Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). Otherwise the study has a reliability of 1 (reliable without restriction). This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment. GLP Guideline study (OECD 471)

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1999
Reference Type:
other: english study summary
Title:
Unnamed
Year:
1999
Reference Type:
publication
Title:
OECD SIDS 2,2'-Azobis (2-methylpropionitrile) (CAS 78-67-1) - SIAM 9
Author:
Anonymous
Year:
2000
Bibliographic source:
UNEP Publications

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,2'-AZOBIS(2-METHYLPROPIONITRILE)
- Physical state: white crystal
- Analytical purity: 99.9%

Method

Species / strainopen allclose all
Species / strain / cell type:
other: TA 98, TA 100, TA 1535, TA 1537 and TA 97
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 mix. Liver S9 homogenate was prepared from rats that have been induced with phenobarbital and 5,6-benzoflavone.
Test concentrations with justification for top dose:
0, 313, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: (see below: details on test system and conditions)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- The test was performed according to the pre-incubation method.

NUMBER AND SELECTION OF DOSES, CONTROLS USED
- Five doses of test substance, together with the appropriate concurrent solvent and positive controls, were tested in triplicate on each tester strain with and without metabolic activation. The experiment was repeated twice except for TA1537 whic was tested four times.
-The dose-levels were the following: 0, 313,625, 1250, 2500 and 5000 µg/plate. All the strains were tested without S9 whereas TA97 was not part
of the experiment performed with S9.
The positive controls were as follows:
without S9 mix:
• sodium azide (NaN3): TA 1535 strain
• 9-Aminoacridine (9AA): TA 1537 and TA97 strains,
• 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide: TA 98 and TA 100 strains,
with S9 mix:
• 2-Aminoanthracene: TA 1535, TA 1537, TA 98 and TA 100 strains,
Evaluation criteria:
Not precised
Statistics:
no

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537 and TA 97
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
See tables below.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 : Number of revertants per strain : 1st experiment

 

TA 98

TA 98

TA100

TA 100

TA 1535

TA1535

TA1537

TA 1537

WP2 uvrA

WP2 uvrA

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

DMSO

26

17

31

31

37

29

134

126

134

135

117

120

8

8

11

7

9

11

11

5

8

20

16

16

21

30

16

37

24

17

313ug

21

30

27

24

37

24

130

125

94

144

160

125

14

5

12

6

10

14

12

10

11

19

15

16

19

14

23

21

21

20

625ug

25

28

23

28

26

24

113

135

127

131

120

142

8

10

13

9

12

15

20

16

17

18

14

12

17

22

19

33

21

28

1250ug

23

35

26

29

34

37

128

141

102

136

134

143

9

11

13

15

15

10

19

15

9

12

15

15

23

23

18

20

22

25

2500ug

12

31

30

26

26

33

109

122

152

107

138

161

10

12

14

9

9

10

13

13

7

11

14

18

13

12

18

28

35

29

5000ug

28

23

27

24

31

28

116

105

115

141

112

110

11

11

14

7

11

9

22

12

10

13

9

12

8

9

11

18

13

16

 Positive controls  (AF2)745  (2AA) 368  (AF2) 605 (2AA) 399 (SA) 337  (2AA) 240  (9AA) 397  (2AA) 310 (AF2) 259  (2AA) 422

 

Table 2 : Number of revertants per strain : 2nd experiment

 

TA 98

TA 98

TA100

TA 100

TA 1535

TA1535

TA1537

TA 1537

WP2 uvrA

WP2 uvrA

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

DMSO

29

18

20

27

31

31

111

91

119

94

118

113

9

11

6

18

8

18

6

5

5

19

16

10

22

23

21

37

40

24

313ug

25

14

15

21

30

32

110

112

121

117

106

113

13

8

8

13

17

11

14

15

21

18

14

18

30

25

31

31

30

28

625ug

15

37

20

24

37

29

105

122

130

138

110

99

15

21

14

14

14

15

13

16

20

18

22

12

29

27

25

27

25

25

1250ug

29

28

23

21

31

28

115

106

122

128

119

116

14

8

17

12

9

13

13

20

7

12

13

17

20

25

15

24

20

29

2500ug

23

19

18

29

23

19

125

108

121

129

114

126

13

12

14

9

10

9

11

9

8

17

16

8

17

16

13

24

25

30

5000ug

15

21

18

22

21

31

113

93

130

130

112

127

13

18

6

11

8

7

10

9

17

7

9

9

19

27

19

18

20

18

 Positive controls  (AF2) 688  (2AA) 347  (AF2) 779 (2AA) 938 (SA) 376  (2AA) 293  (9AA) 10003  (2AA) 436 (AF2) 240  (2AA) 670

§Table 3 : Number of revertants per strain : Confirmation test

 

TA1537

 

- S9

DMSO

6

4

9

313ug

7

15

10

625ug

9

8

10

1250ug

10

12

3

2500ug

14

12

5

5000ug

9

4

2

 Positive controls

 (9AA) 873

Table 4 : Number of revertants per strain: Additional experiment

 

TA1537

 TA 97

 

- S9

 - S9

DMSO

18

8

11

 200194228

313ug

4

4

5

 139167194

625ug

7

7

8

 156165180

1250ug

8

2

7

 183169180

2500ug

10

4

4

 169198209

5000ug

12

11

12

 185173196

 Positive controls

 (9AA) 1363

(9AA) 3130

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
Under the experimental conditions described, the test substance 2,2'-AZOBIS(ISOBUTYRONITRILE) did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella or E. coli.
Executive summary:

The potential of the test item 2,2-AZOBIS(ISOBUTYRONITRILE) to induce reverse mutation in Salmonella typhimurium was evaluated according to OECD 471 guideline in compliance with the Principles of Good Laboratory Practice.

Methods:

The test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with phenobarbital and 5,6-benzoflavone. Both experiments were performed according to the preincubation method. Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 97 were used except for TA 97 which was only used in the experiment without S9. E. coli WP2 uvrA was also tested with and without activation. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test item 2,2-AZOBIS(ISOBUTYRONITRILE) was dissolved in dimethylsulfoxide (DMSO) and the following positive controls were used:

without S9 mix:

"sodium azide (NaN3): TA 1535 strain,

"9-Aminoacridine (9AA): TA 1537and TA 97 strains,

"2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide : TA 98 and TA100 strains,

with S9 mix:

"2-Aminoanthracene: TA 1535, TA 1537, TA 98 and TA 100 strains

Results:

The total maximum dose of 5 mg per plate was selected as the highest dose of the experiment. The selected treatment-levels were 313, 625, 1250, 2500 and 5000 ug/plate for all strains with or without metabolic activation. The test item did not induce any significant increase in the number of revertants, in either experiment, in any of the five strains and no toxicity was observed.

Conclusion:

Under these experimental conditions, the test item 2,2-AZOBIS(ISOBUTYRONITRILE) did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.