2,6-Octadienal, 3,7-dimethyl-, acid-isomerized

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:

18.02.-05.03.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,6-Octadienal, 3,7-dimethyl-, acid-isomerized

EC Number:

291-768-8

EC Name:

2,6-Octadienal, 3,7-dimethyl-, acid-isomerized

Cas Number:

90480-35-6

IUPAC Name:

1-methyl-4-(propan-2-yl)benzene; ethanol

Method

Target gene:

histidine (Salmonella typhimurium), tryptophan (Escherichia coli)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Mammalian Microsomal Fraction S9 Mix

Test concentrations with justification for top dose:

- initial mutagenicity assay at 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 µg/plate with and without S9

- independent confirmatory mutagenicity assay at 16.0, 50.0, 160, 500, 1600, and 5000 µg/plate with S9 and 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 µg/plate without S9

Vehicle / solvent:

DMSO

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Controls 2

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: ICR-191

Remarks:

Without metabolic activation

Controls 3

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with metabolic activation

Controls 5

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Method of application:
plate incorporation methodology

Duration:
- 52 ± 4 hours at 37 ± 2°C

Number of replications: 3

Acceptance criteria::
The following criteria were used to determine a valid assay:
- Salmonella tester strains must exhibit the presence of the his- mutation
- Tester strain WP2uvrA must exhibit the presence of the trp- mutation
- Salmonella tester strains must exhibit the presence of the rfa mutation
- Tester strains TA98 and TA100 must exhibit the presence of the pKM101 plasmid
- All tester strains must exhibit the presence of the uvrA or uvrB mutations
- The average revertants/plate of the vehicle control cultures must be within the acceptance limits of the laboratory historical data
- Each culture must reach a target OD650(1:4) = 0.4 to 0.6, which has been demonstrated to be representative of cultures in the late exponential or early stationary phase with ≥1x10-9 cells/mL
- The positive controls must induce a ≥ 3-fold increase in revertants/plate as compared to the concurrent vehicle controls
- A minimum of three non-toxic doses is required to evaluate assay data
- In the absence of toxicity, at least two plates per treatment level showed evidence of bacterial growth indicating the test strain and all overlay components were added correctly

Rationale for test conditions:

These are according to the OECD guideline 471

Evaluation criteria:

Criteria for a Positive Response:
A test article is considered to have produced a positive response if it induces a dose-dependent increase in revertant frequency that is ≥ 2.0-fold vehicle control values for tester strains TA98, TA100, and WP2uvrA, or ≥ 3.0-fold vehicle control values for tester strains TA1535 and TA1537. In addition, any response should be reproducible.

Criteria for a Negative Response:
A test article is considered to have produced a negative response if no dose-dependent, ≥ 2.0-fold or ≥ 3.0-fold increases are observed in tester strains TA98, TA100, and WP2uvrA, or TA1535 and TA1537, respectively.

Criteria for an Equivocal Response:
Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those instances, the test article may be considered to have produced an equivocal response.

Other criteria may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.).

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the
interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Initial mutagenicity assay:
Precipitate was observed at 5000 μg/plate in the presence and absence of S9.
Toxicity was observed at 5000 μg/plate in the presence of S9 in all tester strains except TA100, where toxicity was observed at ≥ 1600 μg/plate, and WP2uvrA, where no toxicity was observed at any tested dose level.
In the absence of S9, toxicity was observed at ≥ 500 μg/plate in all tester strains except TA98 and WP2uvrA, where toxicity was observed at ≥ 1600 and 5000 μg/plate.

Confirmatory mutagenicity assay:
Precipitate was observed at 5000 μg/plate in all tester strains in the presence and absence of S9 except TA98 and WP2uvrA, where no precipitate was observed at any tested dose level in the absence of S9. Toxicity was observed at 5000 μg/plate in all tester strains in the presence of S9 except TA1535, where toxicity was observed at ≥ 1600 μg/plate. In the absence of S9, toxicity was observed at ≥ 500 μg/plate in all tester strains except WP2uvrA, where toxicity was observed at ≥ 1600 μg/plate.

