2,6,10-trimethylundec-9-enal

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:

17 January 2003 - 10 February 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6,10-trimethylundec-9-enal

EC Number:

205-460-8

EC Name:

2,6,10-trimethylundec-9-enal

Cas Number:

141-13-9

Molecular formula:

C14H26O

IUPAC Name:

2,6,10-trimethylundec-9-enal

Test material form:

liquid

Specific details on test material used for the study:

Batch 9000484970
Expiry date 18 March 2003

Method

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-mix

Test concentrations with justification for top dose:

Dose range finding test (results used as part of the definitve test):
Strains TA100 and TA98; 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (with and without metabolic activation).

Definitive test - preincubation method:
Strains TA1535, TA1537 and TA102; 10, 33, 100, 333, 1000 and 3330 µg/plate (with and without metabolic activation).

Definitive test - direct plate method:
Strains TA1535, TA1537 and TA102; 3, 10, 33, 100, 333 and 1000 µg/plate (with and without metabolic activation).

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: daunomycin; methylmethanesulfonate; 2-aminoanthracene; 1,8 dihydroxy anthraquinone

Details on test system and experimental conditions:

METHOD OF APPLICATION: First experiment, preincubation method; Second experiment, direct plate method.

DURATION
- Preincubation period: 30 minutes by 70 rpm at 37 °C.
- Exposure duration: 48 hours, incubated in the dark at 37 °C.

NUMBER OF REPLICATIONS: Each concentration was tested in triplicate.

NUMBER OF CELLS EVALUATED: The revertant colonies were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

DOSE RANGE FINDER
- Method: All doses were tested in triplicate in the preincubation and the direct plate assay.
- Results used for defining the definitive dosing range: The highest concentration of the test material used in the definitive mutation assay was the level at which the test material exhibited limited solubility. The dose range finding tests were reported as a part of the preincubation and direct plate assays for the definitive tests, respectively.

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 2: Toxicity of the Test Material in the Dose Range Finding Study (preincubation assay)

Strain	Without S9-mix			With S9-mix
	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies
TA98	333-3330 5000	Slight Slight	Slight Moderate	5000	-^1	-^2
TA100	10-5000	Slight	-^2	333-5000	Slight	-^2

-¹= No reduction of the bacterial background lawn

-²= No reduction in the number of revertant colonies.

Table 3: Toxicity to the Test Material in the Preincubation Assay (Definitive Test)

Strain	Without S9-mix			With S9-mix
	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies
TA1535	10	Slight	-^1	333	Slight	-^1
	33-100	Slight	-^2
	333-1000	Moderate	-^2	1000-3330	Moderate	-^1
	3330	Extreme	Microcolonies
TA1537	100	Slight	-^1	333-3330	Slight	-^1
	333	Moderate	Extreme
	1000	Moderate	Moderate
	3330	Extreme	Microcolonies
TA102	333-1000	-^3	-^2	3330	-^3	-^1
	3330	Slight	Moderate

 -¹= No reduction in the number of revertant colonies

-²= Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range

-³= No reduction of the bacterial background lawn

Table 4: Mutagenic Response of the Test Material in the Preincubation Assay

Dose (µg/plate)	Mean number of revertant colonies/ 3 replicate plates (± SD)
	TA1535	TA1537	TA98	TA100	TA102
	Without S9-mix
Positive control	777 ± 42	75 ± 6	377 ± 38	440 ± 24	594 ± 23
Solvent control	11 ± 3	6 ± 3	15 ± 2	139 ± 11	210 ± 4
3			12 ± 3	127 ± 14
10	9 ± 1^1	6 ± 3	14 ± 4	151 ± 15^1	219 ± 21
33	8 ± 2^1	6 ± 3	19 ± 1	186 ± 13^1	206 ± 5
100	5 ± 2^1	7 ± 2^1	10 ± 1	195 ± 14^1	202 ± 22
333	8 ± 3^2	2 ± 1^2	10 ± 1^1	190 ± 13^1	163 ± 7
1000	7 ± 1^2	3 ± 1^2	10 ± 2^1	247 ± 10^1	140 ± 15
3330^SP	MC^3	MC^3	9 ± 1^1	229 ± 27^1	109 ± 15^1
5000^SP			8 ± 3^1	222 ± 4^1
	With S9-mix
Positive control	99 ± 6	139 ± 24	344 ± 65	344 ± 21	693 ± 47
Solvent control	6 ± 2	6 ± 1	20 ± 3	137 ± 12	212 ± 14
3			20 ± 1	149 ± 25
10	9 ± 3	5 ± 2	17 ± 3	142 ± 12	268 ± 9
33	6 ± 4	5 ± 1	19 ± 2	138 ± 3	286 ± 44
100	9 ± 1	5 ± 2	18 ± 3	145 ± 18	230 ± 9
333	6 ± 2^1	6 ± 2^1	11 ± 2	159 ± 11^1	243 ± 12
1000	6 ± 2^2	6 ± 1^1	14 ± 2	159 ± 17^1	245 ± 19
3330^SP	7 ± 4^2	5 ± 1^1	14 ± 3	212 ± 27^1	221 ± 16
5000^SP			15 ± 2	180 ± 36^1

Solvent control: 0.1 mL DMSO

1 = Bacterial background lawn slightly reduced

2 = Bacterial background lawn moderately reduced

3 = Bacterial background lawn extremely reduced

SP = Slight Precipitate

MC = Microcolonies

 

Table 5: Mutagenic Response of the Test Material in the Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/ 3 replicate plates (± SD)
	TA1535	TA1537	TA98	TA100	TA102
	Without S9-mix
Positive control	1067 ± 54	605 ± 92	276 ± 20	998 ± 23	1403 ± 59
Solvent control	14 ± 3	6 ± 1	19 ± 2	140 ± 17	286 ± 22
3	12 ± 2	9 ± 3	20 ± 3	132 ± 6	184 ± 9
10	13 ± 2	7 ± 2	15 ± 3	131 ± 25	183 ± 16
33	14 ± 3	7 ± 2	17 ±3	123 ± 8	190 ± 7
100	12 ± 3	8 ± 3	18 ± 5	124 ± 16	239 ± 31
333	9 ± 1	8 ± 2	18 ± 4	114 ± 4	225 ± 6
1000^SP	7 ± 3	9 ± 1	17 ± 6	119 ± 8	211 ± 13
3330^SP			17 ± 2	124 ± 11
5000^SP			14 ± 1	128 ± 6
	With S9-mix
Positive control	281 ± 27	811 ± 57	1520 ± 39	1073 ± 61	1219 ± 48
Solvent control	13± 6	8 ± 3	22 ± 3	132 ± 10	269 ± 13
3	15 ± 5	9 ± 1	23 ± 6	149 ± 25	202 ± 12
10	15 ± 3	6 ± 2	24 ± 7	138 ± 25	212 ± 8
33	11 ± 2	9 ± 2	21 ± 3	147 ± 16	235 ± 15
100	12 ± 5	9 ± 6	22 ± 1	137 ± 5	205 ± 11
333	7 ± 4	7 ± 3	20 ± 3	123 ± 17	238 ± 19
1000^SP	9 ± 5	6 ± 3	24 ± 2	117 ± 8	214 ± 14
3330^SP			17 ± 4	99 ± 6
5000^SP			21 ± 3	120 ± 6

Solvent control: 0.1 mL DMSO

SP = Slight Precipitate

Applicant's summary and conclusion

Conclusions:

Based on the test results of this study it is concluded that ADOXAL is not mutagenic in the Salmonella Typhimurium reverse mutation assay.

Executive summary:

ADOXAL was tested in the Salmonella typhimurium reverse mutation assay with five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102). The test was performed in two separate experiments in the presence and absence of S9 -mix (Aroclor-1254 induced rat liver S9-mix). ADOXAL did not induce a dose-related increase in the number of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA98, TA100 and TA102) both in the absence and presence of S9 -metabolic activation. These results were confirmed in a separate experiment.