[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Apr - 24 Apr 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Due to growth inhibition in Salmonella, but not in E.coli strains, only low test concentrations used.

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:

yes

Remarks:

low test substance concentrations in Salmonella strains used, cytotoxic effects (growth inhibition) not further specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates

EC Number:

293-170-2

EC Name:

Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates

Cas Number:

91052-13-0

Molecular formula:

C15H26O6, C19H34O6, C21H38O6, C25H46O6

Method

Target gene:

his operon (for S. typhimurium) and trp operon (for E. coli)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbital (PB) and 5,6-benzoflavone (BF)

Test concentrations with justification for top dose:

0.61, 1.2, 2.4, 4.9, 10, 20, 39, and 78 µg/plate without metabolic activation: TA 100, TA1535, TA98 and TA1537
10, 20, 39, 78, 156, and 313 µg/plate with metabolic activation: TA 100, TA1535, TA98 and TA1537
313, 625, 1250, 2500, and 5000 µg/plate with and without metabolic activation: E. coli WP2 uvr A

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test substance was insoluble in water, a solubility test was performed with DMSO and acetone. The test substance was insoluble at 50 mg/mL in DMSO, and was dissolved at 100 mg/mL in acetone, and neither exothermic reaction nor generation of gas was observed. Therefore acetone was used as solvent for preparation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: +S9: 2-Aminoanthracene, 2.0 & 10.0 µg/plate for TA1535 and WP2 uvrA; -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 0.01 & 0.1 µg/plate for TA100, WP2 uvrA & TA98; 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine, 1.0 µg/plate for TA1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation);

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: Two replications each in 3 independent experiments.

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition

Evaluation criteria:

If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates ( based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged postive.

Statistics:

The results at each concentration were demonstrated with the mean and the standard deviation.

Results and discussion

Test results

Test results 1

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 examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Growth inhibition of the tested bacterium by the test substance was observed at concentrations of 10 µg/plate and above in the absence of metabolic activation as well as at concentrations of 156 µg/plate and above in the presence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: insoluble
- Precipitation:Yes, at 1250 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary test, the growth inhibition by the test substance was observed at 20 µg/plate and in S. typhimurim TA98, TA1537 without metabolic activation, and at 78 µg/plate and in S. typhimurim TA100, TA1535 without metabolic activation, and at 313 µg/plate and in S. typhimurim TA strains with metabolic activation. Therefore, as the highest dose level of the test substance in the main tests, the 20 µg/plate dose was selected for S. typhimurim TA98, TA1537 without metabolic activation, and the 78 µg/ plate was selected for S. typhimurim TA100, TA1535 without metabolic activation, and the 313 µg/plate dose was selected for S. typhimurim TA strains with metabolic activation, and these highest dose was diluted 5 times (using a ratio of 2) to provide a total of 6 dose levels. And the 5000 µg/plate dose was selected for E.coli WP2uvrA both with and without metabolic activation, and this highest dose was diluted 4 times (using a common ratio of 2) to provide a total of 5 dose levels.

Any other information on results incl. tables

Table 1: Results of the Ames Test

1st experiment	number of revertants: mean value of negative control	number of revertants: mean value of positive control	max. number of revertants: mean value of test material [µg/plate]
without S9-mix
TA 1535	14 ±2	399 ±25	12±2.5 [39]
TA 100	103±5.0	825 ± 34.6	113 ± 9.7 [4.9]
TA 1537	11±1.5	1989± 52.0	13±0.6 [10]
TA 98	19 ±1.7	561 ± 19	18 ±4.4 [1.2]
WP2uvrA	25 ±4.0	243 ±24.7	30 ±2.6 [5000]
with S9-mix
TA 1535	11± 0.6	399 ±25	12 ±2.5 [20]
TA 100	111 ± 6.1	825 ± 34.6	126 ±7.5 [39]
TA 1537	17 ±4.7	1989± 52.0	20±3.8 [20]
TA 98	29±1.5	561 ± 19	28 ±5.0 [39]
WP2uvrA	28 ±0.6	243 ±24.7	33±7.0 [5000]
2nd experiment	number of revertants: mean value of negative control	number of revertants: mean value of positive control	max. number of revertants: mean value of test material [µg/plate]
without S9-mix
TA 1535	10±2.3	388± 16.4	8 ±0.6 [10]
TA 100	121 ± 6.0	780 ±7.1	114 ±7.8 [20]
TA 1537	11 ±1.0	2057±200.9	15 ±0 [4.9]
TA 98	17 ±1.2	577 ±32.8	20±1.5  [1.2]
WP2uvrA	20 ±2.0	219 ±28:1	27 ±4.6 [1250]
with S9-mix
TA 1535	9 ±2.1	388 ±16.4	11 ±3.2 [78]
TA 100	114 ±12.7	780 ±7.1	117±9.3 [10]
TA 1537	23 ±4.7	2057 ±200:9	21±2.3 [10]
TA 98	25±3.8	577 ±32.8	29 ±2.6 [39]
WP2uvrA	25± 3.2	219 ±28:1	35 ±2.1 [1250]

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative