Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All in vitro studies on 13472-08-7 (two Ames), and on read across substance 78 -67 -1 (Ames, MLA, Chrome ab) are negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
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)
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across source
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
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
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
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'.

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
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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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 473)
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster lung cells (CHL/IU)
Details on mammalian cell type (if applicable):
no data
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:
-S9 mix (continuous treatment) : 0, 0.40, 0.80, 1.6 mg/L
-S9 mix (short-term treatment) : 0, 0.40, 0.80, 1.6 mg/L
+S9 mix (short-term treatment) : 0, 0.40, 0.80, 1.6 mg/L

Vehicle / solvent:
0.5% CMC (Carboxymethylcellulose) sodium solution
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: - S9 : Mitomycin C ; +S9 : Cyclophosphamide
Details on test system and experimental conditions:
Three doses of the test substance, together with the appropriate concurrent solvent and positive controls, were tested with and without metabolic activation for a 6H-period. Given the negative results from this protocol (short-treatment), a continous experiment was conducted for a 24H-period and a 48H-period witout activation.
Evaluation criteria:
Not precised
Statistics:
no
Species / strain:
other: Chinese hamster lung cells (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
See table (below)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Chromosome analysis of Chinese hamster cells continous treatment, without S9 mix.

Group

Concentration

Time of exposure

N° of cells analysed

N° of structural aberrations

Others

N° of cells with aberrations

Polyploid

Trend test

Concurrent cytotoxicity

 

(mg/mL)

(h)

 

gap

ctb

cte

csb

cse

mul

total

 

TAG (%)

TA (%)

(%)

SA

NA

(%)

Control

-

-

200

0

0

0

0

0

0

0

0

0 (0.0)

0 (0.0)

0.25

 

 

-

Vehicle

0

24

200

1

0

0

0

0

0

1

0

1 (0.5)

0 (0.0)

0.13

 

 

100.0

AZDN

0.40

24

200

0

0

0

0

0

0

0

0

0 (0.0)

0 (0.0)

0.13

 

 

85.0

AZDN

0.80

24

200

1

0

0

0

0

0

1

0

1 (0.5)

0 (0.0)

0.38

NT

NT

74.0

AZDN

1.6

24

200

1

1

0

0

0

0

2

0

2 (1.0)

1 (0.5)

0.25

 

 

64.0

MC

0.00005

24

200

7

52

74

2

2

0

137

1

93 (46.5)

89 (44.5)

0.38

 

 

-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Vehicle

0

48

200

1

0

0

0

0

0

1

0

1 (0.5)

0 (0.0)

0.13

 

 

100.0

AZDN

0.40

48

200

1

0

0

0

0

0

1

0

1 (0.5)

0 (0.0)

0.00

 

 

104.5

AZDN

0.80

48

200

1

0

0

0

0

0

1

0

1 (0.5)

0 (0.0)

0.13

NT

NT

128.0

AZDN

1.6

48

200

0

0

1

0

0

0

1

0

1 (0.5)

1 (0.5)

0.13

 

 

159.5

MC

0.00005

48

200

11

40

99

1

1

0

152

0

77 (38.5)

70 (35.0)

0.00

 

 

-

Table 2: Chromosome analysis of Chinese hamster cells short treatment, with and without S9 mix.

Group

Concentration

S9 mix

Time of exposure

N° of cells analysed

N° of structural aberrations

Others

N° of cells with aberrations

Polyploid

Trend test

Concurrent cytotoxicity

 

(mg/mL)

 

(h)

 

gap

ctb

cte

csb

cse

mul

total

 

TAG (%)

TA (%)

(%)

SA

NA

(%)

Control

-

 

-

200

0

0

0

0

0

0

0

0

0 (0.0)

0 (0.0)

0.25

 

 

-

Vehicle

0

-

6 - (18)

200

1

0

0

0

0

0

1

0

1 (0.5)

0 (0.0)

0.25

 

 

100.0

AZDN

0.40

-

6 - (18)

200

1

0

0

0

0

0

1

0

1 (0.5)

0 (0.0)

0.13

 

 

99.5

AZDN

0.80

-

6 - (18)

200

0

2

0

0

0

0

2

0

2 (1.0)

2 (1.0)

0.38

NT

NT

96.5

AZDN

1.6

-

6 - (18)

200

1

0

0

0

0

0

1

0

1 (0.5)

0 (0.0)

0.13

 

 

91.5

CPA

0.005

-

6 - (18)

200

1

0

0

0

0

0

1

0

1 (0.5)

0 (0.0)

0.13

 

 

-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Vehicle

0

+

6 - (18)

200

0

0

0

0

0

0

0

0

0 (0.0)

0 (0.0)

0.00

 

 

100.0

AZDN

0.40

+

6 - (18)

200

0

0

0

0

0

0

0

0

0 (0.0)

0 (0.0)

0.13

 

 

89.0

AZDN

0.80

+

6 - (18)

200

1

2

1

0

0

0

5

0

5 (2.5)

4 (2.0)

0.25

NT

NT

81.0

AZDN

1.6

+

6 - (18)

200

0

0

0

0

0

0

0

0

0 (0.0)

0 (0.0)

0.38

 

 

71.5

CPA

0.005

+

6 - (18)

200

4

25

53

1

0

0

83

0

54 (27.0)

51 (25.5)

0.00

 

 

-

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 mammalian chromosome aberration test.
Executive summary:

The potential of the test item 2,2-AZOBIS(ISOBUTYRONITRILE) to cause structural chromosome aberrations in cultured Chinese hamster lung cells was evaluated according to OECD 473 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. The first experiment was performed for a short period of 6 hours with and without metabolic activation. The second one was a continuous treatment performed for a 24 hour and a 48 hour period but without metabolic activation. Each cultured cell was exposed to three dose-levels of the test item (two plates/dose-level).

The evaluation of the toxicity was performed on the basis of the observation of the increase in the number of cells with chromosome aberrations, in the number of polyploid cells or in the number of cells with endoreduplicated chromosomes.

The test item 2,2-AZOBIS(ISOBUTYRONITRILE) was dissolved in a 0.5% Carboxymethylcellulose sodium solution and the following positive controls were used:

  • without S9 mix: Mytomycin C
  • with S9 mix : Cyclophosphamide

Results:

The selected treatment-levels were 0.40, 0.80 and 1.6 mg/mL with or without metabolic activation. The test item did not induce any significant increase in the number of cells with chromosome aberrations, in the number of polyploid cells nor in the number of cells with endoreduplicated chromosomes in either experiment, and no toxicity was observed.

Conclusion:

Under these experimental conditions, the test item 2,2-AZOBIS(ISOBUTYRONITRILE) did not induce chromosome aberrations in cultured mammalian somatic cells. 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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. Guideine study (OECD 476). No indication of GLP compliance, Restrictions: sample analyzed by MRI but purity not reported, the size of the colony was determined was not reported even for the solvent and positive controls.
Justification for type of information:
see 13.2 for attached read across rationale
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Cells were originally obtained from Dr. Donald Clive, Burroughs Wellcome Co. (Research Triangle Park, NC).
- Cells were grown in Fisher's medium for leukemic cells of mice (Gibco, Grand Island, NY or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI.
- Cells were screened for the presence of mycoplasma after cryopreservation.
- New cultures were initiated at approximately 3 month intervals from cells stored in liquid N2.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 mix. The microsomal enzyme fraction was prepared as described by Clive et al. (1979, Mutat. Res.59, 61-108). Liver S9 homogenate was prepared from male Sprague-Dawley rats that have been injected with Aroclor 1254
Test concentrations with justification for top dose:
600, 700, 800, 900 and 1000 µg/mL

Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO)
True negative controls:
no
Positive controls:
yes
Remarks:
with & without S9
Positive control substance:
other: Without S9 : ethylmethanesulphonate With S9 : 3-methylcholantrene (dimethylbenz[a]anthracene also used)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- The mutagenicity assay was performed according to Clive and Spector (Mutat Res., 31, 17-29).
- A total of 1.2*1E7 cells in duplicate cultures were exposed to the test chemical (as well as to solvent and positive control) for 4 hours at 37±1°C. After washing twice with growth medium, cells were maintained at 37±1°C for 48 hours in log-phase growth to allow recovery and mutant expression. Cells in the culture were adjusted to 3*1E5/mL at 24-hour intervals.
- Cells were cloned to 1*1E6 cells/plate for mutant selection and 200 cells/plate for viable count determinations in soft agar medium (contained Fisher's medium, 20% horse serum, 2mM sodium pyruvate, 0.02% pluronic F-68 and 0.23% granulated agar (BBL Inc., Cockeyville, MD)).
- Resistance to trifluorothymidine (TFT) was determined by adding TFT (3 µg/mL) to the cloning medium for mutant selection. Plates were incubated at 37±1°C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, Dynatech) or Protocol colony counter (Synbiosis, Frederick, MD). Only colonies larger than 0.2 mm diameter (ca) were counted.
- Mutant frequencies were expressed as mutants per 1E6 surviving cells.
- The size of mutant mouse lymphoma colonies were also determined thanks to the Artek 982 colony counter/sizer or Protocol colony counter.The size range used was from 0.2 (ca) to 1.1 mm.