Remarks on result:

other: not mutagenic

Any other information on results incl. tables

Tabulated results

Initial Mutagenicity Assay Results with S9

Strain	Compound	Dose level (mg/plate)	Mean revertants per plate	SD	Ratio treated/ vehicle	Individual revertant colony counts
TA98	Test item	5000	12.7	0.6	0.5	13 P R	13 P M R	12 P M R
		1600	26.7	4	1.1	26 N	31 N	23 N
		500	19	1.7	0.8	18 N	18 N	21 N
		160	19.3	5.5	0.8	14 N	19 N	25 N
		50	23.3	4.5	1	23 N	28 N	19 N
		16	24.3	13.9	1	28 N	9 N	36 N
		5	23.7	4	1	26 N	19 N	26 N
	DMSO		23.7	2.3		25 N	21 N	25 N
TA100	Test item	5000	86.7	6.5	0.6	87 P R	93 P R,	80 P R
		1600	122.3	10.4	0.9	114 R	134 R	119 R
		500	148.3	27.5	1.1	150 N	120 N	175 N
		160	129.7	4.2	0.9	131 N	133 N	125 N
		50	151.7	13.3	1.1	144 N	167 N	144 N
		16	141	12	1	153 N	141 N	129 N
		5	129	7.2	0.9	123 N	137 N	127 N
	DMSO		138.3	5.5		138 N	144 N	133 N
TA1535	Test item	5000	8	2	0.6	10 P M R	6 P R	8 P M R
		1600	13	4	0.9	9 N	17 N	13 N
		500	14	4	1	10 N	14 M N	18 N
		160	16	0	1.1	16 N	16 N	16 N
		50	14	2	1	16 N	12 N	14 N
		16	15.3	2.9	1.1	17 N	17 M N	12 N
		5	13.7	4.5	1	9 N	18 N	14 N
	DMSO		14	5.3		8 N	18 N	16 N
TA1537	Test item	5000	4.3	1.2	0.5	3 P R	5 P R	5 P R
		1600	9	4	1.1	9 N	5 N	13 N
		500	9.3	2.3	1.1	12 N	8 N	8 N
		160	9.7	4	1.2	6 N	14 N	9 N
		50	9	3.6	1.1	12 N	5 N	10 N
		16	8.7	3.5	1	5 N	12 N	9 N
		5	9.7	0.6	1.2	10 N	10 N	9 N
	D		8.3	4		9 N	12 N	4 N
WP2uvrA	Test item	5000	13.3	8.1	0.7	19 P N	4 P N	17 P N
		1600	22.3	8	1.2	14 N	30 N	23 N
		500	15	7.2	0.8	9 N	23 N	13 N
		160	21.7	4	1.1	18 N	21 N	26 N
		50	17	9.6	0.9	10 N	13 N	28 N
		16	14.3	2.5	0.7	14 N	17 N	12 N
		5	14.3	8.5	0.7	6 N	23 N	14 N
	DMSO		19.3	4.7		23 N	21 N	14 N
TA98	BP	2.5	517.7	18.9	21.9	497 N	522 N	534 N
TA100	2AA	2.5	1830.3	20.2	13.2	1812 N	1827 N	1852 N
TA1535	2AA	2.5	249	4.4	17.8	252 N	244 N	251 N
TA1537	2AA	2.5	135.3	25.6	16.2	111 N	133 N	162 N
WP2uvrA	2AA	25	465	74.8	24.1	494 N	521 N	380 N

Initial Mutagenicity Assay Results without S9

Strain	Compound	Dose level (g/plate)	Mean revertants per plate	SD	Ratio treated/ vehicle	Individual revertant colony counts
TA98	Test item	5000	0	0	0	0 P R	0 P R	0 P R
		1600	12.3	6.4	0.8	5 M R	16 R	16 R
		500	14.3	2.3	1	13 N	17 N	13 N
		160	14	1	0.9	13 M N	14 N	15 M N
		50	16	4.4	1.1	14 N	13 N	21 M N
		16	19	3.6	1.3	18 N	23 N	16 N
		5	18.7	3.1	1.2	18 N	16 N	22 N
	DMSO		15	4.6		19 N	10 N	16 N
TA100	Test item	5000	15.3	7	0.1	8 P R	22 P R	16 P R
		1600	75.7	2.1	0.5	75 R	74 R	78 R
		500	114.3	12.1	0.8	105 R	110 R	128 R
		160	125	16.5	0.9	144 N	116 R	115 N
		50	127.3	19.6	0.9	146 N	129 N	107 N
		16	136.7	22.3	0.9	128 N	120 N	162 N
		5	134.7	1.2	0.9	134 N	134 N	136 N
	DMSO		144.7	10.1		133 N	151 N	150 N
TA1535	Test item	5000	0	0	0	0 P R	0 P R	0 P R
		1600	9.7	3.5	0.7	13 R	6 R	10 M R
		500	9	1	0.7	8 R	10 R	9 R
		160	18	1.7	1.4	16 N	19 N	19 N
		50	18.3	2.5	1.4	18 N	16 N	21 N
		16	19.3	2.3	1.5	18 M N	22 N	18 N
		5	20.7	2.5	1.6	21 N	18 N	23 N
	DMSO		13.3	3.2		11 M N	12 N	17 N
TA1537	Test item	5000	0	0	0	0 P R	0 P R	0 P R
		1600	5.3	0.6	0.5	6 R	5 R	5 R
		500	6	2.6	0.6	9 R	4 R	5 R
		160	7	2.6	0.7	5 N	10 N	6 N
		50	6	1.7	0.6	8 N	5 N	5 N
		16	3.7	4.6	0.4	1 N	9 N	1 N
		5	8	5.3	0.8	6 N	14 N	4 N
	DMSO		10	5.3		6 N	16 N	8 N
WP2uvrA	Test item	5000	19.7	3.2	1.3	21 P R	16 P R	22 P R
		1600	13	1	0.9	13 N	14 N	12 N
		500	15.7	6.5	1.1	22 N	9 N	16 N
		160	15.7	1.5	1.1	14 N	16 N	17 N
		50	21.3	5	1.5	26 N	22 N	16 N
		16	16	7	1.1	23 N	16 N	9 N
		5	15.7	1.5	1.1	16 N	14 N	17 N
	DMSO		14.7	2.1		13 N	17 N	14 N
TA98	2NF	1	348	32.4	23.2	321 N	384 N	339 N
TA100	SA	2	1236	106.2	8.5	1206 N	1148 N	1354 N
TA1535	SA	2	862.3	72.2	64.7	781 N	887 N	919 N
TA1537	ICR	2	273.3	25	27.3	295 N	246 N	279 N
WP2uvrA	4NQO	1	75.3	13	5.1	76 N	62 N	88 N

Confirmatory Mutagenicity Assay Results with S9

Strain	Compound	Dose level (g/plate)	Mean revertants per plate	SD	Ratio treated/ vehicle	Individual revertant colony counts
TA98	Test item	5000	21	3	0.9	18 P R M	21 P R M	24 P R M
		1600	26.7	1.2	1.1	26 N	28 N	26 N
		500	19.3	9.6	0.8	21 N	28 N	9 N
		160	16.3	3.8	0.7	12 N	19 N	18 N
		50	20	6.1	0.8	13 N	23 N	24 N
		16	29.3	9.5	1.2	22 N	26 N	40 N
	DMSO		23.7	3.8		28 N	21 N	22 N
TA100	Test item	5000	86.3	9.1	0.6	96 P R	78 P R	85 P R
		1600	115.3	7.1	0.8	109 N	123 N	114 N
		500	119.3	8.1	0.8	124 N	124 N	110 N
		160	138	5.2	1	135 N	135 N	144 N
		50	136.3	11.1	1	126 N	135 N	148 N
		16	145.7	5.9	1	139 N	148 N	150 N
	DMSO		141.7	9.5		131N	145 N	149 N
TA1535	Test item	5000	10.7	2.9	0.7	14 P E M	9 P E M	9 P E M
		1600	5.3	1.2	0.3	6 N	6 M N	4 M N
		500	14.7	2.1	1	17 N	14 N	13 N
		160	16	6.1	1	12 N	13 N	23 N
		50	16.3	3.1	1.1	19 N	17 N	13 N
		16	9.3	4	0.6	10 N	5 N	13 N
	DMSO		15.3	6.5		15 N	9 N	22 N
TA1537	Test item	5000	6	2	0.6	6 P R	8 P R	4 P M R
		1600	8	1.7	0.8	9 N	9 M N	6 N
		500	10.3	1.5	1.1	12 M N	9 N	10 N
		160	6.7	4.6	0.7	12 M N	4 N	4 M N
		50	7.7	0.6	0.8	7 M N	8 N	8 M N
		16	7.7	2.5	0.8	8 N	10 N	5 N
	DMSO		9.7	5.9		12 N	3 N	14 N
WP2uvrA	Test item	5000	13.7	4.5	0.7	14 P R	9 P M R	18 P M R
		1600	15.3	5.5	0.8	21 N	10 N	15 N
		500	19.7	6.7	1	27 N	18 N	14 N
		160	19	3.5	1	21 N	15N	21 N
		50	14.7	2.5	0.7	15 N	17 N	12 N
		16	16.7	3.2	0.8	19 N	18 N	13 N
	DMSO		19.7	5.1		24 N	14 N	21 N
TA98	BP	2.5	450.3	32.6	19	486 N	422 N	443 N
TA100	2AA	2.5	1609.3	53.7	11.4	1615 N	1660 N	1553 N
TA1535	2AA	2.5	238.3	11.6	15.5	226 N	249 N	240 N
TA1537	2AA	2.5	191	20.9	19.8	205 N	167 N	201 N
WP2uvrA	2AA	25	394	71.2	20	348 N	358 N	476 N

Confirmatory Mutagenicity Assay Results without S9

Strain	Compound	Dose level (mg/plate)	Mean revertants per plate	SD	Ratio treated/ vehicle	Individual revertant colony counts
TA98	Test item	5000	6.3	5	0.4	7 R M	1 R M	11 R M
		1600	9.7	2.1	0.6	8 R	12 R	9 R
		500	12.3	3.8	0.8	15 R	14 R	8 R
		160	14.3	2.5	0.9	17 N	12 N	14 N
		50	15.3	5.5	1	15 N	10 N	21 N
		16	14.3	1.2	0.9	13 N	15 N	15 N
		5	17.3	8.1	1.1	22 N	8 N	22 N
	DMSO		16	1.7		14 N	17 N	17 N
TA100	Test item	5000	2.3	2.5	0	5 R P M	2 R P M	0 R P M
		1600	75.3	13.4	0.6	85 R	60 R	81 R
		500	90.3	11.7	0.8	99 R	77 R	95 R
		160	120.3	8	1	128 N	112 N	121 N
		50	115	7.9	1	124 N	109 N	112 N
		16	134	12.1	1.1	127 N	127 N	148 N
		5	115.3	2.3	1	114 N	114 N	118 N
	DMSO		119.3	8.1		118 N	112 N	128 N
TA1535	Test item	5000	0	0	0	0 R P M	0 R P M	0 R P M
		1600	11.3	2.9	0.8	13 R	8 R	13 M R
		500	18.3	1.2	1.3	19 R	19 R	17 R
		160	13	4	1	9 N	17 N	13 N
		50	18.3	5.5	1.3	12 N	22 N	21 N
		16	12.7	3.2	0.9	9 N	14 N	15 N
		5	15.7	5.8	1.1	9 N	19 N	19 N
	DMSO		13.7	2.9		17 N	12 N	12 N
TA1537	Test item	5000	1	1	0.1	2 P R M	1 P R M	0 P R M
		1600	2.7	1.5	0.3	1 R	4 R	3 R
		500	7.7	2.5	0.7	10 R	8 R	5 R
		160	9.3	3.1	0.9	6 N	10 N	12 N
		50	6.3	1.5	0.6	6 N	5 N	8 N
		16	7	4.6	0.7	12 N	6 M N	3 N
		5	8.7	1.2	0.8	8 N	10 N	8N
	DMSO		10.3	3.8		12 N	13 N	6 N
WP2uvrA	Test item	5000	12	3	1	12 R	9 R	15 R
		1600	12.3	3.1	1	13 R	9 R	15 R
		500	11.3	4.7	0.9	13 N	6 N	15 N
		160	16.3	3.8	1.4	12 N	19 N	18 N
		50	15.7	1.2	1.3	15 N	17 N	15 N
		16	14.3	4	1.2	10 N	18 N	15 N
		5	11	3.5	0.9	15 N	9 N	9 N
	DMSO		12	2		14 N	10 N	12 N
TA98	2NF	1	298.3	13.6	18.6	290 N	291 N	314 N
TA100	SA	2	1187	14.9	9.9	1193 N	1170 N	1198 N
TA1535	SA	2	923	46.5	67.5	963 N	872 N	934 N
TA1537	ICR	2	295	7.8	28.5	291 N	304 N	290 N
WP2uvrA	4NQO	1	78.7	6.7	6.6	82 N M	83 N M	71 N M

P = precipitation of test article observed

R = reduced background bacterial lawn

N = normal backgroud bacterial lawn

M = plate counted manually

Applicant's summary and conclusion

Conclusions:

The test item is negative in the Bacterial Reverse Mutation Assay tested up to 5000 µg/plate in the presence or absence of S9 under the conditions of this protocol.

Executive summary:

The objective of this study was to evaluate the test item and/or its metabolites for its ability to induce reverse mutations at the histidine locus in Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537, and at the tryptophan locus of Escherichia coli strain WP2uvrA. Evaluations were conducted in the presence or absence of an exogenous mammalian activation system (S9) containing microsomal enzymes. The study was performed according to OECD TG 471 and in compliance to GLP.

The test item was evaluated in the initial mutagenicity assay in all five tester strains at dose levels of 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate with and without S9. Precipitate was observed at 5000 μg/plate in the presence and absence of S9. Toxicity was observed at 5000 μg/plate in the presence of S9 in all tester strains except TA100, where toxicity was observed at ≥ 1600 μg/plate, and WP2uvrA, where no toxicity was observed at any tested dose level. In the absence of S9, toxicity was observed at ≥ 500 μg/plate in all tester strains except TA98 and WP2uvrA, where toxicity was observed at ≥ 1600 and 5000 μg/plate.

Based on the results of the initial assay, an independent confirmatory mutagenicity assay was conducted in all five tester strains at dose levels of 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate with S9 and 5.00, 16.0, 50.0, 160, 500, 1600, and 5000 μg/plate without S9. Precipitate was observed at 5000 μg/plate in all tester strains in the presence and absence of S9 except TA98 and WP2uvrA, where no precipitate was observed at any tested dose level in the absence of S9. Toxicity was observed at 5000 μg/plate in all tester strains in the presence of S9 except TA1535, where toxicity was observed at ≥ 1600 μg/plate. In the absence of S9, toxicity was observed at ≥ 500 μg/plate in all tester strains except WP2uvrA, where toxicity was observed at ≥1600 μg/plate.

No increase in the mean number of revertant colonies was observed at any tested dose level in any tester strain in the presence or absence of S9 in the initial or the confirmatory assay. All positive and vehicle control values were within the acceptable ranges, and all criteria for a valid study were met.

These results indicate that the test item is negative in the Bacterial Reverse Mutation Assay tested up to 5000 μg/plate in the presence or absence of S9 under the conditions of this protocol.