Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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:
The test material corresponded to the approximately 45% of the registration substance. The given dose refers to the amount of the registration substance.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver S9 homogenate
Test concentrations with justification for top dose:
Preliminary Toxicity Test - (a) 50, (b) 100, (c) 200, (d) 400, (e) 800, (f) 1600, (g) 3200 and (h) 5000 µg/plate
Initial mutation assay - (a) 8, (b) 25, (c) 80, (d) 253 and (e) 800 µg/plate
Confirmatory mutation assay - (a) 41, (b) 86, (c) 181, (d) 380 and (e) 800 µg/plate.
Vehicle / solvent:
One hundred microliters (100 µL) of DMSO was used as the vehicle control.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-Aminoanthracene
Details on test system and experimental conditions:
- Source of the Test System:
-Salmonella typhimurium: Health Protection Agency, National Collection of Type, Cultures (NCTC), 61, Colindale Avenue, London NW9 5EQ, Great Britain
- Escherichia coli: The National Collection of Industrial and Marine Bacteria Ltd. (NCIMB), Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA, Scotland, U.K.

- Storage of Test System
Stock cultures of tester strains were stored in Oxoid nutrient broth No. 2 in the test facility as frozen permanents in liquid nitrogen. Laboratory stocks were maintained on respective minimal glucose agar plates as master plates of each strain, for a maximum period of 2 months and refrigerated at 2 to 8ºC. The master plates prepared on 14 March 2016 were used in the study.

- Genotypic Characterization of Test System
The growth requirements and the genetic identity of strains like histidine or tryptophan requirement, sensitivity to UV radiation, resistance of strains
TA98, TA100 and WP2uvrA (pKM101) to ampicillin and rfa mutation of Salmonella typhimurium strains were checked along with the range of spontaneous revertants after preparation of the master plates
Evaluation criteria:
- Evaluation and Interpretation : To determine a positive result, there should be a dose related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of the test item either in the presence or absence of the metabolic activation system.

The test will be judged positive, if the increase in mean revertants at the peak of the dose response is equal to or greater than 2 times the mean vehicle control value for strains TA98, TA100 and WP2uvrA (pKM101) or equal to or greater than 3 times the mean vehicle control value for strains TA1535 and TA1537.

An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Results of Preliminary Toxicity Test

Treatment

(mg/plate)

TA 100 revertant colonies/plate*

Presence of S9

Absence of S9

Mean

Background lawn

Precipitation

Mean

Background lawn

Precipitation

 

DMSO (100mL)

114

4+

Nil

112

4+

Nil

 

50

108

4+

Nil

107

4+

Nil

 

100

103

4+

Nil

101

4+

Nil

 

200

72

4+

Nil

70

4+

Nil

 

400

56

3+

Nil

51

3+

Nil

 

800

46

3+

Nil

43

3+

Nil

 

1600

37

3+

Nil

33

3+

Nil

 

3200

9

1+

Nil

7

1+

Nil

 

5000

4

0

Nil

3

0

Nil

*: Mean of two replicates

4+ - Normal

3+ - Slightly reduced   

1+ - Severely reduced

0  - Absent

Table 2: Viable Counts of the Overnight Culture of the Tester Strains

Tester Strains

Viable Counts ( x 109CFU/mL*)

Initial Mutation Assay

Confirmatory Mutation Assay

TA98

1.55

1.55

TA100

1.64

1.54

TA1535

1.53

1.52

TA1537

1.55

1.52

WP2uvrA (pKM101)

1.62

1.63

* Required Cell count: 1-2x109Colony Forming Units (CFU)/mL

Table 3: Summary Results of Initial Mutation Assay in the Presence of Metabolic Activation

Treatment

(µg/plate)

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

 

Vehicle control

DMSO

26

2

NA

116

1

NA

14

2

NA

11

2

NA

134

3

NA

8

26

2

1.00

114

1

0.98

13

2

0.93

10

2

0.91

130

3

0.97

25

28

1

1.08

112

1

0.97

11

2

0.79

11

2

1.00

127

3

0.95

80

26

2

1.00

112

2

0.97

14

1

1.00

12

1

1.09

129

2

0.96

253

21

2

0.81

71

2

0.61

9

2

0.64

7

1

0.64

105

3

0.78

800

14

1

0.54

44

4

0.38

2

2

0.14

3

2

0.27

93

2

0.69

Positive controls

545c

6c

20.96c

882c

11c

7.60c

139c

9c

9.93c

126c

1c

11.45c

572d

4d

4.27d

aValues are means of three replicates calculated from Appendix 2 and are rounded off to the nearest whole number

bRatio of treated/vehicle control (mean revertants per plate)

cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate)                         

dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate)

NA: Not applicable                             SD: Standard deviation                       

Table 4: Summary Results of Initial Mutation Assay in the Absence of Metabolic Activation

Treatment

(µg/plate)

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Vehicle control

DMSO

27

1

NA

115

2

NA

13

3

NA

12

2

NA

132

3

NA

8

27

2

1.00

112

2

0.97

13

2

1.00

12

1

1.00

129

4

0.98

25

27

2

1.00

112

3

0.97

14

3

1.08

11

2

0.92

129

3

0.98

80

27

1

1.00

112

1

0.97

14

2

1.08

10

2

0.83

128

2

0.97

253

21

1

0.78

75

3

0.65

9

1

0.69

8

1

0.67

109

2

0.83

800

15

1

0.56

40

1

0.35

2

1

0.15

3

2

0.25

91

2

0.69

Positive controls

241c

5c

8.93c

530d

6d

4.61d

143d

5d

11.00d

128e

2e

10.67e

572f

7f

4.33f

aValues are means of three replicates calculated from Appendix 3 and are rounded off to the nearest whole number 

bRatio of treated/vehicle control (mean revertants per plate)

cTA98: 2-Nitrofluorene (2 µg/plate),             dTA100, TA1535: Sodium azide (1 µg/plate),           

eTA1537: 9-Aminoacridine (50 µg/plate),      fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)            

NA: Not applicable                                         SD: Standard deviation                                   

Table 5: Summary Results of Confirmatory Mutation Assayin the Presence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA (pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Vehicle control

DMSO

25

2

NA

114

2

NA

14

2

NA

11

2

NA

133

4

NA

41

25

2

1.00

112

1

0.98

14

2

1.00

11

2

1.00

135

5

1.02

86

26

1

1.04

111

1

0.97

15

1

1.07

12

1

1.09

131

5

0.98

181

27

1

1.08

96

2

0.84

14

2

1.00

10

1

0.91

130

3

0.98

380

23

3

0.92

63

2

0.55

8

1

0.57

8

2

0.73

102

4

0.77

800

15

2

0.60

44

3

0.39

3

1

0.21

3

1

0.27

91

2

0.68

Positive control

557c

17c

22.28c

876c

10c

7.68c

134c

6c

9.57c

129c

4c

11.73c

580d

6d

4.36d

aValues are means of three replicates calculated from Appendix 4 and are rounded off to the nearest whole number         

bRatio of treated/vehicle control (mean revertants per plate)

cTA98, TA100, TA1535, TA1537: 2-Aminoanthracene (4 µg/plate) dWP2uvrA(pKM101): 2-Aminoanthracene (30 µg/plate)

NA: Not applicable                                                                             SD: Standard deviation                                                           

Table 6: Summary Results of Confirmatory Mutation Assay in the Absence of Metabolic Activation

Treatment

[µg/plate]

No. of revertants/platea

TA98

TA100

TA1535

TA1537

WP2uvrA

(pKM101)

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Mean

SD

Ratiob

Vehicle control

DMSO

25

1

NA

114

2

NA

15

1

NA

12

1

NA

130

4

NA

41

26

1

1.04

111

2

0.97

15

2

1.00

11

2

0.92

131

5

1.01

86

25

1

1.00

111

1

0.97

14

2

0.93

12

1

1.00

128

3

0.98

181

26

1

1.04

96

3

0.84

15

1

1.00

10

1

0.83

127

2

0.98

380

21

1

0.84

64

3

0.56

9

2

0.60

6

1

0.50

99

3

0.76

800

16

3

0.64

43

2

0.38

2

2

0.13

3

1

0.25

90

2

0.69

Positive control

246c

6c

9.84c

537d

3d

4.71d

133d

6d

8.87d

133e

4e

11.08e

584f

5f

4.49f

aValues are means of three replicates calculated from Appendix 5 and are rounded off to the nearest whole number

bRatio of treated/vehicle control (mean revertants per plate)

cTA98: 2-Nitrofluorene (2 µg/plate),             dTA100, TA1535: Sodium azide (1 µg/plate)            

eTA1537: 9-Aminoacridine (50 µg/plate),      fWP2uvrA(pKM101): 4-Nitroquinoline-1-oxide (4 µg/plate)            

NA: Not applicable                                         SD: Standard deviation                                   

Applicant's summary and conclusion

Conclusions:
The genotoxicity of the registration substance was investigated in Bacterial Reverse Muation Test (Ames test) according to the Guideline OECD 471. No mutagenicity was found.
Executive summary:

The genotoxicity of the registration substance was investigated in Bacterial Reverse Muation Test (Ames test) according to the Guideline OECD 471. In the preliminary cytotoxicity test, the S.typhimurium TA 100 cultures were treated up to 5000 µg/plate with and without metabolic activation. Significantly reduced background lawn was observed at 400 µg/plate or higher. Two independent experiments for mutagenicity were performend, each with and without metabolic activation up to the concentration of 800µg/plate. No increase of number of reverstants were found in any of performed experiments.