Reaction mass of 5,9-Undecadienal-2,6,10-trimethyl-, (5E)- and 5,9-Undecadienal-2,6,10-trimethyl-, (5Z)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 and E.coli WP2 uvr A with and without metabolic activation

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Oct - 18 Nov 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(Commission Regulation (EC) No 440/2008, May 2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital/β-Naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: 3, 10, 33, 100, 333 1000, 2500 and 5000 µg/plate
The pre-experiment is reported as Experiment 1.

Experiment 2: 1, 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for experiment 1; preincubation for experiment 2

DURATION
- Preincubation period: 60 min at 37 °C
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: in triplicates in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Rationale for test conditions:

The test conditions were chosen according to OECD 471.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvr A) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold is
regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Mean values and standard deviations were calculated.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +/-S9: starting at 333 µg/plate; Exp. 2: -S9: starting at 100 µg/plate, +S9: starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +/-S9: starting at 333 µg/plate; Exp. 2: -S9: starting at 100 µg/plate, +S9: starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: +/-S9: starting at 333 µg/plate; Exp. 2: -S9: starting at 333 µg/plate, +S9: starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: -S9: starting at 33 µg/plate, +S9: starting at 333 µg/plate; Exp. 2: -S9: starting at 100 µg/plate; +S9: 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. 1: -S9: at 5000 µg/plate, +S9: starting at 2500 µg/plate; Exp. 2: +/-S9: starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was observed in the overlay agar in the test tubes from 1000 µg/plate up to 5000 µg/plate in the presence of metabolic activation in experiment 1 and with and without metabolic activation in experiment 2. Precipitation of the test item on the incubated agar plates were observed from 1000 µg/plate up to 5000 µg/plate in experiment 1 and from 2500 µg/plate up to 5000 µg/plate in experiment 2. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains were tested in the pre-experiment).

HISTORICAL CONTROL DATA: Solvent, negative and positive control were within the range of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed reduced background growth in all strains with and without metabolic activation in both independent experiments. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups.

 Table 1. Test results of experiment 1

EXPERIMENT 1 (plate incubation method)
S9-Mix	Without
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
SC	13 ± 3	14 ± 1	36 ± 2	36 ± 2	51 ± 11
NC	12 ± 2	13 ± 3	30 ± 5	30 ± 5	41 ± 3
3	11 ± 3	14 ± 1	37 ± 4	37 ± 4	47 ± 10
10	12 ± 4	12 ± 0	33 ± 5	33 ± 5	44 ± 10
33	14 ± 1	14 ± 1	32 ± 5	32 ± 5	38 ± 6
100	14 ± 2	11 ± 2	23 ± 7	23 ± 7	38 ± 3
333	11 ± 2 R	6 ± 1 R	18 ± 1	18 ± 1 R	38 ± 5
1000	6 ± 1 R, P	4 ± 1 R, P	16 ± 2	16 ± 2 R, P	46 ± 3 P
2500	3 ± 1 R, P	4 ± 1 R, P	16 ± 3	16 ± 3 R, P	37 ± 7 P
5000	0 ± 0 R, P	5 ± 3 R, P	17 ± 3	17 ± 3 R, P	24 ± 4 P, R
NaN3	1813 ± 78			1945 ± 41
4-NOPD		79 ± 1	306 ± 31
MMS					1025 ± 68
S9-Mix	With
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
SC	17 ± 5	15 ± 5	39 ± 4	136 ± 10	51 ± 9
NC	15 ± 0	16 ± 4	39 ± 10	126 ± 27	48 ± 12
3	16 ± 2	16 ± 1	37 ± 7	122 ± 5	49 ± 8
10	15 ± 1	16 ± 1	40 ± 6	136 ± 14	49 ± 4
33	15 ± 5	16 ± 1	35 ± 10	155 ± 4	54 ± 6
100	16 ± 3	13 ± 4	37 ± 3	158 ± 3	55 ± 3
333	11 ± 1 R	7 ± 1 R	20 ± 2 R	84 ± 9 R	46 ± 3
1000	9 ± 1 R, P	5 ± 2 R, P	26 ± 3 R, P	59 ± 7 R, P	39 ± 4 P
2500	6 ± 3 R, P	4 ± 1 R, P	15 ± 4 R, P	58 ± 8 R, P	24 ± 4 R, P
5000	5 ± 3 R, P	4 ± 1 R, P	15 ± 4 R, P	46 ± 4 R, P	23 ± 5 R, P
2-AA	296 ± 12		2317 ± 83	2225 ± 10	206 ± 12
SC = Solvent Control (DMSO) NC = Negative Control (untreated) R = Reduced background growth P = Precipitate NaN3: sodium azide; 4-NOPD:4-nitro-o-phenylene-diamine; MMS: methylmethanesulfonate; 2-AA: 2-aminoanthracene

 Table 2. Test results of experiment 2

EXPERIMENT 2 (pre-incubation method)
S9-Mix	Without
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
SC	14 ± 6	15 ± 3	21 ± 2	92 ± 23	44 ± 5
NC	13 ± 1	17 ± 6	29 ± 1	132 ± 27	50 ± 3
1	11 ± 3	13 ± 2	28 ± 2	101 ± 6	47 ± 5
3	14 ± 2	12 ± 3	23 ± 4	88 ± 5	42 ± 13
10	16 ± 8	14 ± 2	24 ± 5	88 ± 15	48 ± 7
33	13 ± 4	8 ± 1	26 ± 6	97 ± 15	57 ± 13
100	10 ± 5 R	10 ± 3 R	24 ± 6	56 ± 12 R	44 ± 7
333	10 ± 3 R	8 ± 1 R	22 ± 2 R	42 ± 9 R	44 ± 5
1000	3 ± 5 R	3 ± 2 R	20 ± 4 R	43 ± 6 R	39 ± 8 R
2500	2 ± 4 R, P	3 ± 2 R, P	11 ± 2 R, P	35 ± 7 R, P	36 ± 6 R, P
5000	1 ± 1 P, R	1 ± 1 R, P	8 ± 1 R, P	1 ± 2 R, P	42 ± 2 R, P
NaN3	1605 ± 40			1755 ± 60
4-NOPD		89 ± 5	385 ± 18
MMS					583 ± 8
S9-Mix	With
Test substance (µg/plate)	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
SC	19 ± 6	21 ± 8	46 ± 5	115 ± 17	57 ± 11
NC	20 ± 6	22 ± 5	33 ± 2	113 ± 10	57 ± 2
1	24 ± 3	32 ± 2	37 ± 3	111 ± 4	67 ± 7
3	17 ± 4	20 ± 4	39 ± 10	113 ± 7	59 ± 3
10	19 ± 4	17 ± 4	48 ± 6	120 ± 8	62 ± 7
33	23 ± 2	18 ± 2	42 ± 5	109 ± 6	69 ± 7
100	18 ± 6	22 ± 1	43 ± 14	107 ± 12	64 ± 4
333	23 ± 11	20 ± 6	29 ± 2	96 ± 9	57 ± 9
1000	6 ± 2 R	1 ± 2 R	26 ± 2 R	23 ± 3 R	33 ± 8 R
2500	7 ± 6 R, P	2 ± 2 R, P	21 ± 4 R, P	26 ± 1 R, P	20 ± 4 R, P
5000	7 ± 3 R, P	1 ± 1 R, P	19 ± 2 R, P	30 ± 4 R	25 ± 23 R, P
2-AA	286 ± 17	189 ± 15	1603 ± 86	1508 ± 434	394 ± 20
SC = Solvent Control (DMSO) NC = Negative Control (untreated) R = Reduced background growth P = Precipitate NaN3: sodium azide; 4-NOPD:4-nitro-o-phenylene-diamine; MMS: methylmethanesulfonate; 2-AA: 2-aminoanthracene

 

Conclusions:

Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation up to 5000 µg/plate.

Executive summary:

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2009). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation up to 5000 µg/plate.Precipitation was observed at concentrations ≥ 1000 µg/plate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (2009). In this study the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation up to 5000 µg/plate. Precipitation was observed at concentrations ≥ 1000 µg/plate.

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008.