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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:
key study
Study period:
11 - 20 Mar 1998
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:
1998
Report Date:
1998

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): only trade name given
- Physical state: pale yellow liquid
- Analytical purity: 100% (UVCB)
- Lot/batch No.: N567701
- Storage condition of test material: at room temperature in the dark
- Other: density approximately 860 kg/m³ (25 ºC)

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 Aroclor 1254
Test concentrations with justification for top dose:
10, 33, 100, 333 and 1000 µg/plate, with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, daunomycine, methylmethanesulfonate, 4-nitroquinoline-N-oxide, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (reduction of bacterial background lawn, increase in size of the microcolonies, reduction of the revertant colonies)

OTHER:
the results of the dose range-finding test were included as part of experiment 1
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following
criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory background historical range for each tester strain (see Table 1)
b) The positive control chemicals should produce responses in all tester strains which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants compared to the solvent control value in any of the tester strains, either with or without metabolic activation. However, any plate with a mean plate count of less than 20 colonies is considered to be not significant and the result will be disregarded.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

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:
no cytotoxicity, but tested up to precipitating concentrations
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed in all strains, with and without metabolic activation, from 1000 µg/plate and above.

RANGE-FINDING/SCREENING STUDIES:
A dose range-finding test with strain TA100 and the WP2uvrA strain (both with and without S9- mix) was performed to select suitable doses for the main experiments. Eight concentrations (3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate) were tested in triplicate. The results were reported as a part of the first experiment of the mutation assay. The highest concentration of the test substance used in the main experiments was the level at which the test substance exhibited limited solubility. Precipitation was observed on the plates from 1000 µg/plate and above in both strains.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the spontaneous mutation rate of each tester strain per plate were within the characteristic spontaneous mutation range (see Table 1 under 'Any other information on materials and methods incl. tables'), with the exception of TA 100 without metabolic activation. The value was slightly outside the limit of the range, therefore the validity of the test was considered not to be affected.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity was observed at any concentration level, with or without metabolic activation (see Table 2 and 3).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Results of experiment 1

 

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-S9

ethanol

55 ± 3

12 ± 3

14 ± 6

16 ± 2

6 ± 1

-S9

3

55 ± 6

-

11 ± 2

-

-

-S9

10

63 ± 7

10 ± 4

10 ± 3

15 ± 6

7 ± 2

-S9

33

49 ± 7

8 ± 3

11 ± 1

16 ± 1

7 ± 4

-S9

100

59 ± 2

8 ± 1

12 ± 2

15 ± 3

5 ± 3

-S9

333

57 ± 7

7 ± 2

11 ± 6

17 ± 4

5 ± 3

-S9

1000 SP

70 ± 6

10 ± 3

10 ± 2

16 ± 1

4 ± 1

-S9

3300 SP

77 ± 9

-

12 ± 2

-

-

-S9

5000 SP

59 ± 13

-

10 ± 4

-

-

Positive controls, –S9

Name

MMS

SA

4-NQO

DM

9-AC

Concentrations

(μg/plate)

650

1

10

4

60

 

529 ± 23

121 ± 18

291 ± 26

375 ± 36

185 ± 66

 

 

 

 

 

 

 

+S9

ethanol

71 ± 3

8 ± 2

10 ± 1

26 ± 2

8 ± 1

+S9

3

66 ± 10

 

11 ± 4

 

 

+S9

10

62 ± 3

9 ± 2

9 ± 4

25 ± 4

8 ± 3

+S9

33

64 ± 3

9 ± 3

14 ± 3

22 ± 2

6 ± 2

+S9

100

50 ± 8

9 ± 3

15 ± 3

24 ± 5

6 ± 2

+S9

333

45 ± 5

13 ± 4

10 ± 3

18 ± 4

6 ± 4

+S9

1000 SP

54 ± 8

12 ± 4

12 ± 6

23 ± 5

8 ± 1

+S9

3330 SP

54 ± 9

-

12 ± 1

-

-

+S9

5000 SP

51 ± 9

-

12 ± 2

-

-

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

1

2.5

5

2.5

2.5

 

1182 ± 90

240 ± 8

88 ± 10

1047 ± 57

713 ± 149

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

DM = daunomycine

2-AA = 2-aminoanthracene

SP = slight precipitate

 

Table 3: Results of experiment 2

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± SD)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-S9

ethanol

88 ± 10

10 ± 2

10 ± 6

22 ± 7

5 ± 2

-S9

10

94 ± 13

9 ± 3

9 ± 2

17 ± 2

7 ± 3

-S9

33

93 ± 3

8 ± 3

12 ± 2

17 ± 3

5 ± 1

-S9

100

90 ± 7

10 ± 3

9 ± 2

14 ± 5

5 ± 3

-S9

333

90 ± 9

6 ± 3

11 ± 3

15 ± 6

5 ± 1

-S9

1000 SP

77 ± 5

6 ± 3

9 ± 4

14 ± 4

7 ± 4

Positive controls, –S9

Name

MMS

SA

4-NQO

DM

9-AC

Concentrations

(μg/plate)

650

1

10

4

60

 

714 ± 49

152 ± 10

936 ± 118

598 ± 16

251 ± 100

 

 

 

 

 

 

 

+S9

ethanol

99 ± 8

8 ± 4

11 ± 7

15 ± 1

7 ± 3

+S9

10

98 ± 10

8 ± 3

7 ± 1

17 ± 3

4 ± 2

+S9

33

103 ± 21

6 ± 3

8 ± 2

27 ± 6

7 ± 1

+S9

100

98 ± 9

8 ± 3

13 ± 7

21 ± 7

6 ± 2

+S9

333

93 ± 6

7 ± 5

8 ± 3

20 ± 5

8 ± 2

+S9

1000 SP

112 ± 2

9 ± 3

8 ± 1

23 ± 1

4 ± 1

Positive controls, +S9

Name

2-AA

2-AA

2-AA

2-AA

2-AA

Concentrations

(μg/plate)

1

2.5

5

2.5

2.5

 

2235 ± 60

322 ± 15

406 ± 20

1732 ± 115

703 ± 41

 

MMS = methylmethanesulfonate

SA = sodium azide

4-NQO = 4-nitroquinoline-N-oxide

9-AC = 9-aminoacridine

DM = daunomycine

2-AA = 2-aminoanthracene

SP = slight precipitate

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative