-12-31 to 2000-02-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Eucalyptus dives, ext.

EC Number:

289-839-3

EC Name:

Eucalyptus dives, ext.

Cas Number:

90028-48-1

Molecular formula:

not applicable - UVCB

IUPAC Name:

(5R)-2-methyl-5-(propan-2-yl)cyclohexa-1,3-diene; (6R)-3-methyl-6-(propan-2-yl)cyclohex-2-en-1-one

Test material form:

other: liquid

Method

Target gene:

Histidine (his+)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 fraction induced by Aroclor 1254

Test concentrations with justification for top dose:

15, 50, 150, 500, 1500 and 5000 μg per plate without metabolic activation (-S9 mix)
50, 150, 500, 1500 and 5000 μg per plate with metabolic activation (+S9 mix)

Vehicle / solvent:

DMSO for test material and positive controls 2-aminoanthracene and 2-nitrofluorene
Distilled water for all other positive controls

Controls

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

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Metabolic activation system (S9 mix)
9000g supernatants of rat liver homogenate fractions (S9 mix) were prepared from livers of rats induced by Aroclor 1254 (500 mg/kg bw). S9 mix was prepared for each assay as follows: 0.335 ml distilled water); 0.5ml phosphate buffer pH 7.4 (NADP (0.1M, 0.04 ml), glucose-6-phosphate (1M, 0.005 ml), MgCl2/KCl (0.4M/1.65M, 0.02ml)) and S9 fraction (0.1ml).

Preparation of bacterial strains
TA1535, TA1537, TA98, TA100 and TA102 tester strains of Salmonella typhimurium grown in oxoid nutrient broth No 2 (2.5%) on a shaker for 14-15 hours at 37°C to a density of 0.5-3 x9 cells per ml, then kept at 4°C. Only fresh cultures were used for mutagenicity studies.
For determination of the cell titer, 0.1ml aliquotes of the 1 x 10^-6 dilution were spread over the completed medium plates and incubated overnight at 37°C.

Test procedure
The Salmonella mutagenicity test was performed by the standard plate-incorporation assay, with and without S9 activation. In brief, 0.1ml bacteria was added to 2 ml of top-agar followed by the test substance/vehicle/positive control, and 0.5ml S9 mix or phosphate buffer, for assays with and without metabolic activation, respectively. The test components were mixed throughly then poured immediately onto minimal agar plates and carefully spread to achieve a uniform spread of top agar. The minimal agar plates contained 20-25ml of 1.5% agar in Vogel-Bonner medium E with 2% glucose. Plates were kept for 48-72 hours at 37°C in the dark dark prior to scoring for revertant his+ bacteria colonies. Plates were prepared in triplcate for each experimental point and the experiment ran in full twice, with an interval of 3 days in between each experiment. Plates were also examined for the existence of a normal background lawn and/or precipitates and microscopically for microcolony growth.
Positive controls were run simultaneously per experiment and the genotypes of the tester strains were checked each experiment.

Evaluation criteria:

Spontaneous revertants observed using the five strains were compared to historical data established previously in the laboratory and with data obtaed by Ames.

Statistics:

Mean and standard deviation were calculated.
Chi squared test

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic at the two highest concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound was observed on the plates at 1500 and 5000 μg/plate μg/plate

COMPARISON WITH HISTORICAL CONTROL DATA: The number of spontaneous revertants observed for all five strains were similar to those previously reported in the laboratory and were within the same range as those cited by Ames et al., (1975). Results with the positive controls were also comparable/

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test substance was cytotoxic to strains TA1535, TA1437 and TA102 at 500 μg/plate, to TA98 at 1500μg/plate and TA100 at 5000 μg/plate.

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1
		Number of revertants (Mean± SD)
		TA98		TA100		TA102		TA1535		TA1537
Substance	Doses (μg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Control	0	35±3	55±13	154±16	148±21	305±8	348±23	56±7	27±5	13±5	23±7
Solvent control	0	34±6	51±12	157±5	150±18	304±7	325±13	48±2	24±6	12±8	25±5
Test item	15
	50	36±3	51±11	142±14	138±7	284±18	313±16	48±9	24±7	12±1	24±4
	150	28±9	45±4	163±19	150±4	270±41	315±35	38±7	25±8	13±3	26±3
	500	28±9	47±7	159±7	125±8	216±1*	282±29*	35±13*	22±5*	11±3*	23±4*
	1500**	35±5	50±7	150±10	148±19	217±11*	305±24*	35±10*	23±3*	11±2*	24±2*
	5000**	25±4*	37±5*	57±19*	111±28*	28±33*	114±35*	35±3*	14±5*	1±3*	10±4*
NaN3	0.7			434±10				688±123
2-NF	2.5	427±14
9-AA	59									153±26
Mitomycin C	0.15			810±67		810±67
2-AA	0.7		1496±126		1330±122
2-AA	1.5						1799±243		794±47		191±39
*bacteriotoxic
**precipitation seen
Experiment 2
		Number of revertants (Mean± SD)
		TA98		TA100		TA102		TA1535		TA1537
Substance	Doses (μg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Control	0	23±3	32±7	157±5	150±10	245±28	248±10	582±2	26±4	11±3	17±5
Solvent control	0	20±5	33±3	162±6	148±14	272±37	246±7	62±10	28±4	11±3	16±5
Test item	15									10±4
	50	26±3	31±8	132±9	137±12	227±8	233±30	227±8	27±5	13±5	19±3
	150	15±3	29±3	132±8	146±20	182±5	194±50	182±5	18±3	6±2	17±3
	500	12±3	35±2	121±11	35±2	137±29*	132±3*	137±29*	16±3*	8±3*	13±3*
	1500**	13±3*	32±3*	135±13	32±3	181±27*	159±6*	181±27*	18±4*	5±3*	13±5*
	5000**	0±0*	26±2*	34±19*	26±2*	0±0*	3±3*	0±0*	10±4*	0±0*	5±5*
NaN3	0.7			437±17*				657±29
2-NF	2.5	559±39
9-AA	59									282±29
Mitomycin C	0.15			777±33		777±33
2-AA	0.7		1028±12		1955±337
2-AA	1.5						961±175		557±63		543±29
*bacteriotoxic
**precipitation seen

Applicant's summary and conclusion

Conclusions:

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

The substance, under the conditions of the study, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102. ollowing Regulation (EC) 1272/2008, the substance is not classified as mutagenic.