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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 from supporting substance (structural analogue or surrogate)
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
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 (+9S, all strains) (10 µg/plate for TA 102, 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
ambiguous 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 Salmonellatyphimurium(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.