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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:
experimental study
Adequacy of study:
key study
Study period:
25 Jul - 23 Aug 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

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 (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 57033
- Expiration date of the lot/batch: 25 November 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.

Method

Target gene:
his operon
Species / strain
Species / strain:
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 Aroclor 1254.
Test concentrations with justification for top dose:
Preliminary toxicity test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
First and second experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
Vehicle:
- Vehicle(s)/solvent(s) used: sterile distilled water
- Justification for choice of solvent/vehicle: the test item was fully miscible in sterile distilled water at 50 mg/mL.
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, 2, 3 and 5 µg/plate, -S9, WP2uvrA, TA 100 and TA1535, respectively); 9-Aminoacridine (9AA, 80 µg/plate, -S9, TA 1537), 4-Nitroquinoline-1-oxide (4NQO, 0.2 µg/plate, -S9, TA 98)
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA, 1, 2 and 10 µg/plate, +S9, TA 100, TA 1535 and TA 1537 and WP2uvrA, respectively); Benzo(a)pyrene (BP, 5 µg/plate, +S9, TA 98)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

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

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentration.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS.
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
Mean values and standard deviations were calculated.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Test Results of Experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

WP2uvrA

TA98

TA1537

0

115 ± 7.6

17 ± 9.2

29 ± 5.3

25 ± 4.0

12 ± 7.0

50

111 ± 11.5

17 ± 7.6

26 ± 6.1

23 ± 3.5

15 ± 2.6

150

115 ± 11.0

20 ± 6.1

31 ± 0.0

21 ± 2.1

12 ± 3.6

500

121 ± 14.6

14 ± 4.2

21 ± 0.0

18 ± 2.1

8 ± 4.0

1500

110 ± 7.5

26 ± 4.2

28 ± 8.0

25 ± 3.5

13 ± 1.5

5000

116 ± 4.7

19 ± 9.3

25 ± 7.8

25 ± 10

8 ± 4.5

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations (μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate (average of 3 ± SD)

475 ± 3.1

222 ± 28.4

690 ± 69.2

232 ± 25.2

763 ± 17.3

+

0

107 ± 15.1

14 ± 4.9

37 ± 10.1

19 ± 2.9

15 ± 3.5

+

50

103 ± 7.0

8 ± 0.6

43 ± 1.7

20 ± 1.2

10 ± 4.0

+

150

106 ± 1.7

10 ± 2.1

41 ± 7.4

25 ± 4.7

12 ± 3.6

+

500

109 ± 9.8

10 ± 1.2

42 ± 2.1

27 ± 7.1

14 ± 2.9

+

1500

93 ± 4.6

13 ± 4.0

35 ± 9.3

27 ± 8.4

14 ± 10.3

+

5000

87 ± 10.4

9 ± 1.5

34 ± 1.2

27 ± 3.8

18 ± 4.2

Positive controls, + S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations (μg/plate)

1

2

10

5

2

Mean No. of colonies/plate (average of 3 ± SD)

1760 ±

16.6

278 ± 13.3

345 ± 48.8

166 ± 6.4

145 ± 62.2

ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine

4NGO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

Table 2: Test Results of Experiment 2 (preincubation).

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

WP2uvrA

TA98

TA1537

0

91 ± 15.5

14 ± 4.0

31±2.5

30 ± 8.9

14 ± 3.1

50

84 ± 7.2

12 ± 1

33 ± 9.5

24 ± 8.1

10 ± 5.7

150

87 ± 1.0

16 ± 7.0

29 ± 9.2

29 ± 8.6

16 ± 8.3

500

83 ± 4.6

17 ± 2.3

27 ± 4.5

23 ± 2.5

8 ± 4.5

1500

83 ± 6.7

16 ± 1.0

27 ± 3.2

25 ± 4.7

9 ± 5.1

5000

98 ± 6.9

13 ± 2.5

28 ± 5.8

28 ± 7.5

13 ± 4.0

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Concentrations (μg/plate)

3

5

2

0.2

80

Mean No. of colonies/plate (average of 3 ± SD)

509 ± 120.5

256 ± 45.4

790 ± 12.5

191 ± 9.8

677 ± 25.1

 ±

0

97 ± 16.3

15 ± 2.3

37 ± 5.5

39 ± 6.5

11 ± 1.7

 ±

50

103 ± 4.0

15 ± 4.7

30 ± 9.5

24 ± 6.9

15 ± 1.7

 ±

150

102 ± 10.7

14 ± 3.1

33 ± 6.1

38 ± 6.7

13 ± 1.5

 ±

500

98 ± 15.9

16 ± 2.3

33 ± 8.2

37 ± 5.7

15 ± 2.5

 ±

1500

98 ± 22.9

13 ± 2.1

40 ± 7.6

28 ± 4.5

21 ± 4.9**

 ±

5000

109 ± 6.2

14 ± 1.2

32 ± 7.2

33 ± 9.7

17 ± 5.9

Positive controls, ± S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations (μg/plate)

1

2

10

5

2

Mean No. of colonies/plate (average of 3 ± SD)

2092 ± 559.3

160 ± 20.2

339 ± 22.2

197 ± 20.0

188 ± 53.6

ENNG: N-ethyl-N´-nitro-N-nitrosoguanidine

4NGO: 4-NItroquinoline-1-oxide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

** p≤0.01

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative