4-methyl-3-decen-5-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:

04 September to 03 October 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-methyl-3-decen-5-ol

EC Number:

279-815-0

EC Name:

4-methyl-3-decen-5-ol

Cas Number:

81782-77-6

Molecular formula:

C11H22O

IUPAC Name:

4-methyldec-3-en-5-ol

Test material form:

liquid

Method

Target gene:

Histidine requirement

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking incubator (37 °C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for a test.- Properly maintained: yes stock cultures were stored in liquid nitrogen (-196 °C).- Periodically "cleansed" against high spontaneous background: yes (strains were also checked for other genotypic properties)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate (Rangefinding test I - TA 100 - direct plate assay) both in the presence and absence of metabolic activation3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate (Rangefinding test II - TA 100 - preincubation assay) both in the presence and absence of metabolic activation1, 3, 10, 33, 100, 333 µg/plate (Direct plate assay I - TA 1535, TA 1537, TA 98 and TA 102 - without metabolic activation); 3, 10, 33, 100, 333, 1000 µg/plate (Direct plate assay I - TA 1535, TA 1537, TA 98 and TA 102 - with metabolic activation)100, 333, 1000, 2000 µg/plate (Direct plate assay II - TA 102 - without metabolic activation)0.3, 1, 3, 10, 33, 100 µg/plate (Preincubation assay I - TA 1535, TA 1537, TA 98 and TA 102 - without metabolic activation); 1, 3, 10, 33, 100, 333 µg/plate (Preincubation assay I - TA 1535, TA 1537, TA 98 and TA 102 - with metabolic activation)33, 100, 166, 333 µg/plate (Preincubation assay II - TA 1535 without metabolic activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cumene hydroperoxide

methylmethanesulfonate

other: daunomycin, 2-aminoanthracene, 1,8 dihydroxyanthraquinone

Details on test system and experimental conditions:

The mutagenicity of the test material was evaluated using two methods, the plate incorporation method and the preincubation method.PLATE INCORPORATION ASSAYMETHOD OF APPLICATION: in agar (plate incorporation)0.1 mL of a fresh bacterial culture (10⁹ cells/mL) of one of the tester strains was added to 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL of S9 mix or 0.5 mL 0.1 M phosphate buffer, dependant if the test was with metabolic activation or without. Top agar in top agar tubes was molten and heated to 45 °C. The culture/test material solution was added successively to 3 mL molten top agar. This mixture was then mixed in a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 °C.DURATION- Exposure duration: 48 hoursNUMBER OF REPLICATIONS: triplicateDETERMINATION OF CYTOTOXICITY- Method: Reduction in background lawn, increase in size of microcolonies and reduction of the revertant colonies.PREINCUBATION ASSAYMETHOD OF APPLICATION: preincubation0.1 mL of a fresh bacterial culture (10⁹ cells/mL) of one of the tester strains was added to 0.1 mL of a dilution of the test material in DMSO and either 0.5 mL of S9 mix or 0.5 mL 0.1 M phosphate buffer, dependant if the test was with metabolic activation or without. The solution was preincubated for at 70 rpm and 37 °C. After the preincubation period the solution was added to 3 mL of molten top agar. The ingredients were mixed in a Vortex and incubated at 37 °C.DURATION- Preincubation period: 30 minutes- Exposure duration: 48 hoursNUMBER OF REPLICATIONS: TriplicateDETERMINATION OF CYTOTOXICITY- Method: Reduction in background lawn, increase in size of microcolonies and reduction of the revertant colonies.

Evaluation criteria:

Colonies were counted automatically with a Protos model 50000 colony counter or manually, if there were < 40 colonies per plate.The test is considered negative or not mutagenic if it meets the following criteria:- The total number of revertants in any tester strain at any concentration if not greater than two times the solvent control value, with or without metabolic activation.- The negative response should be reproducible in at least one repeated experiment.The test is considered positive or mutagenic if it meets the following criteria;- It induces at least a 2 fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation (any mean plate count of less that 20 is not considered significant).- The positive response should be reproducible in at least one repeated experiment.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The test material did not induce a dose-related increase in the number of revertant colonies in each of the five tester strains, both in the presence and absence of metabolic activation. These results were confirmed in separate experiments.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With the exception of the TA 102 in the 1st plate incorporation assay and TA 1535 in the preincubation assay in the absence of S9, toxicity was observed. Additional plate incorporation and preincubation assays were performed with a higher dose range

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The test material did not induce a dose-related increase in the number of revertant colonies in each of the five tester strains, both in the presence and absence of metabolic activation. These results were confirmed in separate experiments.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With the exception of the preincubation assay in the absence of S9, toxicity was observed. Additional plate incorporation and preincubation assays were performed with a higher dose range

