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:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP; minor deviation not affecting the reliability of the study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Minor deviation: first mutation assay: strain TA98, no bacteria were plated at the mid dose 164 μg/plate. However, since at least a total of five dose levels were used at up to the recommended dose level 5000μg/plate, study reliability is unaffected.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Minor deviation; see above
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
Minor deviation; see above
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:
yes
Remarks:
Minor deviation; see above
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
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Physical state: liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Other: Clear 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
Experiment 1: 17, 52, 164, 512, 1600, 5000 ug/plate
Experiment 2: 492, 878, 1568, 2800, 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance formed an emulsion in water and ethanol at a concentration of 50 mg/ml. The test substance was fully soluble in dimethyl sulfoxide (DMSO) at a concentration of 50 mg/ml.
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.

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 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Based on the results of the earlier assays.
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:
cytotoxicity
Remarks:
Cytotoxicity was only observed at the top dose of 5000 μg/plate in tester strain TA1535 and TA100 (absence and presence of S9-mix) and TA1537 and TA98 (absence of S9-mix) in experiment 2.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

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: 17, 52, 164, 512, 1600 and 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 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   

 

WP2uvrA

 

 

Without S9-mix

 

 

 

 

Positive control

1057

± 86

1257

± 41

Solvent control

139

± 26

29

± 5

1.7

146

± 15

35

± 3

5.4

128

± 12

37

± 8

17

130

± 15

35

± 6

52

134

± 6

38

± 3

164

129

± 5

34

± 4

512

126

± 6

36

± 6

1600

121

± 17 n

36

± 3 n

5000

95

± 9 s NP

38

± 15 s NP

With S9-mix #1

 

 

 

 

Positive control

1420

± 45

275

± 33

Solvent control

132

± 19

38

± 7

1.7

121

± 8

44

± 5

5.4

128

± 9

45

± 6

17

126

± 15

48

± 8

52

123

± 4

44

± 5

164

149

± 38

48

± 4

512

122

± 7

42

± 6

1600

112

± 15 n

37

± 8 n

5000

83

± 12 s NP

28

± 5 s NP

#1          Plate incorporation assay (5% S9)

NP         No precipitate

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

 

Without S9-mix

 

 

 

 

 

 

Positive control

562

± 37

541

± 36

924

± 114

Solvent control

25

± 7

9

± 5

17

± 2

17

29

± 0

9

± 5

19

± 1

52

26

± 5

5

± 2

15

± 5

164

30

± 3

10

± 2

#2

 

512

30

± 8

9

± 2

16

± 6

1600

27

± 5

4

± 1

19

± 0

5000

21

± 1 n NP

9

± 4 n NP

14

± 1 n NP

With S9-mix #1

 

 

 

 

 

 

Positive control

251

± 27

259

± 41

715

± 48

Solvent control

16

± 3

6

± 1

23

± 1

17

18

± 6

8

± 3

24

± 2

52

18

± 7

13

± 5

20

± 6

164

18

± 4

13

± 10

#2

 

512

18

± 4

8

± 2

26

± 6

1600

15

± 4

7

± 4

26

± 2

5000

11

± 1 n NP

8

± 1 n NP

19

± 1 n NP

#1          Plate incorporation assay (5% S9)

#2          No bacteria plated

NP         No precipitate

n            Normal bacterial background lawn

 

Table 3 Experiment 2: 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 different strains of Salmonella typhimurium and one Escherichia coli strain

 

 

TA1535

 

TA1537

 

 

TA98

 

TA100

 

WP2uvrA

 

Without S9-mix

 

 

 

 

 

 

 

 

 

 

Positive control

753

± 28

710

± 95

867

± 63

901

± 42

1154

± 106

Solvent control

22

± 5

8

± 4

22

± 6

133

± 8

35

± 11

492

26

± 3

9

± 4

29

± 7

124

± 23

29

± 17

878

26

± 4

5

± 5

21

± 3

131

± 16

33

± 11

1568

22

± 7

7

± 4

25

± 4

123

± 2

32

± 6

2800

24

± 8 n

6

± 2 n

22

± 9 n

107

± 3

26

± 6

5000

17

± 4 s NP

9

± 2 s NP

13

± 6 s NP

93

± 25 s NP

23

± 3 n NP

With S9-mix #1

 

 

 

 

 

 

 

 

 

 

Positive control

166

± 10

443

± 138

1047

± 68

1275

± 218

153

± 41

Solvent control

13

± 2

14

± 2

39

± 2

134

± 13

42

± 13

492

17

± 6

15

± 3

42

± 2

130

± 16

41

± 8

878

13

± 3

7

± 4

34

± 5

129

± 21

46

± 9

1568

17

± 8

15

± 4

36

± 6

121

± 12

40

± 11

2800

12

± 6 n

4

± 1

33

± 14

103

± 11 n

37

± 5

5000

11

± 4 s NP

13

± 6 n NP

33

± 6 n NP

77

± 2 s NP

21

± 2 n NP

#1          Plate incorporation assay (10% S9)

NP         No precipitate

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the conditions of this study the test substance is not considered to be mutagenic. Negative and strain specific positive control values were within laboratory historical control data.
Executive summary:

The study was performed to OECD 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). 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. Cytotoxicity, as evidenced by a decrease of the bacterial background lawn, was observed at the top dose of 5000 μg/plate in both tester strains. 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 17 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In an independent repeat of the assay with additional parameters, the test substance was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity, as evidenced by a decrease of the bacterial background lawn, was only observed at the top dose of 5000 μg/plate in the tester strains TA1535 and TA100 (absence and presence of S9-mix) and TA1537 and TA98 (absence 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 tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges 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.