Fatty acids, essential, Et esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

June 15th, 2010 - August 9th, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance ethyl linoleate (CAS No. 544-35-4). In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

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:

Ethyl linoleate

EC Number:

208-868-4

EC Name:

Ethyl linoleate

Cas Number:

544-35-4

Molecular formula:

C20H36O2

IUPAC Name:

ethyl octadeca-9,12-dienoate

Details on test material:

- CAS No. of test material (as cited in study report): 544-35-4
- Physical state: hazy yellow liquid
- Analytical purity: 75% active substance
- Lot/batch No.: S300660001
- Expiration date of the lot/batch: 31 March 2011
- Storage condition of test material: stable at RT in the dark
- Molecular weight: 308.50

Method

Target gene:

Histidine operon for S. typhimurium and Tryptophan operon for E. 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:

rat liver S9-mix

Test concentrations with justification for top dose:

3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9-mix

Migrated to IUCLID6: 5 µg/plate for TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9-mix

Migrated to IUCLID6: 60 µg/plate for TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9-mix

Migrated to IUCLID6: 10 µg/plate for TA98

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix

Migrated to IUCLID6: 650 µg/plate for TA100

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix

Migrated to IUCLID6: 10 µg/plate for WP2 uvrA

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene: 1 µg/plate for TA98 and TA100; 2.5 µg/plate for TA1535 and TA1537; 10 µg/plate for WPA2uvrA

Remarks:

with 5% S9-mix

Controls 7

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene: 1 µg/plate for TA98; 2.5 µg/plate for TA100 and TA1535; 5µg/plate for TA1537; 10 µg/plate for WPA2uvrA

Remarks:

with 10% S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: Two independent experiments were performed in triplicates each.

DETERMINATION OF CYTOTOXICITY
- Method: The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically.

In the first experiment S9-mix at 5% (v/v) was used. In the second experiment S9-mix at 10% (v/v) was used.

The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present.
If more than 40 colonies were present, these were counted automatically with a Biocount 4000 Pro-S-colony counter.
Plates with abundant test article precipitate which interfered with automated colony counting were counted manually and the evidence of test substance precipitate on the plates was recorded.

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the
concurrent control, and the total number of revertants in tester strains TA1535, TA1537,
TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated
experiment.
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA1OO is greater than two (2) times the
concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98
or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the
tester strains, the positive response should be reproducible in at least one independently
repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final
evaluation decision.

Results and discussion

Test results

Test results 1

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test substance slightly precipitates at concentrations ≥1000 µg/plate

RANGE-FINDING/SCREENING STUDIES: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed based on preliminary tests in TA 100 and WP2uvrA up to 5000 µg/plate in triplicate. The the highest concentration the test substance exhibited limited solubility.

COMPARISON WITH HISTORICAL CONTROL DATA: positive and solvent controls are in range of historical control data

Any other information on results incl. tables

Table 1: Maximum number of revertants at the first experiment with 5% S9 -mix

	Maximum number of revertants
	solvent Control		positive control		treatment (at dose level [µg/mL])
Strain	With S9	Without S9	With S9	Without S9	With S9	Without S9
TA 1535	8	12	132	893	12 (100)	9 (33)
TA 1537	6	7	417	325	10 (33)	9 (1000)
TA 98	28	18	963	1025	34 (33)	21 (333)
TA 100	77	69	890	1248	81 (33)	86 (100)
WPA­2uvrA	23	19	465	1159	35 (5000)	32 (3330)

Table 2: Maximum number of revertants at the second experiment with 10% S9 -mix

	Maximum number of revertants
	solvent Control		positive control		treatment (at dose level [µg/mL])
Strain	With S9	Without S9	With S9	Without S9	With S9	Without S9
TA 1535	7	11	134	838	12 (10)	13 (100)
TA 1537	6	6	413	154	6 (333)	8 (100)
TA 98	25	16	517	981	28 (33)	21 (100)
TA 100	88	89	1418	925	83 (10)	101 (10)
WPA­2uvrA	23	18	200	1195	31 (33)	27 (10)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative