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Toxicological information

Genetic toxicity: in vitro

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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:
weight of evidence
Study period:
09 Apr - 24 Apr 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions. 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:
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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Acetic and fatty acid esters of glycerol
- CAS No. of test material (as cited in study report): 91052-13-0
- Molecular weight: 358.47
- Substance type: light yellow, clear liquid
- Physical state: liquid
- Analytical purity: 100%
- Lot/batch No.: K010722
- Stability under test conditions: stable
- Storage condition of test material: room temperature
- Other information (as cited in study report): melting Point: -7.2°C, solubility- Water: insoluble, DMSO: insoluble, Acetone: 100 mg/ mL and more

Method

Target gene:
his operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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 (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 metablic 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.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9: Benzo(a)pyrene (5.0 µg/plate for TA100, TA98 and TA1537); 2-Aminoanthracene (2.0 and 10.0 µg/plate for TA1535 and WP2 uvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (0.01 and 0.1 µg/plate for TA100, WP2 uvrA and TA98, respectively); 2-Methoxy-6-chloro-9-(3-(2-chloroethyl)aminopropylamino)acridine.2HCl (1.0 µg/plate for TA1537); sodium azide (0.5 µg/plate for TA1535)
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 resultsopen allclose all
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
Positive controls validity:
valid
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:
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
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.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

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 (migrated information):
negative