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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:
10 July - 10 Aug 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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)
Version / remarks:
2008
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

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/β-naphthoflavone (80/100 mg/kg bw/day for 3 days)
Test concentrations with justification for top dose:
Range-finding study:
TA100 and WP2 uvrA: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment 1: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment 2:
TA100, TA98, TA1537 and WP2 uvrA: 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation
TA1535: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate without metabolic activation
TA100 and WP2 uvrA: 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation
TA1535, TA98 and TA1537: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was incompatible with water but was fully soluble in DMSO at 50 mg/mL.
Controls
Untreated negative controls:
yes
Remarks:
without S9 mix
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-aminoanthracene (2-AA)
Remarks:
+S9: 2-AA (1 µg/plate, TA100; 2 µg/plate, TA1535, TA1537; 10 µg/plate, WP2 uvrA); B[a]P (5 µg/plate, TA98); -S9: ENNG (2 µg/plate, WP2 uvrA; 3 µg/plate, TA100; 5 µg/plate, TA1535); 9-AA (80 µg/plate, TA1537); 4-NQO (0.2 µg/plate, TA98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

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

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods can also be used as an aid to evalutation, however, statistical significance will not be the only determining factor for a positive response.
A test substance will be considered non-mutagenic in the test system if the above criteria are not met.

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:
(exp. 2: -S9: starting at 500 µg/plate in TA 1535; +S9: starting at 1500 µg/plate in TA 1535 and at 5000 µg/plate in TA 98 and TA 1537)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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
Remarks:
but tested up to the precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: An oily precipitate was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: In order to select appropriate dose levels for the main study, a range-finding study was carried out using TA 100 and WP2 uvrA. Test substance concentrations from 0.15 to 5000 µg/plate were tested with and without metabolic activation. The test substance was non-toxic to the tested strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In experiment 1, the test material caused no visible reduction in the growth of the bacterial background lawns at any dose level either with or without S9 mix. In experiment 2, the test substance caused a visible reduction in the growth of the bacterial background lawns and/or substantial decrease in the revertant colony frequency of several of the strains both with and without metabolic activation, initially at 500 µg/plate. The results were not indicative of toxicity sufficiently severe enough to prevent the test material being tested up to the maximum recommended dose level of 5000 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. Test results of experiment 1 (plate incorporation).

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

TA100

TA1535

WP2 uvrA

TA98

TA1537

0

95

18

22

17

12

0 (DMSO)

98 ± 1.2

16 ± 1.7

24 ± 2.3

19 ± 3.5

14 ± 0.6

50

93 ± 10.3

14 ± 1.5

23 ± 4.0

18 ± 2.5

14 ± 0.6

150

85 ± 5.6

16 ± 2.5

23 ± 3.5

19 ± 3.6

14 ± 2.1

500

87 ± 2.3

15 ± 4.0

19 ± 1.5

15 ± 2.3

13 ± 3.1

1500

84 ± 5.6

16 ± 3.8

23 ± 6.7

17 ± 4.0

15 ± 2.9

5000

90 ± 1.5P

18 ± 1.5P

23 ± 4.5P

16 ± 4.0P

14 ± 1.2P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4-NQO

9-AA

Concentration

[μg/plate]

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

492 ± 29.2

115 ± 30.7

145 ± 0.0

119 ± 4.0

400 ± 58.8

+

0 (DMSO)

90 ± 7.8

12 ± 0.6

21 ± 2.5

24 ± 4.4

13 ± 2.0

+

50

97 ± 6.2

11 ± 3.6

20 ± 1.5

21 ± 3.0

13 ± 1.2

 

150

94 ± 4.9

12 ± 1.0

24 ± 4.0

21 ± 1.7

13 ± 2.1

+

500

73 ± 3.2

12 ± 2.3

21 ± 2.9

16 ± 1.5

14 ± 4.0

+

1500

68 ± 6.2

9 ± 3.5

22 ± 3.1

22 ± 4.0

8 ± 1.0

+

5000

68 ± 3.2P

8 ± 3.0P

20 ± 0.6P

12 ± 1.2P

6 ± 2.1P

Positive controls, +S9

Name

2-AA

2-AA

2-AA

B[a]P

2-AA

Concentration

[μg/plate]

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

2506 ± 248.1

179 ± 29.1

184 ± 36.9

197 ± 11.7

247 ± 30.5

DMSO: dimethylsulphoxide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

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

4-NQO: 4-nitroquinoline-N-oxide

B[a]P: benzo(a)pyrene

P: precipitate

Table 2. Test results of experiment 2 (preincubation).

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

TA100

TA1535

WP2 uvrA

TA98

TA1537

0

118

15

29

20

13

0 (DMSO)

102 ± 13.1

21 ± 2.0

26 ± 5.9

22 ± 1.2

15 ± 1.5

5

n.d.

22 ± 1.5

n.d.

n.d.

n.d.

15

n.d.

23 ± 1.5

n.d.

n.d.

n.d.

50

89 ± 9.2

25 ± 2.1

24 ± 8.5

23 ± 5.2

12 ± 2.9

150

88 ± 10.3

24 ± 2.6

29 ± 3.6

26 ± 0.6

11 ± 3.5

500

93 ± 12.3

21 ± 2.1#

22 ± 1.7

21 ± 1.0

10 ± 5.0

1500

84 ± 8.1

19 ± 4.6#

27 ± 3.6

26 ± 1.2

12 ± 1.5

5000

81 ± 3.8P,#

26 ± 5.8P,#

24 ± 2.3P

20 ± 2.5P

11 ± 2.6P

Positive controls, –S9

Name

ENNG

ENNG

ENNG

4-NQO

9-AA

Concentration

[μg/plate]

3

5

2

0.2

80

Mean No. of colonies/plate

(average of 3 ± SD)

309 ± 12.8

432 ± 50.8

462 ± 40.3

228 ± 15.9

1426 ± 535.2

+

0 (DMSO)

107 ± 14.0

13 ± 2.6

24 ± 4.9

19 ± 1.0

12 ± 1.5

+

5

n.d.

15 ± 1.5

n.d.

21 ± 3.0

9 ± 1.7

+

15

n.d.

11 ± 0.6

n.d.

19 ± 1.0

13 ± 3.5

+

50

99 ± 15.9

12 ± 2.3

29 ± 5.8

19 ± 3.1

10 ± 1.0

 

150

109 ± 13.8

12 ± 2.1

23 ± 1.0

19 ± 1.7

11 ± 4.6

+

500

85 ± 10.1

11 ± 1.0

23 ± 0.6

18 ± 1.0

8 ± 0.6

+

1500

69 ± 3.2P

6 ± 2.1#

21 ± 1.0

17 ± 1.0

8 ± 0.6

+

5000

47 ± 15.3P

0 ± 0.0P,#

13 ± 2.1P

7 ± 3.5P

3 ± 3.5P

Positive controls, +S9

Name

2-AA

2-AA

2-AA

B[a]P

2-AA

Concentration

[μg/plate]

1

2

10

5

2

Mean No. of colonies/plate

(average of 3 ± SD)

118

15

29

20

13

DMSO: dimethylsulphoxide

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

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

4-NQO: 4-nitroquinoline-N-oxide

B[a]P: benzo(a)pyrene

n.d.: not determined

P: precipitate

#: partial absence of bacterial background lawn

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA1535, TA1537, TA98, TA100 and WP2 uvrA) tested with and without metabolic activation.