Methyl ricinoleate

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:

15 Jun - 09 Aug 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, safflower-oil, Et esters

EC Number:

293-106-3

EC Name:

Fatty acids, safflower-oil, Et esters

Cas Number:

91051-53-5

Molecular formula:

not applicable, substance is UVCB

IUPAC Name:

Fatty acids, safflower-oil, ethyl esters

Method

Target gene:

his operon for S. typhimurium and trp 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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone.

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene: + S9: 1, 2.5, 5 or 10 µg/plate for all strains

Remarks:

Before use, all S9 batches were characterised with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA 98 at concentrations of 5 μg/plate and 1 μg/plate, respectively.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Exposure duration: 48 hNUMBER 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 TA100 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 finalevaluation decision.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

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 applicable

Positive controls validity:

valid

Test results 2

Key result

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: the test substance slightly precipitated at concentrations ≥1000 µg/plateRANGE-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. At the highest concentration the test substance exhibited limited solubility. COMPARISON WITH HISTORICAL CONTROL DATA: positive and solvent controls were in the 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:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.