Reaction mass of (9E)-9-Undecenal and (9Z)-9-Undecenal and undec-10-enal

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: March 2013; signature: May 2013

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(9E)-undec-9-enal; (9Z)-undec-9-enal; undec-10-enal

EC Number:

941-821-2

Molecular formula:

C11H20O

IUPAC Name:

(9E)-undec-9-enal; (9Z)-undec-9-enal; undec-10-enal

Test material form:

liquid

Details on test material:

- Physical state: liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Other: Clear colourless

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

Dose range finding study: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 ug/plateExperiment 1: TA1535, TA1537 and TA98: 0.5, 1.7, 5.4, 52, 164, 512 ug/plate
Experiment 2: TA1535, TA1537 and TA100 and TA98 strains: 9,16, 30, 51, 92, 164 ug/plate; WP2uvrA strain: 492, 1568, 2800, 5000 ug/plate
Experiment 3: TA1535 and TA98 strains only: 92, 164, 492 and 878 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was soluble in dimethyl sulfoxide (DMSO).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted. The revertant colonies were counted automatically with a Colony Counter.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn) and reduction in the number of revertants

OTHER:
- Dose range finding test on TA100 and WP2urvA with and without 5% (v/v) S9-mix; First mutation assay on TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix.
- To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed on all strains, in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 164 μg/plate in strains TA1535, TA1537, TA98, TA100 and 5000 μg/plate in strain WP2uvrA.- Since no toxicity and no precipitate on the plates was observed in tester strains TA1535 and TA98 in the presence of S9-mix in experiment 2, an additional mutation experiment was performed in the presence of 10% (v/v) S9-mix. The following dose range was selected for the third mutation assay: 92, 164, 492 and 878 μg/plate.

Evaluation criteria:

See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.

Statistics:

No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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:

Cytotoxicity was observed at the top two dose levels dose of 5000 μg/plate in tester strain TA1535 and TA100 (absence and presence of S9-mix) and TA1535 and TA98 (with and without S9-mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES:
Test substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. The highest concentration the test substance used in the subsequent mutation assay was 512 μg/plate (experiment 1) based on the results of this assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain
	TA100		WP2uvrA
Without S9-mix
Positive control	853	±	1221	± 76
Solvent control	117	±	30	± 1
1.7	108	±	33	± 5
5.4	123	±	36	± 4
17	121	±	33	± 8
52	111	±	33	± 4
164		e MC	27	± 4
512	0	± 0 a	32	± 9
1600	0	± 0 a	24	± 5 n
5000	0	± 0 a NP	15	± 5 s NP
With S9-mix #1
Positive control	1249	± 53	141	± 27
Solvent control	116	± 21	46	± 10
1.7	94	± 6	43	± 9
5.4	115	± 9	39	± 9
17	117	± 20	37	± 7
52	114	± 16 n	42	± 5
164	61	± 8 m	37	± 3
512		e MC	33	± 9
1600	0	± 15 n	25	± 7
5000	0	± 12 s NP	2	± 2 n NP

#1          Plate incorporation assay (5% S9)

MC        Microcolonies

NP         No precipitate

a            Bacterial lawn absent

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

 

Table 2 Experiment 1: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA1535		TA1537		TA98
Without S9-mix
Positive control	641	± 35	310	± 11	716	± 33
Solvent control	24	± 2	8	± 2	17	± 4
0.5	22	± 8	7	± 4	21	± 14
1.7	30	± 6	11	± 4	15	± 1
5.4	28	± 2	9	± 7	10	± 2
17	26	± 9 n	8	± 3 n	21	± 8 n
52	13	± 3 s	5	± 3 m	13	± 6 m
164		e MC	0	± 0 a	0	± 0 a
512	0	± 0 a NP	0	± 0 a NP	0	± 0 a NP
With S9-mix #1
Positive control	289	± 9	351	±	769	± 59
Solvent control	20	± 9	12	±	25	± 4
0.5	20	± 6	13	±	26	± 4
1.7	13	± 5	11	±	21	± 8
5.4	22	± 9	12	±	24	± 7
17	19	± 1	7	±	24	± 5
52	11	± 3	9	±	20	± 4 n
164		e MC		e MC		e MC
512		e NP MC	0	± 0 NP		e MC

#1          Plate incorporation assay (5% S9)

MC        Microcolonies

NP         No precipitate

a            Bacterial lawn absent

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

 

Table 3 Experiment 2: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535		TA1537		TA98		TA100		WP2uvrA
Without S9-mix
Positive control	713	± 21	445	± 23	823	± 60	981	± 46	1168	± 355
Solvent control	19	± 4	7	± 6	18	± 5	115	± 5	18	± 6
9	22	± 3	7	± 4	19	± 2	114	± 15	-	-
16	27	± 4	9	± 3	22	± 3	114	± 4	-	-
30	23	± 3 n	5	± 1 n	19	± 4 n	123	± 22 n	-	-
51	15	± 5 m	7	± 2 m	17	± 2 s	103	± 5 s	-	-
92		e MC		e MC		e MC		e MC	-	-
164		e NP MC	0	± 0 a NP	0	± 0 a NP	0	± 0 a NP	-	-
492	-	-	-	-	-	-	-	-	20	± 2
878	-	-	-	-	-	-	-	-	17	± 4
1568	-	-	-	-	-	-	-	-	21	± 9 n
2800	-	-	-	-	-	-	-	-	21	± 3 s
5000	-	-	-	-	-	-	-	-	10	± 3 m NP
With S9-mix #1
Positive control	169	± 5	473	± 10	409	± 27	1279	± 57	163	± 10
Solvent control	12	± 6	15	± 6	27	± 4	114	± 23	33	± 5
9	16	± 8	9	± 3	27	± 4	102	± 14	-	-
16	14	± 2	13	± 2	29	± 2	108	± 10	-	-
30	20	± 2	15	± 2	25	± 7	106	± 14	-	-
51	11	± 6	12	± 5	27	± 8	107	± 10	-	-
92	16	± 3	9	± 2 n	17	± 6	115	± 8 n	-	-
164	7	± 4 n NP	0	± 0 a NP	19	± 5 n NP	81	± 5 m NP	-	-
492	-	-	-	-	-	-	-	-	20	± 5
878	-	-	-	-	-	-	-	-	26	± 5
1568	-	-	-	-	-	-	-	-	22	± 8
2800	-	-	-	-	-	-	-	-	20	± 5
5000	-	-	-	-	-	-	-	-	27	± 11 n NP

#1          Plate incorporation assay (10% S9)

MC        Microcolonies

NP         No precipitate

a            Bacterial lawn absent

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

-             Not tested

 

Table 4 Experiment 3: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.
	TA1535		TA98
With S9-mix #1
Positive control	176	± 15	472	± 74
Solvent control	16	± 8	22	± 10
92	15	± 7 n	14	± 3
164	10	± 2 m	27	± 4 n
492		e MC		e MC
878		e NP MC		e NP MC

#1          Plate incorporation assay (10% S9)

MC        Microcolonies

NP         No precipitate

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation. Negative and strain specific positive control values were within laboratory historical control data.

Executive summary:

The study was performed to OECD 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP; to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat). In the dose range finding test, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 164 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at the dose level of 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test the test substance was tested in the first mutation assay at a concentration range of 0.5 to 512 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains. In an independent repeat of the assay with additional parameters, the test substance was tested at a concentration range of 9 to 164 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strain WP2uvrA. In the second mutation assay, in the absence of S9-mix, toxicity was observed in all tester strains. In the presence of S9-mix, toxicity was only observed in tester strains TA1537 and TA100. Since in the second experiment in the presence of S9-mix, insufficient toxicity without precipitate on the plates was observed in tester strains TA1535 and TA98, an additional mutation experiment was performed in the presence of 10% (v/v) S9-mix. The test substance was tested up to the dose level of 878 μg/plate. In the third mutation assay, toxicity was observed in both tester strains. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the conditions of this study it is concluded that that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.