Octan-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

Study period:

09 October 2009 to 13 November 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octan-2-ol

EC Number:

204-667-0

EC Name:

Octan-2-ol

Cas Number:

123-96-6

Molecular formula:

C8H18O

IUPAC Name:

octan-2-ol

Test material form:

liquid

Method

Target gene:

Not applicable.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: These mutant strains of Salmonella are incapable of synthesising histidine

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: Escherichia coli (WP2uvrA-) requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence. This strain also has a deletion in an excision repair gene.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/ ß­naphthoflavone induced rate liver S9

Test concentrations with justification for top dose:

Mutation Test - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Mutation Test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was expanded to 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
Additional dose levels (1.5, 5 and 15 µg/plate) and an expanded dose range were selected for Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test material.

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Other positive controls were: 9-Aminoacridine (9AA), 4-Nitroquinoline-1-oxide (4NQO), 2-Aminoanthracene (2AA) and Benzo(a)pyrene

Details on test system and experimental conditions:

See below

Evaluation criteria:

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, as recommended by the UKEMS 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.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

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

Remarks:

In the second (pre-incubation) experiment, the test material caused a visible reduction in the growth of the bacterial background lawn in all tester strains, both with and without metabolic activation, initially at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the second (pre-incubation) experiment, the test material caused a visible reduction in the growth of the bacterial background lawn in all tester strains, both with and without metabolic activation, initially at 500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Test:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
In Experiment 1, the test material did not cause a visible reduction in the growth of the bacterial background lawn at any dose level. However, in the second (pre-incubation) experiment, the test material caused a visible reduction in the growth of the bacterial background lawn in all tester strains, both with and without metabolic activation, initially at 500 µg/plate. The test material was, therefore, either tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on the Experiment number. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
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 or exposure method.
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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From: 17 October 2009					To: 20 October 2009
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^-			TA98		TA1537
-	0	117 128 125	(123) 5.7#	30 28 30	(29) 1.2	19 27 37		(28) 9.0	22 25 16	(21) 4.6	10 10 20		(13) 5.8
-	50	117 120 132	(123) 7.9	35 27 31	(31) 4.0	21 24 24		(23) 1.7	25 21 24	(23) 2.1	10 16 15		(14) 3.2
-	150	106 113 124	(114) 9.1	31 25 29	(28) 3.1	32 29 30		(30) 1.5	25 25 12	(21) 7.5	14 12 21		(16) 4.7
-	500	134 108 117	(120) 13.2	24 20 24	(23) 2.3	23 22 18		(21) 2.6	23 29 16	(23) 6.5	10 15 10		(12) 2.9
-	1500	102 106 142	(117) 22.0	20 33 28	(27) 6.6	20 21 21		(21) 0.6	19 19 20	(19) 0.6	13 8 8		(10) 2.9
-	5000	126 124 117	(122) 4.7	26 28 29	(28) 1.5	15 13 16		(15) 1.5	20 19 21	(20) 1.0	10 9 11		(10) 1.0
Positive controls   S9-Mix   -	Name Concentration (µg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		675 662 705	(681) 22.1	574 526 471	(524) 51.5	867 883 907		(886) 20.1	164 168 150	(161) 9.5	866 398 617	(627) 234.2

 

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Test Results: Experiment 1 – With Metabolic Activation

Test Period		From: 17 October 2009					To: 20 October 2009
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^-			TA98		TA1537
+	0	121 139 123	(128) 9.9#	16 22 15	(18) 3.8	35 24 27		(29) 5.7	27 29 32	(29) 2.5	12 18 16	(15) 3.1
+	50	118 121 102	(114) 10.2	21 16 13	(17) 4.0	25 31 21		(26) 5.0	26 23 23	(24) 1.7	15 18 16	(16) 1.5
+	150	92 122 114	(109) 15.5	22 18 13	(18) 4.5	27 29 34		(30) 3.6	26 26 23	(25) 1.7	19 15 13	(16) 3.1
+	500	101 98 107	(102) 4.6	18 24 21	(21) 3.0	29 26 21		(25) 4.0	26 26 23	(25) 1.7	9 12 10	(10) 1.5
+	1500	87 86 106	(93) 11.3	20 15 15	(17) 2.9	16 24 29		(23) 6.6	25 20 27	(24) 3.6	13 15 11	(13) 2.0
+	5000	97 107 87	(97) 10.0	8 15 14	(12) 3.8	15 23 16		(18) 4.4	20 18 20	(19) 1.2	15 10 7	(11) 4.0
Positive controls   S9-Mix   +	Name Concentration (µg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1344 2044 1499	(1629) 367.7	139 198 197	(178) 33.8	265 282 249		(265) 16.5	192 197 199	(196) 3.6	301 407 448	(385) 75.9

