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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 17 March 2020 to 16 April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The test was conducted in accordance with the relevant OECD test guideline and in accordance with GLP. All validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
liquid
Details on test material:
Storage conditions: At room temperature protected from light
Physical description: Light orange liquid

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9:
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and
2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively
- method of preparation of S9 mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per
10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg
glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.
Test concentrations with justification for top dose:
In the first mutation experiment, the test item was tested up to concentrations of
1600 μg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
In the second mutation experiment, the test item was tested up to concentrations of
1600 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to 5000 μg/plate in the tester strain WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (Merck, Darmstadt, Germany)
Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands), used for TA1535 without metabolic activation
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 4-nitroquinoline N-oxide; 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E9 cells/mL
- Test substance added in medium; in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 ± 2 minutes (pre-incubation assays only)
- Exposure duration/duration of treatment: 48 ± 4 h
- Harvest time after the end of treatment (sampling/recovery times): Not specified


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- background growth inhibition; decreased in number of revertants and/or presence of microcolonies
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- dose-related increase in the number of revertant colonies compared to the solvent control

Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Dose-Range Finding Test:  Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Direct Plate Assay


Dose

(µg/plate)


Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.

 


TA100


WP2uvrA

 



 

Without S9-mix

Positive control

773

±

176

 

1418

±

112

 

 

 

 

 

Solvent control

120

±

10

 

13

±

1

 

 

 

 

 

1.7

112

±

14

 

17

±

5

 

 

 

 

 

5.4

118

±

25

 

23

±

8

 

 

 

 

 

17

123

±

14

 

20

±

7

 

 

 

 

 

52

123

±

23

 

19

±

5

 

 

 

 

 

164

115

±

25

 

26

±

4

 

 

 

 

 

512

113

±

8

n

20

±

3

 

 

 

 

 

1600

 

 

 

e MC

18

±

4

n

 

 

 

 

5000

0

±

0

a NP

 

 

 

e NP  MC

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

With S9-mix

Positive control

1104

±

304

 

297

±

84

 

 

 

 

 

Solvent control

88

±

8

 

22

±

7

 

 

 

 

 

1.7

121

±

30

 

22

±

6

 

 

 

 

 

5.4

119

±

18

 

20

±

2

 

 

 

 

 

17

110

±

30

 

20

±

9

 

 

 

 

 

52

117

±

6

 

20

±

2

 

 

 

 

 

164

93

±

26

 

26

±

4

 

 

 

 

 

512

118

±

34

n

27

±

6

 

 

 

 

 

1600

 

 

 

e MC

39

±

6

n

 

 

 

 

5000

0

±

0

a NP

25

±

5

s NP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MC

Microcolonies

NP

No precipitate

a

Bacterial background lawn absent

e

Bacterial background lawn extremely reduced

n

Normal bacterial background lawn

s

Bacterial background lawn slightly reduced

 

Table 2          
Experiment 1:  Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay

Direct Plate Assay


Dose

(µg/plate)


Mean number of revertant colonies/3 replicate plates (± S.D.) with
different strains of Salmonella typhimurium.

 


TA1535


TA1537

 


TA98

 

Without S9-mix

Positive control

1014

±

53

 

1120

±

9

 

1292

±

136

 

Solvent control

9

±

3

 

6

±

5

 

10

±

1

 

5.4

11

±

8

 

2

±

2

 

13

±

5

 

17

8

±

2

 

6

±

4

 

9

±

4

 

52

9

±

5

 

4

±

4

 

8

±

2

 

164

10

±

2

 

9

±

7

 

13

±

3

 

512

11

±

9

n

6

±

4

n

23

±

5

n

1600

5

±

2

m NP

4

±

1

m NP

 

 

 

e NP  MC

 

 

 

 

 

 

 

 

 

 

 

 

 

With S9-mix

Positive control

354

±

24

 

311

±

23

 

1656

±

337

 

Solvent control

15

±

11

 

5

±

3

 

16

±

3

 

5.4

11

±

4

 

6

±

6

 

21

±

3

 

17

15

±

8

 

7

±

3

 

16

±

2

 

52

14

±

4

 

6

±

4

 

17

±

5

 

164

9

±

4

 

7

±

5

 

18

±

3

 

512

16

±

6

n

7

±

0

n

26

±

7

n

1600

7

±

4

m NP

 

 

 

e NP  MC

27

±

13

s NP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MC

Microcolonies

NP

No precipitate

e

Bacterial background lawn extremely reduced

m

Bacterial background lawn moderately reduced

n

Normal bacterial background lawn

s

Bacterial background lawn slightly reduced



Table 3          
Experiment 2:  Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Pre-incubation Assay


Dose

(µg/plate)


Mean number of revertant colonies/3 replicate plates (± S.D.) with
different strains of Salmonella typhimurium and one Escherichia coli strain.

 


TA1535


TA1537

 


TA98


TA100


WP2uvrA

Without S9-mix

Positive control

907

±

31

 

136

±

13

 

1879

±

59

 

558

±

68

 

1818

±

34

 

Solvent control

4

±

0

 

7

±

1

 

12

±

2

 

92

±

9

 

15

±

4

 

5.4

8

±

0

 

7

±

2

 

5

±

4

 

83

±

9

 

 

-

 

 

17

7

±

6

 

7

±

4

 

6

±

3

 

103

±

13

 

16

±

7

 

52

8

±

5

 

10

±

3

 

20

±

6

 

192

±

11

 

15

±

4

 

164

12

±

5

n

5

±

0

n

40

±

6

n

274

±

39

n

23

±

4

 

512

15

±

4

s

5

±

2

s

16

±

3

s

83

±

30

s

46

±

8

n

1600

 

 

 

a NP

 

 

 

e NP  MC

 

 

 

a NP

 

 

 

a NP

9

±

6

s

5000

 

-

 

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

 

a NP

With S9-mix

Positive control

154

±

14

 

61

±

15

 

403

±

20

 

1088

±

28

 

536

±

16

 

Solvent control

7

±

2

 

4

±

3

 

13

±

6

 

56

±

4

 

18

±

6

 

5.4

11

±

6

 

6

±

2

 

19

±

4

 

83

±

20

 

 

-

 

 

17

7

±

4

 

9

±

2

 

18

±

10

 

78

±

8

 

18

±

2

 

52

8

±

4

 

7

±

3

 

30

±

10

 

100

±

8

 

23

±

8

 

164

13

±

7

n

4

±

3

n

58

±

4

 

184

±

60

n

31

±

4

 

512

13

±

2

s

3

±

1

s

46

±

15

n

96

±

6

s

52

±

4

n

1600

 

 

 

e NP  MC

 

 

 

e NP  MC

 

 

 

e NP  MC

 

 

 

a NP

7

±

4

s

5000

 

-

 

 

 

-

 

 

 

-

 

 

 

-

 

 

 

 

 

a NP

 

 

MC

Microcolonies

NP

No precipitate

a

Bacterial background lawn absent

e

Bacterial background lawn extremely reduced

n

Normal bacterial background lawn

s

Bacterial background lawn slightly reduced

-

Not tested

 

 

 

 

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The objective of this study was to determine the potential of the test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonellatyphimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay.  The test item did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of
1600 µg/plate in the strains TA1535, TA1537 and TA98.  The test item did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 

In the second mutation experiment, the test item was tested up to concentrations of
1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to 5000 µg/plate in the tester strain WP2uvrA in the pre-incubation assay.  The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate, and that the metabolic activation system functioned properly.

In the second experiment (pre-incubation method), the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in tester strains TA98, TA100 and WP2uvrA

In tester strain TA98, the increases observed where up to 3.3- fold and 4.5- fold the concurrent control, in the absence and presence of S9-mix, respectively.

In tester strain TA100, the increases observed where up to 3.0- fold and 3.3- fold the concurrent control, in the absence and presence of S9-mix, respectively.

In tester strain WP2uvrA, the increases observed where up to 3.1- fold and 2.9- fold the concurrent control, in the absence and presence of S9-mix, respectively.

Although the increases in tester strains TA98 and WP2uvrA were within the laboratory historical control data range, the responses were more than two- and three-fold the concurrent controls in strains WP2uvrA and TA98, respectively. In addition, the increases in tester strain TA100 were outside the historical control data range and more than two-fold the concurrent controls. Based on these observations, the increases were considered biologically relevant. 

In conclusion, based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

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