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Toxicological information

Genetic toxicity: in vitro

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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:
1999-02-12 to 1999-03-16
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: in accordance with "Standards for Toxicity Investigations" .
Version / remarks:
Ministry of Labor, Notification No.77, September'l, 1988 and Notification No.67, June 2, 1997
GLP compliance:
yes
Remarks:
This study was conducted in compliance with "Standards to be observed by Testing Institutions for Toxicity Investigations" (Ministry of Labor, Notification No.76, September 1, 1988).
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: white crystalline
Details on test material:
- Name of test material (as cited in study report): ABT or (2-Benzothiazolylthio)acetic acid
- Physical state: solid
- Appearance: white crystalline
- Analytical purity: 99%
- Purity test date: n.a.
- Lot/batch No.: 99001
- Expiration date of the lot/batch: n.a.
- Stability under test conditions: stable in ordinary temperature
- Storage condition of test material: stred at a cool and dark place

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Dose finding test: with and without S9 mix: 4.88, 19.5, 78.1, 313, 1250, 5000 (µg/plate)
Main test with and without S9 mix: 313, 625, 1250, 5000 (µg/plate)
Confirmation test: with strain TA1537 with and without S9 mix: 313, 625, 1250, 5000 (µg/plate)

Positive controls without S9 mix:
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide: 0.01 µg/plate (TA100, WP2uvrA), 0.1 µg/plate (TA98)
NaN3: Sodium azide: 0.5 µg/plate (TA1535)
ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropy1amino]acridine' 2HCl: 1 µg/plate (TA 1537)

Positive controls with S9 mix:
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide: 0.01 µg/plate (TA100, WP2uvrA), 0.1 µg/plate (TA98)
NaN3: Sodium azide: 0.5 µg/plate (TA1535)
ICR-191: 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropy1amino]acridine' 2HCl: 1 µg/plate (TA 1537)
2AA: 2-Aminoanthracene: 0.5 µg/plate (TA98), 1 µg/plate (TA100), 2 µg/plate (TA1535, TA1537), 10 µg/plate (WP2uvrA)
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Since the solubility of the test substance was less than 50 mg/mL in water but more than 50 mg/mL in DMSO, DMSO was selected as a solvent. It was confirmed that there was no change in the 50 mg/mL solution in DMSO, including color change and heat generation, until 2 hours after preparation.
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
The solvent used in the tests was employed as a negative control
Positive controls:
yes
Positive control substance:
sodium azide
other: 2(2-Furyl)3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Aminoanthracene (2AA)
Details on test system and conditions:
This study was performed by the pre-incubation method with and without S9 mix. Triplicate plates were used for the negative control group and duplicate plates per dose for the test substance treatment groups and the positive control groups. The test code number, name of test strain, presence or absence of S9 mix and dose level were noted on each plate.
Evaluation criteria:
The test substance was judged to be positive when the number of revertant colonies of the test substance treatment groups increased to twice or more that in the negative control with a concentration-dependent manner.
Statistics:
Any statistical methods were not used.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: TA 1535, TA 98, TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: salmonella typhimurium
Remarks:
Migrated from field 'Test system'.
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Result of Confirmation test: Number of revertant colonies in TA1537 strain was less than twice than in the negative control (with and without S9 mix). Increase of numbers of revertants in main test is juded accidential as value of negative group was low.
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: salmonella typhimurium
Remarks:
Migrated from field 'Test system'.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: E. coli
Remarks:
Migrated from field 'Test system'.
Additional information on results:
Confirmation test:
The number of revertant colonies in the test substance treatment groups was less than twice than in the negative control and in the groups with and without S9 mix in TA1537. Therefore, the increase of the number of revertant colonies in the main test was not confirmed dose-dependend and reproducible and due to the value of negative controls were low in the main test it was judged as an accidental increase result. The number of revertant colonies of the test substance treatment group in the main test was within the range of the historical data. From the above results, mutagenisity was judged to be negative in TA1537.

Any other information on results incl. tables

Table 1: Summary of mean number of revertants in Salmonella typhimurium strains and E. coli WP2uvrA strain with and without metabolic activation (mean of 3 plates for vehicle controls and 2 plates for test item treated or positive controls)

Results of dose finding Test

Concentration

TA100

TA1535

WP2uvrA

TA98

TA1537

[µg/plate]

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Negative Control

159

131

7

8

19

19

19

28

4

9

4.88

137

140

6

7

20

25

18

37

3

13

19.5

131

143

7

6

17

27

17

29

3

10

78.1

131

141

13

11

17

23

17

24

3

7

313

111

136

6

10

18

28

20

28

3

10

1250

117

113

10

5

19

22

21

25

4

13

5000

104

97

7

6

15

32

20

21

4

11

positive Control

AF-2

2AA

NaN3

2AA

AF-2

2AA

AF-2

2AA

ICR-191

2AA

448

718

328

147

155

1050

585

291

2778

97

Results of Main Test

Concentration

TA100

TA1535

WP2uvrA

TA98

TA1537

[µg/plate]

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Negative Control

133

119

9

7

17

26

18

28

3

5

313

116

122

9

9

18

30

17

32

7

10

625

111

121

11

6

19

25

17

26

5

13

1250

106

114

7

7

21

23

19

24

8

17

2500

102

111

6

7

15

23

19

27

7

12

5000

92

91

5

7

19

23

20

20

6

11

positive Control

AF-2

2AA

NaN3

2AA

AF-2

2AA

AF-2

2AA

ICR-191

2AA

449

766

386

163

149

1046

554

394

2599

99

AF-2:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide = positive control –S9 mix for strains TA100, WP2uvrA and TA98 [selected doses of 0.01, 0.01 and 0.1 µg/plate, respectively]

NaN3: Sodium azide= positive control –S9 mix for strain TA1535 [selected dose:0.5 µg/plate]

ICR- 191:2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine-2HCl = positive control –S9 mix for strain TA1537 [selected dose:1 µg/plate]

2AA: 2-Aminoanthracene = positive control + S9 mix for all tested bacteria strains[selected doses of 1, 2, 10, 0.5 and 2 µg/plate for strain TA100, TA1535, WP2uvrA, TA98 and TA1537, respectively]

 

Table 2: Confirmation Test: Summary of mean number of revertants in Salmonella typhimurium strain TA1537 with and without metabolic activation (mean of 3 plates for vehicle controls and 2 plates for test item treated or positive controls)

Results of confirmation Test

Concentration

TA1537

[µg/plate]

- S9

+ S9

Negative Control

8

10

313

10

10

625

9

11

1250

7

11

2500

10

11

5000

8

11

positive Control

 

ICR-191

2AA

2753

110

ICR- 191:2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine'2HCl [Dose:1 µg/plate]

2AA: 2-Aminoanthracene[Dose: 2 µg/plate]

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the above results, it is concluded that the registered substance has no ability to induce mutation under the conditions of the present study.
Executive summary:

The ability of registered substance to induce mutations was investigated using Salmonella typhimurium strains TAl00, TA1535, TA98 and TAl537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix).

As a result, increase in the numbers of revertant colonies was not observed at any doses of the test substance treatment group in all test strains with and without S9 mix.

The numbers of revertant colonies in the negative and positive controls were confirmed to be within the range of the historical data in the testing facility. Based on the above results, it is concluded that the registered substance has no ability to induce mutation under the conditions of the present study.