Reaction mass of methyl (9E,12E,15Z)-9,12,15-octadecatrienoate and methyl (9E,12Z,15E)-9,12,15-octadecatrienoate and methyl (9Z)-9-octadecenoate and methyl (9Z,12E,15E)-9,12,15-octadecatrienoate and methyl (9Z,12Z)-9,12-octadecadienoate

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:

2015

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:

2015

Report date:

2015

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:

methyl (9E,12E,15Z)-octadeca-9,12,15-trienoate; methyl (9E,12Z,15E)-octadeca-9,12,15-trienoate; methyl (9Z)-octadec-9-enoate; methyl (9Z,12E,15E)-octadeca-9,12,15-trienoate; methyl (9Z,12Z)-octadeca-9,12-dienoate

Molecular formula:

not applicable (reaction mass of constitutional isomers)

IUPAC Name:

methyl (9E,12E,15Z)-octadeca-9,12,15-trienoate; methyl (9E,12Z,15E)-octadeca-9,12,15-trienoate; methyl (9Z)-octadec-9-enoate; methyl (9Z,12E,15E)-octadeca-9,12,15-trienoate; methyl (9Z,12Z)-octadeca-9,12-dienoate

Test material form:

gas under pressure: refrigerated liquefied gas

Details on test material:

- Physical state: Liquid.
- Storage condition of test material: In refrigerator (2-8°C protected from light, container flushed with nitrogen
- Other: Colourless to pale yellow

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 μg/plate
Experiment 1: 52, 164, 512, 1600, 5000 μg/plate
Experiment 2: 275, 492, 878, 1568, 2800 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed. The test substance formed an emulsion in dimethyl sulfoxide. The test substance was soluble in ethanol. Based on this solubility test ethanol was the solvent of preference.

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. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

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 2800 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Test substance was either tested up to precipitating concentrations or the limit concentration (5000 μg/plate).

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:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Test substance was either tested up to precipitating concentrations or the limit concentration (5000 μg/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Range-finding test and Experiment 1: The test substance precipitated on the plates as oily droplets at the start of the incubation period at concentrations of 512 µg/plate and upwards and at 1600 µg/plate and above at the end of the incubation period.
Experiment 2: Oily droplets of the test substance were observed on the plates at the start of the incubation period at concentrations of 275 µg/plate and upwards (absence of S9-mix) or 878 µg/plate and upwards (presence of S9-mix) in tester strains TA1535, TA1537, TA98 and TA100. In tester strain WP2uvrA precipitate was observed at concentrations of 878 µg/plate and upwards both in the absence and presence of S9-mix. At the end of the incubation period, precipitate was observed on the plates at 878 µg/plate and above (absence of S9-mix) or 2800 µg/plate (presence of S9-mix).

RANGE-FINDING/SCREENING STUDIES:
The test substance was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
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.
Tester strain TA100, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the absence of S9-mix in the range finder, Experiment 1 and Experiment 2. This was highest in Experiment 2 at the dose level of 2800 µg/plate. However, since the increase was not two-fold (a maximum of 1.7-fold was reached), this increase was not considered to be relevant. See 'any other information on materials and methods incl. tables' for more information on the acceptability/interpretation criteria of the 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
Without S9-mix	TA100			WP2uvrA
Positive control	668	± 75		955	± 45
Solvent control	97	± 11		27	± 4
1.7	94	± 17		29	± 4
5.4	91	± 11		25	± 6
17	88	± 12		36	± 8
52	78	± 9		29	± 3
164	97	± 12		26	± 1
512	101	± 20	NP	30	± 2	NP
1600	93	± 9	SP	39	± 6	SP
5000	155	± 26	n SP	33	± 4	n SP
With S9-mix #1	TA100			WP2uvrA
Positive control	1132	± 111		183	± 7
Solvent control	102	± 17		34	± 11
1.7	102	± 15		30	± 7
5.4	103	± 11		37	± 6
17	108	± 8		40	± 7
52	84	± 6		32	± 11
164	96	± 3		30	± 4
512	78	± 13	NP	41	± 9	NP
1600	81	± 2	SP	39	± 6	SP
5000	107	± 14	n SP		± 4	n SP

#1: Plate incorporation assay (5% S9)

NP: No precipitate

SP: Slight Precipitate (oily droplets)

n: Normal bacterial background lawn

 

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
Without S9-mix	TA1535			TA1537			TA98
Positive control	718	± 36		235	± 18		873	± 63
Solvent control	16	± 2		12	± 3		27	± 7
52	14	± 2		9	± 3		18	± 4
164	14	± 6		6	± 5		25	± 4
512	18	± 6	NP	9	± 5	NP	26	± 2	NP
1600	17	± 3	SP	11	± 4	SP	24	± 9	SP
5000	13	± 3	n SP	9	± 3	n SP	31	± 12	n SP
With S9-mix #1	TA1535			TA1537			TA98
Positive control	306	± 3		457	± 46		1075	± 214
Solvent control	18	± 5		11	± 1		29	± 3
52	16	± 5		6	± 2		34	± 0
164	18	± 2		6	± 2		36	± 2
512	20	± 5	NP	11	± 4	NP	27	± 3	NP
1600	17	± 5	SP	11	± 7	SP	24	± 10	SP
5000	17	± 6	n SP	8	± 6	n SP	27	± 7	n SP

#1: Plate incorporation assay (5% S9)

NP: No precipitate

SP: Slight Precipitate (oily droplets)

N: Normal bacterial background lawn

 

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
Without S9-mix	TA1535			TA1537			TA98			TA100			WP2uvrA
Positive control	682	±25		756	± 123		690	± 209		746	± 16		1619	± 121
Ethanol	13	± 6		4	± 2		18	± 6		96	± 7		29	± 14
275	13	± 1		6	± 3		26	± 15		94	± 7		25	± 6
492	11	± 4	NP	6	± 2	NP	14	± 8	NP	124	± 6	NP	30	± 3	NP
878	13	± 4	SP	8	± 5	SP	30	± 23	SP	105	± 5	SP	32	± 6	SP
1568	8	± 3	SP	8	± 1	SP	19	± 5	SP	147	± 12	SP	32	± 4	SP
2800	14	± 4	n SP	12	± 2	n SP	16	± 3	n SP	163	± 10	n SP	26	± 6	n SP
With S9-mix #1	TA1535			TA1537			TA98			TA100			WP2uvrA
Positive control	222	± 26		443	± 38		462	± 72		1185	± 39		334	± 23
Ethanol	24	± 6		10	± 7		36	± 6		91	± 14		33	± 3
275	18	± 5		8	± 2		25	± 3		82	± 12		31	± 8
492	18	± 3		16	± 3		22	± 2		79	± 8		28	± 2
878	18	± 0		3	± 2		21	± 6		102	± 9		33	± 3
1568	16	± 6	NP	4	± 4	NP	26	± 4	NP	87	± 12	NP	34	± 3	NP
2800	20	± 12	n SP	6	± 3	n SP	18	± 6	n SP	94	± 7	n SP	35	± 3	n SP

#1: Plate incorporation assay (10% S9)

NP: No precipitate

SP: Slight Precipitate (oily droplets)

n: Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study the test substance is not considered to be mutagenic. Negative and strain specific positive control values were within laboratory historical control data.

Executive summary:

The study was performed to OECD TG 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). Within 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. Oily droplets of the test substance on the plates were observed at dose levels of 1600 and 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first mutation assay. 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 52 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment of the assay with additional parameters, the test substance was tested at a concentration range of 275 to 2800 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. In the first mutation assay, oily droplets of the test substance on the plates were observed at dose levels of 1600 and 5000 μg/plate. In the second mutation assay, oily droplets of the test substance on the plates were observed at dose levels of 878 μg/plate and upwards (absence of S9-mix) and 2800 µg/plate (presence of S9-mix). The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test substance did not induce a biologically relevant or 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control substances which 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.