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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
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): glycerol monooleate
- Physical state: light yellow pellets with characteristic odour
- Analytical purity: 99.93%
- Stability under test conditions: verified after test

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E. coli strain)
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 and 5,6-benzoflavone
Test concentrations with justification for top dose:
-S9 mix:
4.88, 9.77, 19.5, 39.1, 78.1, 156 μg/plate (TA100, TA98)
156, 313, 625, 1250, 2500, 5000 μg/plate (TA1535, WP2 uvrA)
0.153, 0.305, 0.610, 1.22, 2.44, 4.88, 9.77 μg/ plate (TA1537)

+S9 mix:
9.77, 19.5, 39.1, 78.1, 156, 313, 625 μg/plate (TA100)
156, 313, 625, 1250, 2500, 5000 μg/plate (TA1535, WP2 uvrA, TA98)
9.77, 19.5, 39.1, 78.1, 156, 313 μg/plate (TA1537)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was not soluble in water.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF2; 0.01 or 0.1 µg/plate, TA100, TA98, E. coli); sodium azide (SA; 0.5 µg/plate, TA1535); 9-aminoacridine (9AA; 80 µg/plate, TA1537); +S9: 2-Aminoanthracene (2AA; 0.5-10 µg/plate, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Pre-incubation method

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

NUMBER OF REPLICATIONS: 3 replications in one experiment

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn




Evaluation criteria:
Revertant colonies were counted for 3 plates per bacterial strain. The test result was considered positive, if the mean value out of 3 plates was at least twice as high as the negative vehicle control and a dose-dependency was found.
Statistics:
Mean values and standard deviation were calculated.

Results and discussion

Test resultsopen allclose all
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:
-S9: at 3.13 (TA1537), 78.1 (TA98) and 100 (TA100) µg/plate and above; +S9: at 128 (TA1537) and 156 (TA100) µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance was not soluble in water.
- Precipitation: in the dose-finding studies and the main study, precipitation was observed at the end of the exposure period at ≥ 625 and ≥ 1250 µg/plate in the presence and absence of metabolic activation, respectively.

RANGE-FINDING/SCREENING STUDIES:
The highest concentrations analysed in the main study were selected based on the solubility and/or cytotoxicity of the test substance in the cell culture medium. Three dose-finding studies were performed:

[Dose-finding study]
-S9 mix: 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
+S9 mix: 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
[Dose-finding restudy]
-S9 mix: 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
+S9 mix: 8.19, 20.5, 51.2, 128, 320, 800, 2000, 5000 μg/plate (all strains)
[Dose-finding additional study]
-S9 mix
6.25, 12.5, 25.0, 50.0, 100, 200 μg/plate (TA100),
3.13, 6.25, 12.5, 25.0, 50.0, 100 μg/plate (TA98),
0.0977, 0.195, 0.391, 0.781, 1.56, 3.13, 6.25, 12.5 μg/plate (TA1537)
+S9 mix:
6.25, 12.5, 25.0, 50.0, 100, 200 μg/plate (TA1537)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test substance was not cytotoxic to tester strains TA1535 and WP2uvrA at any concentration with and without metabolic activation.
In the dose-finding studies and in the main study, the minimum cytotoxic concentrations determined for the tester strains TA100, TA98 and TA1537 were as follows:

[Dose-finding study]
-S9 mix: 8.19 (TA1537) and 128 (TA100) μg/plate
+S9 mix: 128 (TA1537) and 320 (TA100) μg/plate
[Dose-finding restudy]
-S9 mix: 8.19 (TA1537), 51.2 (TA98) and 128 (TA100) μg/plate
+S9 mix: 128 (TA1537), 320 (TA100) μg/plate
[Dose-finding additional study]
-S9 mix: 3.13 (TA1537) and 100 (TA100, TA98) μg/plate
+S9 mix: 200 (TA1537) μg/plate
[Main study]
-S9 mix: 4.88 (TA1537), 78.1 (TA98) and 156 (TA100) µg/plate
+S9 mix: 156 (TA100) and 313 (TA1537) 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. Results of the bacterial reverse mutation test

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

WP2urvA

TA98

TA1537

0

129 ± 7

13 ± 2

25 ± 5

21 ± 2

9 ± 1

0.153

 

 

 

 

9 ± 2

0.305

 

 

 

 

7 ± 1

0.610

 

 

 

 

9 ± 1

1.22

 

 

 

 

12 ± 1

2.44

 

 

 

 

7 ± 2

4.88

129 ± 5

 

 

21 ± 1

10 ± 2*

9.77

139 ± 4

 

 

18 ± 3

10 ± 3*

19.5

125 ± 5

 

 

20 ± 4

 

39.1

134 ± 3

 

 

17 ± 2

 

78.1

142 ± 6

 

 

18 ± 3*

 

156

131 ± 6 *

12 ± 2

16 ± 3

24 ± 6*

 

313

10 ± 3

22 ± 3

 

 

625

12 ± 2

21 ± 4

 

 

1250 (+)

10 ± 2

16 ± 2

 

 

2500 (+)

10 ± 2

16 ± 2

 

 

5000 (+)

 

9 ± 1

13 ± 3

 

 

Positive controls,

–S9

Name

AF2

SA

AF2

AF2

9AA

Concentrations

(μg/plate)

0.01

0.5

0.01

0.1

80

Mean No. of colonies/plate

(average of 3 plates ± SD)

886 ± 5

549 ± 8

148 ± 19

689 ± 53

200 ±7

+

0

121 ± 4

13 ± 1

26 ± 5

29 ± 1

25 ± 3

+

9.77

132 ± 7

 

 

 

20 ± 2

+

19.5

130 ± 6

 

 

 

20 ± 2

+

39.1

125 ± 8

 

 

 

21 ± 2

+

78.1

121 ± 4

 

 

 

13 ± 4

+

156

123 ± 4*

14 ± 0

25 ± 4

24 ± 3

12 ± 2

+

313

118 ± 5*

12 ± 2

19 ± 5

25 ± 3

10 ± 2*

+

625 (+)

106 ± 8*

13 ± 2

23 ± 4

26 ± 6

 

+

1250 (+)

 

8 ± 2

16 ± 2

19 ± 3

 

+

2500 (+)

 

9 ± 1

16 ± 2

19 ± 1

 

+

5000 (+)

 

11 ± 1

16 ± 2

12 ± 2

 

Positive controls, +S9

Name

2AA

2AA

2AA

2AA

2AA

Concentrations

(μg/plate)

1

2

10

0.5

2

Mean No. of colonies/plate

(average of 3 plates ± SD)

1018 ± 35

336 ± 19

732 ± 65

370 ± 10

142 ± 5

(+): Visible precipitation was observed at the end of exposure period.

*: Growth inhibition was observed

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative