Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Apr - 21 Apr 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
(duplicate instead of triplicate plating was performed)

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
yes
Remarks:
duplicate instead of triplicate plating was performed
Qualifier:
according to
Guideline:
other: SystemsGuidelines for Japanese Industrial Safety and Health Act and Chemical Substance Control Law
Version / remarks:
Criteria for Mutagenicity Test in Bacterial Systems (Ministry of Labour, Japan, Notification No.77 dated September 1, 1988 and Notification No.67 dated J une 2, 1997)

Japanese Ministry of Health, Labor and Welfare, Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of the Environment, Heisei 23.3.31 Yakushokuhatsu Notification No. 0331-7, Heisei 23.3.29 Seikyoku Notification No. 5, Kanpokihatsu Notification No. 110331009
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: ≥ 50 mg/mL in water, stable for 20 hours at room temperature and prepared just before use

FORM AS APPLIED IN THE TEST: liquid

Method

Target gene:
his operon, tryp operon
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 male rats, treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
First experiment (dose-finding study, all strains): 4.88 - 5000 µg/plate with and without metabolic activation (tested up to the limit concentration)
Second experiment (main assay, all strains): 156 - 5000 µg/plate with and without metabolic activation (tested up to the limit concentration)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubiulity properties and its nontoxicity for the bacteria.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
sterilized purified water
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: The dose-finding assay and main assay were carried out in duplicate plating for each dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial plate
Evaluation criteria:
Acceptance criteria
When at least one plate count in the solvent and positive control group is within the acceptance control range (mean ± 3SD, calculated from the historical control data, see table 3), the assay is judged to be acceptable.

Evaluation criteria
When the test substance shows a dose-dependent increase in the number of revertant colonies to at least twice as many as that of the solvent control, the response is judged to be positive 4 5). The study director judges the result with scientific consideration of biological relevance. When the positive response is reproducible, the test substance is judged to have mutagenic potential.
Statistics:
No statistics were performed.

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:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
number of revertants is close to or within historical control date range
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
number of revertants is close to or within historical control date range
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
number of revertants is close to or within historical control date range
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
number of revertants is close to or within historical control date range
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
number of revertants is close to or within historical control date range
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
number of revertants is close to or within historical control date range
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
decrease in number of revertants in exp. 2, starting at 1250 µg/mL, with S9 mix (around 32-45%)
Vehicle controls validity:
valid
Remarks:
number of revertants is close to or within historical control date range
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
number of revertants is close to or within historical control date range
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Remarks:
number of revertants is close to or within historical control date range
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
number of revertants is close to or within historical control date range

Any other information on results incl. tables

Table 1: Summary of test results (experiment 1; dose-finding assay)

With or without S9-Mix Test substance concentration (μg/plate) Mean number of revertant colonies per plate (average of 2 plates) 
Frameshift type Base-pair substitution type
TA98 TA1537 TA100 TA1535 WP2 uvrA
Solvent control (water) 20 7 104 9 22
4.88 21 7 110 8 19
19.5 23 4 105 7 23
78.1 20 9 106 8 19
313 15 6 102 5 14
1250 18 9 108 11 21
5000 15 5 111 13 19
Positive controls (concentrations (µg/plate))  AF-2
(0.1)
9AA
(80)
AF-2
(0.01)
SA
(0.5)
AF-2
(0.01)
Mean No. of colonies/plate (average of 2 plates) 432 341 646 306 107
Solvent control (water) 29 15 104 6 24
4.88 34 14 110 10 21
19.5 34 14 126 8 18
78.1 32 13 115 7 26
313 43 13 111 9 21
1250 36 13 112 7 24
5000 46 10 126 7 18
Positive controls (concentrations (µg/plate)) 2AA
(0.5)
2AA
(2) 
2AA
(1)
2AA
(2)
2AA
(10)
Mean No. of colonies/plate (average of 2 plates) 259 147 708 206 264

SA = sodium azide

AF-2=2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

9AA= 9-Aminoacridine

2AA = 2-Aminoanthracene

SD = standard deviation

Table 2: Summary of test results (experiment 2; main assay)

With or without S9-Mix

Test substance concentration (μg/plate)

Mean number of revertant colonies per plate (average of 2 plates)

Frameshift type

Base-pair substitution type

TA98

TA1537

TA100

TA1535

WP2 uvrA

Solvent control (water)

20

11

106

10

21

156

29

14

114

11

27

313

27

11

106

12

16

625

27

10

112

13

22

1250

23

6

113

12

17

2500

30

11

110

7

23

5000

27

15

105

9

22

Positive controls (concentrations (µg/plate))

AF-2
(0.1)

9AA
(80)

AF-2
(0.01)

SA
(0.5)

AF-2
(0.01)

Mean No. of colonies/plate (average of 2 plates)

370

388

633

347

84

+

Solvent control (water)

43

22

115

12

24

+

156

43

19

109

9

23

+

313

42

19

119

9

18

+

625

43

22

119

14

23

+

1250

41

15

115

9

24

+

2500

43

16

119

8

23

+

5000

44

12

126

14

31

+

Positive controls (concentrations (µg/plate))

2AA
(0.5)

2AA
(2)

2AA
(1)

2AA
(2)

2AA
(10)

Mean No. of colonies/plate (average of 2 plates)

249

137

727

206

297

SA = sodium azide

AF-2 =2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

9AA= 9-Aminoacridine

2AA = 2-Aminoanthracene

SD = standard deviation

Table 3: Historical control data (Jan - Dec 2013)

 

 

Revertant colonies

Test strain

S9 Mix

Solvent control

Positive control(1)

 

 

Mean ± SD

Number of data

Mean ± SD

Number of data

TA100

-

90.7 ± 8.93

203

574.5 ± 68.48

199

TA1535

-

10.5 ± 2.56

162

287.4 ± 40.55

162

WP2 uvrA

-

18.1 ± 3.91

159

96.4 ± 13.59

159

TA98

-

20.7 ± 3.95

158

353.0 ± 34.85

158

TA1537

-

9.0 ± 2.69

162

445.2 ± 75.08

162

TA100

+

92.2 ± 10.32

193

566.1 ± 55.17

193

TA1535

+

10.3 ± 2.54

157

165.5 ± 16.44

157

WP2 uvrA

+

21.6 ± 4.69

161

270.3 ± 49.98

161

TA98

+

30.7 ± 5.29

196

192.6 ± 23.40

192

TA1537

+

15.7 + 3.93

157

99.9 ± 18.23

157

Mean ± 3SD were set as the acceptable control range.

 

(1) Positive control substances

Test strain

S9 Mix

Chemical

Dose [µg/plate]

TA100

-

2-(2-fury 1)-3-(5-nitro-2-fury 1) acrylamide

0.01

TA1535

-

sodium azide

0.5

WP2 uvrA

-

2-(2-fury 1)-3-(5-nitro-2-fury 1) acrylamide

0.01

TA98

-

2-(2-fury 1)-3-(5-nitro-2-fury 1) acrylamide

0.1

TA1537

-

9-aminoacridine

80

TA100

+

2-aminoanthracene

1

TA1535

+

2

WP2 uvrA

+

10

TA98

+

0.5

TA1537

+

2

 

Applicant's summary and conclusion