2,6-dimethyloct-7-en-2-ol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to generally valid and/or internationally accepted testing guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report Date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Test guideline 3

Qualifier:

according to

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 4

Qualifier:

according to

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Test guideline 5

Qualifier:

according to

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. certificate)

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): Dihydromyrcenol
- Physical state: Clear, colorless slightly viscous liquid
- Analytical purity: 99.2% (COA)
- Purity test date: 29 October 2010
- Lot/batch No.: 9SFD04
- Expiration date of the lot/batch: 27 October 2011
- Storage condition of test material: Room temperature in the dark under nitrogen
- Other: Information relating to the identity, purity and stability of the test material was the responsibility of the Sponsor

Method

Target gene:

Salmonella typhimurium:
TA 1535, TA 100: hisG46
TA 1537: hisC3076
TA 98: hisD3052
Escherichia coli (WP2uvrA/pKM101):
contains ochre mutation; deficient in DNA repair system 9uvrA); contains the pKM101 plasmid

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:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test
0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 microg/plate

Experiment 1 (Range-finding)
15, 50, 150, 500, 1500 and 5000 microg/plate

Mutation Test (Main Test) - with preincubation
TA100 (with/without S9), TA1535 and TA1537 (without S9) and E. coli (without S9): 1.5, 5, 15, 50, 150, 500 and 1500 microg/plate

TA98 (with/without S9), TA1535 and TA1537 (with S9) and E. coli (with S9): 15, 50, 150, 500, 1500 and 5000 microg/plate

Vehicle / solvent:

dimethyl sulphoxide

The test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Controls 2

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Pos. Control for TA98 at 5 microg/plate (+S9)

Controls 3

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

Pos. Control for TA100 at 1 microg/plate (+S9); TA1535 and TA1537, 2 microg/plate (+S9); and E. coli WP2uvrA- at 10 microg/plate (+S9)

Controls 4

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Pos. Control for E. coli WP2uvrA- at 2 microg/plate (-S9); TA100 at 3 microg/plate (-S9); TA1535 at 5 microg/plate (-S9)

Controls 5

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Pos. Control for TA1537 at 80 microg/plate (-S9)

Controls 6

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Pos. Control for TA98 at 0.2 microg/plate (-S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A standard plate incorporation method was employed.

Preliminary Toxicity Test:
In order to select appropriate dose levels for use in the main test, a preliminary test were conducted in the presence or absence of S9. Ten dose levels and controls were tested up to and including 5,000 microg/plate at approximately half-log intervals. The assay was conducted by mixing 0.1 ml the bacterial culture (TA100 or WP2uvrA-), and 0.1 ml of the vehicle or test chemical mixture, 0.5 mL of S9-mix or phosphate buffer and 2.0 ml of molten agar supplemented with trace histidine or tryptophan and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 deg C, the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Experiment 1 (Range-finding Test):
Six concentrations of the test material were assayed with or without S9-mix against each tester strain using the above described direct plate incorporation method. An additional dose level and expanded dose range were selected in order to achieve both four non-toxic doses and the toxic limit of the test material.

Experiment 2 (Main Test):
A second experiment was performed with fresh bacterial cultures, test material and control solutions. The dose ranges selected were based on the range-finding test and an aborted main test (data not shown). Preincubation was employed. Thus, measured aliquots (0.1 mL) of each bacterial culture were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of vehicle or test material formulation and incubated for 20 minutes at 37 deg C with shaking at approximately 130 rpm prior to addition of 2 mL of molten trace histidine or tryptophan supplemented top agar. The contents of the tubes were mixed and poured onto the surface of Vogel-Bonner Minimal agar plates. This procedure was repeated for each bacterial strain either with or without S9.

NUMBER OF REPLICATIONS:
3 replicates/strain

DETERMINATION OF CYTOTOXICITY
Any toxic effects of the test substance would be detected by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.

Evaluation criteria:

Acceptance Criteria:

The following criteria must be met for acceptance:
- All tester strain cultures must exhibit a characteristic number of spontaneous revertants per plate in vehicle and untreated controls.
- The appropriate characteristics of each tester strain must be confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor.
- All tester strain cultures should be in the range of 1 to 9.9 x 10^9 bacteria per mL.
- Each mean positive control value should be at least 2x the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains and the integrity of the S9-mix
- The test should include a minimum of four non-toxic dose levels.

