Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 Jul - 05 Aug 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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:
Commission Directive No. 2000/32/EC
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Aug 1998
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)
Test concentrations with justification for top dose:
Pre-experiment: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without mtabolic activation for TA 100 and WP2 uvr A

Main Experiment 1:
50, 150, 500, 1500 and 5000 µg/plate with metabolic activation for WP2 uvr A
5, 15, 50, 150, 500, 1500 and 500 µg/plate with and without metabolic activation for remaining strains and without metabolic activation for WP2 uvr A

Main Experiment 2:
50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation for WP2 uvr A
5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation for remaining strains

Vehicle / solvent:
- Vehicle/solvent used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was fully soluble in dimethyl sulphoxide at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was not evaluated as a potential vehicle in this test system as information supplied by the sponsor suggested that the test material is insoluble in water.
Controls
Untreated negative controls:
yes
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 (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: 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, as recommended by UKEMS (Kirkland ( 1989) Sub-committee on Guidelines for mutagenicity Testing, Report-Part III, Cambridge University Press) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response. A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Acceptance criteria:
The reverse mutation assay may be considered valid if the following criteria are met:
Spontaneous revertants of tester strain cultures for vehicle and negative control should be in the range of historical control data. The appropriate characteristics for each tester strain have been confirmed (eg rfs cell-wall-mutation, pKM101 plasmid R-factor etc.). All tester strain cultures should be in the approximate range of 1 to 9.9 x 10E09 bacteria per mL. Each mean positive control value should be at least two times the respective vehicle control for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and integrity of the S9-mix. There should be a minimum of four non-toxic test material dose levels. There should be no evidence of excessive contamination.
Statistics:
Mean value and standard deviations were calculated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate (+/- S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 5000 µg/plate (+/- S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate (+/- S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at and above 1500 µg/plate (+/- S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate (- S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the dose levels tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: The test material was toxic at and above 1500 μg/plate to TA100 (with and without S9) and WP2uvrA- (without S9) and non-toxic to WP2uvrA- with S9. The test material formulation and S9-mix used in this experiment were both shown to be sterile.

HISTORICAL CONTROL DATA: Positive and vehicle controls were within the normal range of historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test material caused a visible reduction in the growth of the bacterial background lawns of the majority of tester strains, initially from 1500 μg/plate in both the presence and absence of S9. No toxicity was exhibited to Escherichia coli strain WP2 uvr A, in the presence of S9 only, at any test substance dose level. The sensitivity of the tester strains to the toxicity of the test substance varied slightly between strain type, exposure with or without S9 and experiment number. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

Any other information on results incl. tables

Table 1. Results of Experiment 1

 

Number of revertant colonies (mean of 3 plates ± SD)

S9-Mix

Without

Test substance (µg/plate)

TA 100

TA 1535

WP2 uvr A

TA 98

TA1537

SC

128 ± 4.6

26 ± 2.6

30 ± 3.8

23 ± 5.5

10 ± 4.9

5

128 ± 7.2

27 ± 2.1

19 ± 7.4

23 ± 8.3

10 ± 6.0

15

130 ± 6.5

25 ± 3.0

22 ± 3.8

20 ± 4.0

6 ± 1.2

50

127 ± 8.6

19 ± 2.6

25 ± 3.2

22 ± 4.4

12 ± 1.5

150

135 ± 12.5

23 ± 6.8

22 ± 7.5

17 ± 5.5

13 ± 2.1

500

126 ± 10.4

28 ± 9.0

25 ± 8.0

13 ± 1.7

13 ± 2.0

1500

75 ± 0.6 s

28 ± 4.2 s

23 ± 4.5

13 ± 3.6 s

7 ± 2.5

5000

0 ± 0.0 v

0 ± 0.0 v

16 ± 2.3 s

0 ± 0.0 v

0 ± 0.0 v

Positive Control

ENNG

ENNG

ENNG

4NQO

9AA

Dose (µg/plate)

3

5

2

0.2

80

Number of revertant colonies/plate

443 ± 67.0

168 ± 32.1

540 ± 27.3

101 ± 4.5

659 ± 69.6

S9-Mix

With

Test substance (µg/plate)

TA 100

TA 1535

WP2 uvr A

TA 98

TA1537

SC

128 ± 4.4

13 ± 2.1

29 ± 6.1

22 ± 1.5

15 ± 2.0

5

107 ± 9.3

13 ± 1.5

N/T

28 ± 7.8

12 ± 2.5

15

102 ± 6.8

13 ± 0.0

N/T

26 ± 4.9

13 ± 3.2

50

108 ± 8.4

11 ± 2.1

28 ± 2.0

21 ± 1.5

10 ± 2.3

150

111 ± 10.2

12 ± 3.1

23 ± 3.0

22 ± 0.6

13 ± 7.8

500

98 ± 14.4

14 ± 1.0

31 ± 9.8

22 ± 7.0

10 ± 3.6

1500

68 ± 14.5 s

9 ± 3.5 s

27 ± 4.0

16 ± 0.6 s

8 ± 1.2

5000

0 ± 0.0 v

5 ± 1.2 s

25 ± 0.6

14 ± 5.1 s

7 ± 0.6 s

Positive Control

2AA

2AA

2AA

BP

2AA

Dose (µg/plate)

1

2

10

5

2

Number of revertant colonies/plate

458 ± 154.0

166 ± 25.1

168 ± 7.0

262 ± 17.0

354 ± 48.8

SC: Solvent Control (dimethyl sulphoxide)

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

N/T: Not tested at this dose level

s: sparse bacterial background lawn

v: very weak bacterial background lawn

Table 2: Results of Experiment 2

 

Number of revertant colonies (mean of 3 plates ± SD)

S9-Mix

Without

Test substance (µg/plate)

TA 100

TA 1535

WP2 uvr A

TA 98

TA1537

SC

115 ± 18.7

18 ± 2.5

26 ± 3.2

21 ± 1.0

8 ± 0.6

5

123 ± 9.2

19 ± 7.0

N/T

19 ± 3.5

10 ± 2.9

15

118 ± 10.1

20 ± 1.2

N/T

15 ± 3.5

15 ± 6.7

50

122 ± 11.2

19 ± 5.0

27 ± 3.0

17 ± 8.5

10 ± 6.0

150

117 ± 7.0

24 ± 1.7

21 ± 1.5

15 ± 4.6

13 ± 5.7

500

116 ± 14.8

19 ± 3.8

28 ± 4.7

20 ± 7.8

10 ± 1.0

1500

100 ± 4.9 s

25 ± 2.5 s

25 ± 5.7

14 ± 4.2 s

7 ± 4.0

5000

77 ± 13.3 s

17 ± 3.6 v

23 ± 2.5 s

6 ± 1.5 v

8 ± 3.6 s

Positive Control

ENNG

ENNG

ENNG

4NQO

9AA

Dose (µg/plate)

3

5

2

0.2

80

Number of revertant colonies/plate

302 ± 14.2

179 ± 6.0

400 ± 14.7

100 ± 15.0

219 ± 31.7

S9-Mix

With

Test substance (µg/plate)

TA 100

TA 1535

WP2 uvr A

TA 98

TA1537

SC

101 ± 2.5

10 ± 2.1

29 ± 5.8

19 ± 5.9

11 ± 2.0

5

115 ± 20.6

10 ± 2.0

N/T

23 ± 6.1

14 ± 0.6

15

98 ± 12.3

8 ± 1.0

N/T

21 ± 3.1

10 ± 2.5

50

88 ± 8.0

8 ± 3.5

28 ± 8.1

22 ± 7.1

15 ± 3.1

150

98 ± 15.0

9 ± 2.1

31 ± 7.5

17 ± 3.8

16 ± 5.5

500

96 ± 6.4

11 ± 3.1

24 ± 8.0

20 ± 6.0

11 ± 2.1

1500

83 ± 4.4

8 ± 2.5

31 ± 5.9

17 ± 5.1

10 ± 1.5

5000

62 ± 8.0 s

7 ± 3.1

23 ± 2.3

13 ± 3.2 s

13 ± 6.2

Positive Control

2AA

2AA

2AA

BP

2AA

Dose (µg/plate)

1

2

10

5

2

Number of revertant colonies/plate

525 ± 118.2

220 ± 24.5

340 ± 37.0

223 ± 16.4

218 ± 27.4

SC: Solvent Control (dimethyl sulphoxide)

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

N/T: Not tested at this dose level

s: sparse bacterial background lawn

v: very weak bacterial background lawn

Applicant's summary and conclusion

Conclusions:
Under the conditions of the Ames test the test substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvr A) tested with and without metabolic activation up to the limit dose of 5000 µg/plate.