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

With the exception of the TA 102 in the 1st plate incorporation assay and TA 1535 in the preincubation assay in the absence of S9, toxicity was observed. Additional plate incorporation and preincubation assays were performed with a higher dose range

Cytotoxicity / choice of top concentrations:

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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

With the exception of the TA 102 in the 1st plate incorporation assay and TA 1535 in the preincubation assay in the absence of S9, toxicity was observed. Additional plate incorporation and preincubation assays were performed with a higher dose range

Cytotoxicity / choice of top concentrations:

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

With the exception of the TA 102 in the 1st plate incorporation assay and TA 1535 in the preincubation assay in the absence of S9, toxicity was observed. Additional plate incorporation and preincubation assays were performed with a higher dose range

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The test material did not induce a dose-related increase in the number of revertant colonies in each of the five tester strains, both in the presence and absence of metabolic activation. These results were confirmed in separate experiments.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With the exception of the TA 102 in the 1st plate incorporation assay, toxicity was observed. Additional plate incorporation and preincubation assays were performed with a higher dose range

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: The test material precipitated in the top agar at concentrations above 333 µg/plate in the plate incorporation assays and the first preincubation assay. In the first plate incorporation assay, precipitate of the test material was noticed on top of the agar at 1000 µg/plate and in the second plate incorporation assay at 1000 and 2000 µg/plate. This was not observed in the preincubation assays.RANGE-FINDING/SCREENING STUDIES:- The toxicity of the test material was assessed in TA 100 with and without S9, to identify a suitable range of concentrations for the main test for which the test material did not inhibit bacterial growth. These tests were performed in triplicate at 8 concentrations. The test was performed according to the direct plate assay and the preincubation assay methods.- Test I: The test material precipitated on the plates at dose levels of 3330 and 5000 µg/plate. Toxicity was observed at dose levels of 333 µg/plate and above.- Test II: The test material did not precipitate on the plates at dose levels up to 5000 µg/plate. Toxicity was observed at dose levels of 33 and 100 µg/plate and upwards in the absence and presence of metabolic activation, respectively.COMPARISON WITH HISTORICAL CONTROL DATA:All control results were within the historical control range. ADDITIONAL INFORMATION ON CYTOTOXICITY:The second direct plate incorporation assay was performed due to the low of toxicity observed in the TA 102 strain in the absence of S9, a higher dose range was selected for the second assay. The second preincubation assay was performed due to the low toxicity observed in the TA 1535 strain in the absence of S9 mix in the first assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2. Mutagenic Response in the Direct Plate Assay 1

	Dose (µg/plate)	Mean number of revertant colonies/3 replicates plates (± S.D.)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
Without S9	Positive Control	610 ± 39	255 ± 58	369 ± 37	433 ± 54	528 ± 22
	Solvent Control	12 ± 2	4 ± 2	34 ± 4	101 ± 12	180 ± 3
	1	12 ± 3	6 ± 3	36 ± 7		184 ± 18
	3	10 ± 3	6 ± 4	36 ± 11	112 ± 12	183 ± 10
	10	13 ± 4	5 ± 1	23 ± 3	108 ± 7	186 ± 23
	33	8 ± 2 s	6 ± 2 s	16 ± 3	119 ± 10	163 ± 23
	100	9 ± 2 s	3 ± 2 m	24 ± 6	109 ± 11	155 ± 17
	333	MC, e	4 ± 2 m	19 ± 7 s	MC e	164 ± 12
	1000				0 ± 0 a
	3330 SP				0 ± 0 a
	5000 SP				0 ± 0 a
With S9	Positive Control	112 ± 5	396 ± 34	665 ± 74	454 ± 23	763 ± 155
	Solvent Control	9 ± 5	8 ± 2	37 ± 11	117 ± 19	210 ± 5
	3	12 ± 5	5 ± 3	35 ± 4	95 ± 10	192 ± 10
	10	7 ± 2	5 ± 1	35 ± 8	107 ± 5	184 ± 18
	33	11 ± 3	5 ± 2	29 ± 4	91 ± 12	193 ± 7
	100	8 ± 2	5 ± 2	32 ± 5	97 ± 9	183 ± 16
	333	10 ± 2 s	4 ± 3 s	43 ± 4	60 ± 8 s	184 ± 26
	1000	9 ± 1 m	3 ± 2 m	16 ± 4 s	MC e	100 ± 16 s
	3330 SP				0 ± 0 a
	5000 SP				0 ± 0 a
s = Bacterial background lawn slightly reduced m = Bacterial background lawn moderately reduced e = Bacterial background lawn extremely reduced a = Bacterial background lawn absent SP = Slight Precipitate MC = Microcolonies - Toxicity was observed in all tester strains, 3except tester strain TA 102 in the absence of metabolic activation.

 

Table 3. Mutagenic Response in the Direct Plate Assay 2

 

Dose µg/plate	Mean number of revertant colonies / 3 replicate plates ( ± S.D.) with TA 102 without S9 mix
Positive control	676 ± 33
Solvent control	297 ± 21
100	296 ± 27
333	230 ± 31
1000 SP	168 ± 15 s
2000 SP	148 ± 15 s
s = Bacterial background lawn slightly reduced SP = Slight Precipitate - Toxicity was observed in this strain.

 

Table 4.Mutagenic Response in the Preincubation Assay 1

	Dose (µg/plate)	Mean number of revertant colonies/3 replicates plates (± S.D.)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
Without S9	Positive Control	718 ± 70	82 ± 7	311 ± 44	586 ± 75	861 ± 21
	Solvent Control	9 ± 6	5 ± 2	17 ± 5	120 ± 9	282 ± 18
	0.3	8 ± 1	5 ± 3	19 ± 4		241 ± 53
	1	8 ± 1	5 ± 2	18 ± 1		237 ± 27
	3	9 ± 1	6 ± 3	17 ± 5	105 ± 14	231 ± 23
	10	8 ± 1	5 ± 3	18 ± 6	105 ± 4	239 ± 28
	33	6 ± 1	6 ± 2 s	14 ± 5	97 ± 10 s	225 ±26
	100	8 ± 2	0 ± 0 a	4 ± 2 m	MC e	109 ± 26
	333				0 ± 0 a
	1000				0 ± 0 a
	3330				0 ± 0 a
	5000				0 ± 0 a
With S9	Positive Control	105 ± 20	199 ± 18	526 ± 33	362 ± 41	901 ± 58
	Solvent Control	10 ± 4	8 ± 4	20 ± 5	115 ± 9	282 ± 7
	1	7 ± 2	6 ± 3	29 ± 5		274 ± 16
	3	11 ± 2	6 ± 1	20 ± 3	110 ± 11	280 ± 8
	10	14 ± 1	8 ± 5	23 ± 5	96 ±1 12	277 ± 21
	33	12 ± 2	9 ± 2	22 ± 6	98 ± 10	265 ± 33
	100	9 ± 2	4 ± 2	13 ± 2	89 ± 9 m	239 ± 28
	333	7 ± 2 m	MC e	7 ± 4 m	MC e	155 ± 47
	1000				0 ± 0 a
	3330				0 ± 0 a
	5000				0 ± 0 a
s = Bacterial background lawn slightly reduced m = Bacterial background lawn moderately reduced e = Bacterial background lawn extremely reduced a = Bacterial background lawn absent SP = Slight Precipitate MC = Microcolonies - Toxicity was observed in all test strains except TA 1535 in the absence of metabolic activation

 

Table 5. Mutagenic Response in the Preincubation Assay 2

Dose µg/plate	Mean number of revertant colonies / 3 replicate plates ( ± S.D.) with TA 1535, without S9 mix
Positive Control	536± 27
Solvent Control	7 ± 3
33 s	6 ± 3
100 m	MC
166 m	MC
333 m	0 ± 0
s = Bacterial background lawn slightly reduced m = Bacterial background lawn moderately reduced e = Bacterial background lawn extremely reduced MC = Microcolonies - Toxicity was observed in this strain

 

Mutagenicity

The test material did not induce a dose-related increase in the number of revertant colonies in each of the five tester strains, both in the presence and absence of metabolic activation. These results were confirmed in separate experiments. Based on these results, it is concluded that the test material is non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):negative with and without metabolic activationUnder the conditions of the study, the test material was non-mutagenic in the Salmonella typhimurium reverse mutation assay both in the presence and in the absence of metabolic activation.

Executive summary:

In a GLP compliant genetic toxicity study conducted in accordance with standardised guidelines OECD 471 and EU Method B. 13/14 the mutagenicity of the test material was determined in an Ames test using both the direct plate and preincubation methods. Salmonella typhimurium strains TA98, TA 100, TA 102, TA 1535 and TA 1537 were exposed to varying concentrations of the test material in the presence and absence of metabolic activation. Under the conditions of the study, the test material was non-mutagenic in the Salmonella typhimurium reverse mutation assay both in the presence and in the absence of metabolic activation.