Evaluation Criteria:

A dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain either with or without metabolic activation. Biological relevance of the response is to be considered first, as recommended by the UKEMS sub-committee on Guidelines for Mutagenicity Testing (1989). Statistical methods can be used as an aid to evaluation but may not be the only determining factor for a positive response.

A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Mean values with standard deviations were reported.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Please refer to Tables 1 to 5 for details of results.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was toxic at 5000 microg/plate to tester strains TA100 and WP2uvrA-. The results are summarized in Table 1.

Table 1: Numbers of revertant colonies in the preliminary toxicity test

With (+) or without (-) S9-mix	Strain	Dose (mg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	93	100	120	101	123	133	132	106	128	152	113*
+	TA100	62	107	77	82	81	88	94	84	84	74	0*
-	WP2uvrA-	18	15	16	18	22	33	24	14	19	20	17*
+	WP2uvrA-	28	32	22	27	22	20	24	25	25	31	15*

* Partial absence of bacterial background lawn

Table 2: Range-finding test without metabolic activation

	Revertant colony counts (mean 3 replicates)
Addition (mg)	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	101	24	18	17	11
15	81	30	19	15	10
50	86	28	19	19	10
150	91	23	17	20	9
500	99	27	18	18	11
1500	92	27	20	14	10
5000	82*	15*	13	12*	2*
ENNG (3)	421
ENNG (5)		907
ENNG (2)			596
4NQO (0.2)				167
9AA (80)					354

* Partial absence of background lawn in all replicate plates.

Abreviations: ENNG, N-ethyl-N’-nitro-N-nitrosoguanidine; 9AA, 2-aminoacridine; BP, benzo(a)pyrene; 2AA, 2-aminoanthracene; 4NQO, 4-nitroquinoline-1-oxide

Table 3: Range-finding test with metabolic activation

	Revertant colony counts (mean 3 replicates)
Addition (mg)	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	88	14	22	19	14
15	90	15	22	21	14
50	87	14	21	15	14
150	88	10	19	21	10
500	74	11	23	19	10
1500	84	9	17	16	9
5000	72*	2*	17	12*	3*
2AA (1)	760
2AA (2)		212			195
2AA (10)			244
BP (5)				119

* Partial absence of background lawn in all replicate plates.

Table 4: Main Test without metabolic activation

	Revertant colony counts (mean 3 replicates)
Addition (mg)	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	106	28	26	11	12
1.5	107	24	26	N/T	14
5	103	27	17	N/T	12
15	108	29	20	15	9
50	100	25	21	10	8
150	107*	28	26	15	9
500	76*	27*	25	10	8*
1500	0*	0*	0*	0*	0*
5000	N/T	N/T	N/T	0*	N/T
ENNG (3)	406
ENNG (5)		291
ENNG (2)			498
4NQO (0.2)				100
9AA (80)					937

* Partial absence of background lawn in all replicate plates.

N/T - not tested at this dose level.

Table 5: Main Test with metabolic activation

	Revertant colony counts (mean 3 replicates)
Addition (mg)	TA100	TA1535	WP2uvrA-	TA98	TA1537
0	114	10	18	19	9
1.5	102	N/T	N/T	N/T	N/T
5	115	N/T	N/T	N/T	N/T
15	90	11	17	22	11
50	102	11	21	25	8
150	103	14	19	20	10
500	95*	11	15	18	7
1500	0*	0*	0*	0*	0*
5000	N/T	0*	0*	0*	0*
2AA (1)	813
2AA (2)		230			199
2AA (10)			212
BP (5)				248

* Partial absence of background lawn in all replicate plates.

N/T - not tested at this dose level.

Applicant's summary and conclusion

Conclusions:

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

The test material was considered non-mutagenic under the conditions of this test.

Executive summary:

In the first experiment, the test material caused a visible reduction in the growth f the bacterial background lawns of all the Salmonella stains at 5000 microg/plate in both the presence and absence of S9. No toxic response to Escherichia coli strain WP2uvrA- was noted. In the second experiment (with pre-incubation) the test material reduced the bacterial background lawns of all the tester strains in both the presence and absence of S9. The sensitivity of the various stains varied with the presence or absence of S9 and the strain involved; therefore, the test material was tested up to the maximum recommended dose level. No test material precipitate was observed on the plates at any of the doses tested either with or without S9.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9 -mix and the sensitivity of the bacterial strains.

The test material was considered to be non-mutagenic under the conditions of this test.