rel-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]hexan-3-ol

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:

18 January 2006 - 17 February 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

21 November 2005

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

rel-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]hexan-3-ol

EC Number:

814-113-5

Cas Number:

253454-23-8

Molecular formula:

C15H30O

IUPAC Name:

rel-1-[(1R,6S)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Test material form:

liquid

Specific details on test material used for the study:

Hysandol called Timberol in the test report may be a multi of the cis and trans-isomer, while Hysandol is a mono-trans isomer. The cis isomer is expected to have the same results for this endpoint because it is stereo isomer.

Method

Target gene:

Histidine

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:

S9 from livers of male Sprague-Dawley rats which had received phenobarbitone/β-naphtoflavone

Test concentrations with justification for top dose:

Dose range finding study: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Main study (with and without S9 mix): 50, 150, 500, 1500 and 5000 µg/plate for both tests.
Top dose was choosen according toe OECD guideline 471.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test substance was fully soluble in DMSO compared to insoluble in sterile distilled water at the same concentration (50 mg/mL).

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

with S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

with S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

with S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

with S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

without S9

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar

DURATION
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION: Top agar was prepared using 0.6% Difco Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin solution added to each 100 mL of top agar.

DETERMINATION OF CYTOTOXICITY
- Method: growth of bacterial background lawn using a Domino colony counter.

Evaluation criteria:

According to OECD guideline 471 there are several criteria for determining a positive result, such as 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 with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods can also be used as an aid to evaluation, however, statistical significance will 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.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at and above 1500 µg/plate

RANGE-FINDING/SCREENING STUDIES: yes, using concentrations of 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. In addition 0.1 mL of the maximum concentration of the test material on 2mLof molten, trace histidine supplemented top agar was overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test material.

ACCEPTABILITY CRITERIA:
- Results of solvent and positive controls were within historical data.
- Other acceptance criteria were met.

Any other information on results incl. tables

Tabel 1 Test results

S9 mix	Substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate ± standard deviation)
		Base-pair substitution type						Frameshift type
		TA100		TA1535		TA102		TA98		TA1537
Experiment 1
	Negative controls	87	110	19	18	313	330	36	31	19	15
		117		13		381		29		16
		126		21		297		27		11
-	0	120	(114 ±7.4)	27	(23 ±4.0)	345	(332 ±14.1)	20	(21 ±5.6)	16	(16 ±3.5)
		117		19		317		16		19
		106		23		334		27		12
-	50	118	(109 ±19.5)	23	(22 ±0.6)	330	(293 ±39.7)	32	(24 ±6.7)	16	(13 ±3.0)
		87		22		251		21		13
		123		22		297		20		10
-	150	101	(99 ±3.8)	26	(21 ±4.6)	304	(256 ±41.6)	14	(22 ±6.8)	16	(15 ±3.6)
		102		18		231		27		18
		95		18		233		24		11
-	500	80	(96 ±17.7)	25	(21 ±4.7)	269	(265 ±9.3)	25	(19 ±5.5)	9	(12 ±2.3)
		93		23		254		14		13
		115		16		271		19		13
-	1500	131 P	(105 ±22.7)	23 P	(20 ±4.4)	278 P	(242 ±34.1)	21 P	(20 ±2.1)	12 P	(13 ±4.6)
		95 P		15 P		239 P		18 P		18 P
		89 P		22 P		210 P		22 P		9 P
-	5000	98 P	(85 ±11.0)	24 P	(23 ±1.2)	298 P	(264 ±33.0)	20 P	(21 ±2.1)	13 P	(12 ±2.3)
		78 P		22 P		232 P		22 P		9 P
		80 P		22 P		262 P		20 P		13 P
Positive controls	Name (concentration(µg/plate))	ENNG (3)		ENNG (5)		MMC (0.5)		4NQO (0.2)		9AA (80)
S9 mix	Number of colonies per plate	432	(479 ±50.8)	406	(382 ±25.7)	1739	(1739 ±6.5)	264	(275 ± 16.8)	566	(756 ±230.1)
-		473		355		1745		266		691
		533		386		1732		294		1012
+	0	92	(88 ±6.4)	14	(14 ±0.0)	341	(328 ±14.2)	37	(32 ±5.0)	23	(19 ±3.5)
		81		14		331		33		16
		92		14		313		27		19
+	50	86	(93 ±6.5)	15	(14 ±1.7)	307	(336 ±30.1)	33	(32 ±1.7)	23	(18 ±4.7)
		99		15		333		30		16
		93		12		367		33		14
+	150	91	(89 ±18.6)	13		307	(285 ±23.1)	36	(30 ±5.5)	22	(20 ±2.1)
		107		9		287		26		19
		70		9		261		27		18
+	500	88	(88 ±12.5)	10	(10 ±2.3)	307	(308 ±19.0)	25	(28 ±5.5)	21	(14 ±6.7)
		75		12		328		34		12
		100		18		290		24		8
+	1500	90 P	(83 ±7.6)	13 P	(13 ±4.2)	265 P	(296 ±27.1)	30 P	(23 ± 5.9)	11 P	(11 ±0.6)
		85 P		9 P		312 P		21 P		11 P
		75 P		9 P		312 P		19 P		10 P
+	5000	79 P	(89 ±9.6)	15 P	(10 ±2.3)	274 P	(286 ±11.0)	25 P	(26 ±2.3)	9 P	(12 ±3.5)
		91 P		10 P		295 P		29 P		12 P
		98 P		13 P		290 P		25 P		16 P
Positive controls	Name (concentration(µg/plate))	2AA (1)		2AA (2)		DAN (10)		BP (5)		2AA (2)
S9 mix	Number of colonies per plate	2820	(2762 ±93.6)	125	(174 ±42.2)	1127	(1010 ±101.7)	200	(158 ± 36.7)	228	(210 ±19.6)
+
		2654		199		946		142		189
		2812		197		456		132		212
Experiment 2
	Negative controls	112	(109)	25	(22)	296	(298)	19	(18)	17	(13)
		101		27		317		17		11
		115		15		280		17		10
-	0	107	(92 ±13.9)	23	(25 ±5.3)	294	(293 ±33.0)	20	(21 ±1.0)	15	(13 ±5.9)
		88		21		260		22		17
		80		31		326		21		6
-	50	82	(98 ±17.1)	22	(19 ±5.2)	341	(321 ±17.6)	19	(14 ±5.7)	16	(13 ±3.8)
		116		13		307		16		15
		96		22		316		8		9
-	150	109	(103 ±5.3)	20	(19 ±2.6)	285	(284 ±8.5)	11	(11 ±3.5)	14	(11 ±2.6)
		99		21		275		15		9
		101		16		292		8		10
-	500	93	(91 ±13.1)	9	(9 ±3.5)	280	(272 ±16.2)	11	(12 ±4.0)	8	(8 ±1.5)
		103		6		282		8		6
		77		13		253		16		9
-	1500	84 P	(78 ±7.4)	17 P	(12 ±4.2)	311 P	(288 ±22.5)	9 P	(9 ±0.6)	11 P	(11 ±1.5)
		81 P		9 P		286 P		9 P		13 P
		70 P		11 P		266 P		8 P		10 P
-	5000	70 P	(83 ±14.2)	20 P	(14 ±5.1)	275 P	(274 ±7.5)	6 P	(8 ±2.0)	18 P	(17 ±2.3)
		80 P		10 P		266 P		8 P		14 P
		98 P		13 P		281 P		10 P		18 P
Positive controls	Name (concentration(µg/plate))	ENNG (3)		ENNG (5)		MMC (0.5)		4NQO (0.2)		9AA (80)
S9 mix	Number of colonies per plate	712	(673 ±76.4)	929	(859 ±123.0)	1269	(1341 ±95.8)	149	(128 ±28.4)	322	(323 ±32.5)
-		585		931		1305		140		356
		722		717		1450		96		291
+	0	73	(84 ±12.7)	9	(11 ±2.1)	275	(310 ±30.7)	18	(15 ±4.2)	11	(11 ±3.5)
		98		12		326		10		7
		82		13		330		16		14
+	50	81	(84 ±9.8)	15	(17 ±2.0)	303	(315 ±10.1)	16	(17 ±1.2)	7	(10 ±3.1)
		95		19		321		16		13
		76		17		320		18		9
+	150	77	(81 ±4.0)	13	(9 ±4.0)	269	(283 ±16.5)	27	(20 ±6.1)	6	(7 ±2.1)
		82		5		301		16		5
		85		10		278		17		9
+	500	52	(61 ±7.8)	11	(11 ±0.0)	296	(291 ±20.4)	21	(17 ±5.1)	8	(8 ±1.0)
		67		11		269		11		9
		63		11		309		18		7
+	1500	61 P	(57 ±5.5)	15 P	(10 ±4.2)	255 P	(268 ±12.1)	16 P	(14 ±2.6)	9 P	(11 ±2.9)
		51 P		9 P		269 P		15 P		9 P
		60 P		7 P		279 P		11 P		14 P
+	5000	66 P	(59 ±7.0)	11 P	(10 ±2.3)	270 P	(272 ±12.6)	18 P	(14 ±3.6)	14 P	(10 ±3.6)
		52 P		7 P		260 P		13 P		9 P
		60 P		11 P		285 P		11 P		7 P
Positive controls	Name (concentration(µg/plate))	2AA (1)		2AA (2)		DAN (10)		BP (5)		2AA (2)
S9 mix	Number of colonies per plate (± standard deviation)	1411	(1536 ±162.9)	114	(174 ±52.5)	748	(733 ±22.6)	192	(173 ±17.7)	515	(568 ±46.2)
+		1476		199		707		157		589
		1720		210		744		170		600

 

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 and according to GLP principles. The test was performed in two independent direct plate assays. The dose levels were 50, 150, 500, 1500 and 5000 µg/plate. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102), both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.