Registration Dossier

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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected March 2013; signature: May 2013
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Liquid.
- Storage condition of test material: At room temperature protected from light.
- Other: Colourless

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Dose range finding study: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 ug/plate (Strains: TA100 and WP2uvrA)
Experiment 1: 0.17, 0.54, 1.7, 5.4, 17, 52 ug/plate (Strains: TA1535, TA1537, TA98 and TA100)
Experiment 2: 0.17, 0.54, 1.7, 5.4, 17, 52 and 164 ug/plate (Strains: TA1535, TA1537, TA98 and TA100) and; 5.4, 17, 52, 164, 512, 1600 and 5000 ug/plate (Strains: WP2uvrA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: A solubility test was performed. The test substance could not be dissolved in water. The test substance was soluble in DMSO.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted. The revertant colonies were counted automatically with a Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually and evidence of test article precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn) and reduction in the number of revertants

OTHER:
Dose range finding test on TA100 and WP2urvA with and without 5% (v/v) S9-mix; First mutation assay on TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix. To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed on all strains, in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 164 μg/plate in strains TA1535, TA1537, TA98 and TA100. Whereas WP2uvrA was tested up to 5000 μg/plate.
Evaluation criteria:
See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.
Statistics:
No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
In tester strain TA100, fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the mid doses of 492 µg/plate and 878 µg/plate. This reduction was not considered to be caused by toxicity.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Dose range finding test: Precipitation of the test substance on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 µg/plate. No precipitate was observed at the end of the incubation period. Mutation experiments 1 and 2: Precipitation of the test substance on the plates was not observed at the start or at the end of the incubation.

RANGE-FINDING/SCREENING STUDIES:
Test substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

COMPARISON WITH HISTORICAL CONTROL DATA:
Historical control data from experiments was presented within the study report.
1. The negative control values were within the laboratory historical control data ranges, except for the response of TA100 in the absence of S9-mix (dose range finding) and TA1537 in the presence of S9-mix (second experiment). The value of 64 in tester strain TA100 was just below the limit of the range (69) and the value of 28 in tester strain TA1537 was above the limit of the range (21) of the historic control range. The mean plate counts were just outside the limit of the ranges, therefore this deviation in the mean plate count of the solvent control had no effect on the results of the study.
2. The positive control substance of tester strain TA100 (second experiment, absence of S9-mix) showed a response (mean plate count) which was not within the laboratory historical range. The value (1436) was above the limit of the range (1394). The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.
The integrity of the study was not considered to be impacted by the above minor deviations from historic control values.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose

(µg/plate)

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

 

TA100

TA100

Without S9-mix

 

 

 

 

 

 

 

 

Positive control

750

±

15

 

1177

±

89

 

Solvent control

64

±

5

 

28

±

4

 

1.7

80

±

9

 

28

±

5

 

5.4

75

±

13

n

27

±

4

 

17

44

±

2

m

27

±

3

 

52

 

 

 

e MC

25

±

5

 

164

 

 

 

e MC

15

±

4

n

512

 

 

 

a

14

±

6

s

1600

 

 

 

a NP

9

±

3

m

5000

 

 

 

a NP

3

±

2

m NP

With S9-mix1

 

 

 

 

 

 

 

 

Positive control

1161

±

144

 

244

±

20

 

Solvent control

83

±

10

 

37

±

9

 

1.7

78

±

10

 

33

±

8

 

5.4

71

±

13

 

28

±

10

 

17

88

±

12

n

34

±

1

 

52

67

±

9

s

25

±

4

 

164

 

 

 

e MC

30

±

7

 

512

 

 

 

a

11

±

4

 

1600

 

 

 

a

9

±

2

n

5000

 

 

 

a NP

2

±

1

s NP

1: Plate incorporation assay (5% S9)

MC: Microcolonies

NP: No precipitate

a: Bacterial background lawn absent

e: Bacterial background lawn extremely reduced

m: Bacterial background lawn moderately reduced

n: Normal bacterial background lawn

s: Bacterial background lawn slightly reduced

 

Table 2 Experiment 1: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay

Dose

(µg/plate)

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

 

TA1535

TA1537

TA98

TA100

Without S9-mix

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive control

889

±

53

 

4

±

4

 

1162

±

60

 

968

±

26

 

Solvent control

18

±

2

 

5

±

6

 

19

±

4

 

93

±

20

 

0.17

25

±

11

 

5

±

3

 

19

±

1

 

93

±

4

 

0.54

17

±

5

 

8

±

3

 

13

±

4

 

96

±

5

 

1.7

18

±

3

 

9

±

8

 

18

±

5

 

94

±

1

 

5.4

17

±

6

n

7

±

3

n

17

±

6

n

95

±

3

n

17

9

±

1

m

5

±

1

m

15

±

1

s

94

±

10

m

52

 

 

 

e NP MC

 

 

 

a NP

 

 

 

e NP MC

-

 

 

a NP

With S9-mix1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Positive control

257

±

14

 

486

±

78

 

1089

±

66

 

1752

±

370

 

Solvent control

13

±

7

 

9

±

2

 

23

±

1

 

93

±

5

 

0.54

12

±

5

 

11

±

5

 

32

±

4

 

120

±

8

 

1.7

16

±

5

 

12

±

2

 

28

±

6

 

92

±

9

 

5.4

14

±

6

 

10

±

4

 

32

±

9

 

89

±

8

 

17

13

±

3

n

13

±

12

n

26

±

7

 

97

±

12

n

52

6

±

2

m

 

 

 

e

20

±

4

n

73

±

18

m

164

 

 

 

a NP

 

 

 

a NP

 

 

 

e NP MC

-

 

 

a NP

1: Plate incorporation assay (5% S9)

MC: Microcolonies

NP: No precipitate

a: Bacterial background lawn absent

e: Bacterial background lawn extremely reduced

m: Bacterial background lawn moderately reduced

n: Normal bacterial background lawn

s: Bacterial background lawn slightly reduced

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative
Under the conditions of this study the test substance is not considered to be mutagenic. Negative and strain specific positive control values were in general within laboratory historical control data ranges indicating that the test conditions were adequate.
Executive summary:

The study was performed to OECD TG 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP; to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat). In the dose range finding test, the test substance was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 17 μg/plate and above in the absence of S9-mix and at 52 μg/plate and above in the presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at dose levels of 512 μg/plate and above in the absence and presence of S9-mix. Results of this dose range finding test were reported as part of the first mutation assay. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 0.17 to 52 µg/plate in the absence of S9-mix and at 0.54 to 164 µg/plate in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. Toxicity was observed in all four tester strains in the absence and presence of S9-mix. In a follow-up experiment of the assay with additional parameters, the test substance was tested at a concentration range of 0.17 to 52 µg/plate in the absence of S9-mix and at a concentration range of 0.54 to 164 µg/plate in the presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and TA100 and at 5.4 to 5000 µg/plate in tester strain WP2uvrA in the absence and presence of 10% (v/v) S9-mix. Toxicity was observed in all tester strains in the absence and presence of S9-mix. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. The negative control values were within the laboratory historical control data ranges, except for TA100 in the absence of S9-mix (dose range finding) and TA1537 in the presence of S9-mix (second experiment). However, the validity of the test was considered to be not affected. The strain-specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the conditions of this study it is concluded that that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.