Fatty acids, C10-18 and C12-22-unsatd., C14-18 and C16-18-unsatd. alkyl 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:

From 1999-03-08 To 1999-04-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 1a: The study was performed according to OECD 406 guideline and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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, C16-18 and C18-unsatd., Me esters

EC Number:

267-015-4

EC Name:

Fatty acids, C16-18 and C18-unsatd., Me esters

Cas Number:

67762-38-3

IUPAC Name:

67762-38-3

Details on test material:

- Name of test material (as cited in study report): Esterol C
- Analytical purity: 78.09 % (amount of C18 esters)
- Composition of test material, percentage of components: Mixture of methylesters of saturated and unsaturated C16 to C20 fatty acids, no data
- Lot/batch No.: 9906501
- Acid number: 9.30 mgKOH/g
- Expiration date of the lot/batch: December 1999
- Physical state: light yellow liquid
- Storage condition of test material: in dark and at room temperature

Method

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 from induced Aroclor 1254 Rat liver

Test concentrations with justification for top dose:

0, 62.5, 125, 250, 500 and 1000 µg/plate, for all tested strains in the first experiment.
0, 312.5, 625, 1250, 2500 and 5000 µg/plate, for all tested strains in the second experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (-S9, TA 1535 and TA 100) (1 µg/plate); 9-aminoacridine (-S9, TA 1537) (50 µg/plate); 2-nitrofluorene (-S9, TA 98) (0.5 µg/plate); Mitomycin C (-S9, TA 102) (0.5 µg/plate); 2-Anthramine (+S9, all strains) (10 µg/plate for TA102, 2 µg/plate f

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation only for the second experiment with S9 mix.
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours

DETERMINATION OF CYTOTOXICITY
- Method: other: The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn during a preliminary toxicity test.

Evaluation criteria:

A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.

Statistics:

No statistical analysis was used.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants of the vehicle and positive controls was consistent with historical
data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiments without S9 mix:
Except for a slight thinning of the bacterial lawn noted in the TA strain at 1000 µg/plate, no toxicity was noted in the first experiment. In the second
experiment, a slight to marked toxicity was noted in the TA 1537 and TA 100 strains at dose-levels >= 625 µg/plate. In the TA1535 and TA 98
strains, a slight to moderate toxicity was mainly noted at dose-levels>=1250 µg/plate.
- Experiments with S9 mix:
No toxicity was noted in the first experiment in all tester strains. In the second experiment (preincubation method) a slight to moderate toxicity was induced in the TA 1535, TA 1537 and TA 100 strains mainly at dose-levels >= 1250 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Number of revertants per plate (first experiment) (mean of 3 plates)

	TA 1535		TA 1537		TA 98		TA 100		TA 102
Conc. [unit]	- MA	+MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
0	9	14	7	10	17	28	79	90	154	188
62.5	9	14	8	8	12	28	79	92	175	238
125	12	13	9	8	13	27	79	100	174	228
250	11	15	5	9	19	29	75	107	176	235
500	10	14	7	9	17	24	81	91	174	202
1000	10	11	7	8	15	26	72	93	162	241
Positive control	471	332	314	110	160	937	564	1904	932	1735

MA: metabolic activation

Table 2: Number of revertants per plate (second experiment) (mean of 3 plates)

	TA 1535		TA 1537		TA 98		TA 100		TA 102
Conc. [unit]	- MA	+MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
0	17	18	9	12	26	34	99	115	168	190
62.5	14	19	9	7	17	31	111	89	196	142
125	11	15	5	7	22	31	86	91	211	199
250	11	15	8	5	15	27	80	77	159	191
500	11	10	6	5	17	34	67	81	158	177
1000	7	10	2	3	15	32	58	65	129	146
Positive control	385	242	385	112	161	1031	405	1106	823	1646

MA: metabolic activation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Esterol C does not show any mutagenic activity in the bacterial reverse mutation test on Salmonella typhimurium strains.

Executive summary:

In a reverse gene mutation assay in bacteria (Haddouk, 1999) strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to Esterol C (batch 99.06.501) at concentration of, 0, 62.5, 125, 250, 500 and 1000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. No noteworthy increase in the number of revertants was induced in all tested strains with and without metabolic activation.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.