Cyclohexanepropanol, 2,2,3,6-tetramethyl-.alpha.-propyl-

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:

21-02-2007 to 16-03-2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: August 2005 ; signature: November 2005

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

478-330-3

EC Name:

-

Cas Number:

95851-08-4

Molecular formula:

C16H32O

IUPAC Name:

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

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Approximately 4°C in the dark under nitrogen
- Other: colourless slightly viscous liquid

Method

Target gene:

histidine or tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity test (TA100 and WP2urvA): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 1 (plate incorporation method): All strains: 0, 50, 150, 500, 1500, 5000 µg/plate
Experiment 2 (plate incorporation method): All strains: 0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed. DMSO was selected as the vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (plate incorporation)

DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Experiment 2. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.

Statistics:

Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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, but tested up to precipitating concentrations

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The current Positive HCD dataset is presented in the full study report.
- Negative (solvent/vehicle) historical control data: The current background spontaneous revertant counts in concurrent untreated controls and/or or vehicle controls ; historic negative controls are presented in the full study report.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Table 1 : Test Results: Experiment 1: with and without metabolic activation and results of concurrent positive controls

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	96 101 86	(96) 4.5#	18 23 20	(20) 2.5	26 18 30	(25) 6.1	24 18 32	(25) 7.0	8 17 8	(11) 5.2
	50 µg	93 85 81	(86) 6.1	17 22 21	(20) 2.6	21 19 23	(21) 2.0	20 13 23	(19) 5.1	9 10 14	(11) 2.6
	150 µg	116 91 73	(93) 21.6	19 22 18	(20) 2.1	21 17 23	(20) 3.1	8 20 18	(15) 6.4	18 10 11	(13) 4.4
	500 µg	85 76 82	(81) 4.6	24 19 18	(20) 3.2	21 17 23	(22) 5.5	19 25 20	(21) 3.2	8 11 7	(9) 2.1
	1500 µg	81 91 82	(85) 5.5	15 20 9	(15) 5.5	24 20 21	(22) 2.1	23 18 23	(21) 2.9	7 2 13	(7) 6.0
	5000 µg	81 P 54 P 85 P	(73) 16.9	18 P 14 P 14 P	(15) 2.3	21 P 18 P 16 P	(18) 2.5	28 P 24 P 23 P	(25) 2.6	14 P 14 P 10 P	(13) 2.3
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		796 786 792	(791) 5.0	373 348 344	(355) 15.7	382 430 417	(410) 24.8	250 269 259	(259) 9.5	653 933 845	(810) 143.2
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	91 90 85	(89) 3.2#	8 16 10	(11) 3.8	27 29 23	(26) 3.1	25 28 30	(28) 2.5	14 7 8	(12) 2.3
	50 µg	104 84 102	(97) 11.0	15 10 10	(12) 2.9	25 31 25	(27) 3.5	19 29 30	(26) 6.1	6 4 13	(8) 4.7
	150 µg	96 108 96	(100) 6.9	10 11 10	(10) 0.6	25 23 24	(24) 1.0	21 15 26	(21) 5.5	13 10 9	(11) 2.1
	500 µg	99 90 89	(93) 5.5	14 11 11	(12) 1.7	29 24 28	(27) 2.6	15 21 17	(18) 3.1	6 8 7	(7) 1.0
	1500 µg	73 86 66	(75) 10.1	8 8 11	(9) 1.7	25 20 24	(23) 2.6	13 29 22	(21) 8.0	9 7 9	(8) 1.2
	5000 µg	82 P 82 P 87 P	(84) 2.9	9 P 9 P 13 P	(10) 2.3	25 P 16 P 25 P	(22) 5.2	24 P 22 P 16 P	(21) 4.2	5 P 6 P 5 P	(5) 0.6
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		1585 1479 1661	(1575) 91.4	180 179 193	(184) 7.8	542 576 588	(569) 23.9	113 104 155	(124) 27.2	349 375 313	(346) 31.1

 

Table 2 : Test Results: Experiment 2: with and without metabolic activation and results of concurrent positive controls

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	127 110 138	(125) 14.1#	17 21 14	(17) 3.5	26 25 22	(24) 2.1	33 33 28	(21) 4.7	7 14 7	(9) 4.0
	50 µg	115 105 124	(115) 9.5	17 13 21	(17) 4.0	29 13 29	(24) 9.2	32 26 24	(24) 7.6	8 5 6	(6) 1.5
	150 µg	98 104 99	(100) 3.2	9 24 15	(16) 7.5	10 24 22	(19) 7.6	37 24 28	(17) 5.7	7 5 6	(6) 1.0
	500 µg	129 136 158	(141) 15.1	10 14 15	(13) 2.6	18 13 18	(16) 2.9	16 28 24	(19) 5.5	7 8 4	(6) 2.1
	1500 µg	95 78 75	(83) 10.8	10 18 15	(14) 4.0	23 28 8	(20) 10.4	26 29 27	(20) 5.6	7 3 8	(6) 2.6
	5000 µg	79 P 80 P 74 P	(78) 3.2	16 P 13 P 19 P	(16) 3.0	19 P 18 P 18 P	(18) 0.6	17 P 26 P 27 P	(21) 4.2	2 P 5 P 9 P	(5) 3.5
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		416 447 471	(445) 27.6	267 266 367	(300) 58.0	235 357 331	(308) 64.3	425 330 306	(364) 62.9	1338 1402 1382	(1374) 32.7
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	65 74 94	(78) 14.8#	10 9 10	(10) 0.6	21 22 23	(22) 1.0	29 34 22	(28) 6.0	9 10 8	(9) 1.0
	50 µg	69 64 81	(71) 8.7	9 10 9	(9) 0.6	16 16 23	(18) 4.0	28 29 32	(30) 2.1	2 8 5	(5) 3.0
	150 µg	78 97 76	(84) 11.6	8 8 10	(9) 1.2	23 29 24	(25) 3.2	25 25 22	(24) 1.7	8 6 5	(6) 1.5
	500 µg	73 71 77	(74) 3.1	8 9 9	(9) 0.6	24 17 24	(22) 4.0	14 14 15	(14) 0.6	6 3 3	(4) 1.7
	1500 µg	66 73 76	(72) 5.1	8 9 8	(8) 0.6	17 28 21	(22) 5.6	17 19 20	(19) 1.5	2 2 4	(3) 1.2
	5000 µg	61 P 70 P 73 P	(68) 6.2	8 P 13 P 9 P	(10) 2.6	21 P 23 P 19 P	(21) 2.0	14 P 24 P 20 P	(19) 5.0	7 P 8 P 11 P	(9) 2.1
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		1879 2004 1965	(1949) 64.0	328 296 340	(321) 22.7	527 503 541	(524) 19.2	371 643 903	(639) 266.0	424 518 310	(417) 104.2

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

N/T: Not tested at this dose level

S: partial absence of bacterial background lawn

P : precipitate

#: Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B.13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test item the Ames plate incorporation method at up to five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined based on the results of a preliminary toxicity assay. The dose levels were 50 to 5000 µg/plate for all Salmonella strains: TA98, TA100, TA1535, TA1537 and for E.coli strain WP2uvrA-. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test item formulations. The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate or based on absence of cytotoxicity (reduction in growth of the bacterial background law) in all tester strains. A item item (oily in appearance) precipitate was observed on the plates at 5000 μg/plate. This did not prevent scoring of the revertant colonies. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9 mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.