 

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

Experiment 2 – Without Metabolic Activation

Test Period		From: 10 November 2009					To: 13 November 2009
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^-			TA98		TA1537
-	0	106 121 119	(115) 8.1#	20 23 27	(23) 3.5	23 26 23		(24) 1.7	22 19 19	(20) 1.7	14 13 12		(13) 1.0
-	1.5	101 121 95	(106) 13.6	21 21 22	(21) 0.6	24 23 26		(24) 1.5	20 20 20	(20) 0.0	15 16 12		(14) 2.1
-	5	119 98 136	(118) 19.0	22 27 23	(24) 2.6	25 24 27		(25) 1.5	24 19 20	(21) 2.6	11 13 14		(13) 1.5
-	15	98 102 90	(97) 6.1	24 22 24	(23) 1.2	26 22 26		(25) 2.3	19 24 15	(19) 4.5	11 15 11		(12) 2.3
-	50	99 120 110	(110) 10.5	26 22 18	(22) 4.0	22 20 18		(20) 2.0	23 20 20	(21) 1.7	14 15 13		(14) 1.0
-	150	107 102 106	(105) 2.6	20 26 24	(23) 3.1	27 22 22		(24) 2.9	18 23 20	(20) 2.5	13 15 11		(13) 2.0
-	500	89 S 71 S 85 S	(82) 9.5	20 S 19 S 15 S	(18) 2.6	0 V 0 V 0 V		(0) 0.0	11 S 13 S 3 S	(9) 5.3	10 S 2 S 3 S		(5) 4.4
-	1500	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T		(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 V 0 V 0 V		(0) 0.0
Positive controls   S9-Mix   -	Name Concentration (µg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		771 739 551	(687) 118.9	267 278 332	(292) 34.8	1027 967 975		(990) 32.6	122 95 122	(113) 15.6	690 338 370	(466) 194.6

 

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

S        Sparse bacterial background lawn

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

Test Results: Experiment 2 – With Metabolic Activation

Test Period		From: 10 November 2009					To: 13 November 2009
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		WP2uvrA^-			TA98		TA1537
+	0	88 102 91	(94) 7.4#	15 21 18	(18) 3.0	31 36 32		(33) 2.6	24 22 22	(23) 1.2	14 13 15	(14) 1.0
+	1.5	84 82 102	(89) 11.0	15 22 18	(18) 3.5	30 22 30		(27) 4.6	21 23 19	(21) 2.0	15 11 11	(12) 2.3
+	5	101 90 89	(93) 6.7	12 15 12	(13) 1.7	27 34 36		(32) 4.7	20 14 19	(18) 3.2	15 15 10	(13) 2.9
+	15	89 103 101	(98) 7.6	20 14 18	(17) 3.1	33 30 29		(31) 2.1	19 19 21	(20) 1.2	14 13 15	(14) 1.0
+	50	95 87 85	(89) 5.3	14 13 15	(14) 1.0	35 27 30		(31) 4.0	18 19 18	(18) 0.6	16 15 13	(15) 1.5
+	150	99 113 97	(103) 8.7	14 21 19	(18) 3.6	34 25 23		(27) 5.9	21 18 19	(19) 1.5	13 13 16	(14) 1.7
+	500	98 82 90	(90) 8.0	15 S 15 S 14 S	(15) 0.6	21 29 24		(25) 4.0	19 24 22	(22) 2.5	11 15 11	(12) 2.3
+	1500	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T		(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
Positive controls   S9-Mix   +	Name Concentration (µg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		1393 1269 1372	(1345) 66.4	200 205 172	(192) 17.8	503 486 514		(501) 14.1	173 139 123	(145) 25.5	388 374 383	(382) 7.1

 

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

S        Sparse bacterial background lawn

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

Applicant's summary and conclusion

Conclusions:

Interpretation of results :
negative

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

Executive summary:

The test material was considered to be non-mutagenic under the conditions of this test conducted according to EU Method B13/14.