NUMBER AND SELECTION OF DOSES, CONTROLS USED
- The toxicity of each chemical was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats.
- The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study. Cells at concentration of 6*1E5/mL were exposed for 4 hours to a range of concentrations from 0.0005 to 10000 µg/mL. The cells where then washed, resuspended in growth medium and incubated at 37°C (±1°C) for 48 hours. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding 0 - 90% cytotoxicity. For each assay, there were 2-4 solvent controls, a positive control of ethyl methylsulfonate at 4.7*1E-6M (or methyl methanesulfonate at 10-20 µg/mL) for the test without metabolic activation, and a positive control of 3-methylcholantrene at 1.86*1E-5M (or dimethylbenz[a]anthracene at 0.5-4 µg/mL) for the test with metabolic activation.
Evaluation criteria:
For a test substance to be considered positive, it had to induce at least a doubling of the mutant frequency over the concurrent solvent-treated control value. This increase in the mean mutant frequency had to be accompanied by a dose response to increasing concentrations of the test substance. Only doses yielding total growth values of 10% or greater were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.
Statistics:
No statistics were performed
Species / strain:
mouse lymphoma L5178Y cells
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 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'.

Table 1: Mouse lymphoma test results both with and without activation

 

Non-activated cultures

 

S9-activated cultures

Dose (µg/mL)

Average TFT

Average VC

Mutat frequency

RTG

 

Average TFT

Average VC

Mutat frequency

RTG

Solvent

21

180

0.23

 

 

23

158

0.29

 

600

22

155

028

77

 

28

144

0.39

102

 

24

153

0.31

76

 

28

155

0.36

100

700

27

150

0.36

71

 

30

156

0.38

113

 

20

171

0.23

85

 

28

151

0.37

100

800

22

166

0.27

83

 

23

153

0.3

96

 

30

156

0.38

66

 

28

146

0.38

102

900

25

142

0.35

66

 

18

133

0327

90

 

24

159

0.3

70

 

22

158

0.28

105

1000

33

171

0.39

70

 

26

156

0.33

117

 

25

179

0.28

77

 

24

156

0.31

101

Positive

394

77

10.23

26

 

315

155

4.06

62

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 did not show any mutagenic activity in the mouse lymphoma assay.
Executive summary:

The potential of the test item 2,2-AZOBIS(ISOBUTYRONITRILE) to induce gene mutation was evaluated in a mouse lymphoma mutagenicity assay using L5178Y TK+/- cells. The test method used was similar to OECD 476 guideline but purity of the product nor compliance with the Principles of Good Laboratory Practice were reported by the authors of the publication.

The test item was tested in an experiment with and without a metabolic activation system, the S9 mix. From the cytotoxicity results of the preliminary test, the total maximum dose of 1000 µg/mL was selected as the highest dose of the experiment. The selected treatment-levels were 600, 700, 800, 900 and 1000 µg/mL. Duplicate cultures of 1.2*1E7 cells were exposed to the test or control items for 4 hours at 37±1°C. At the end of the exposure period, cells were washed and maintained for 48 hours at 37±1°C to allow for the mutant expression, then cultured in appropriate medium with trifluorothymidine for 10 -12 days at 37±1°C to determine survival and to allow for the mutant selection (expression of the mutant phenotype). Mutant colonies and relative total growth were then scored.

The evaluation of the toxicity was performed on the basis of the observation of an increase in the number of mutant frequencies and/or a reduction in the growth rate (small colonies). The test item did not induce any significant increase in the mutation frequency and no toxicity was observed. Under these experimental conditions, the test item 2,2-AZOBIS(ISOBUTYRONITRILE) did not show any mutagenic activity in the in vitro Mammalian Cell Gene Mutation Test using L5178Y mouse lymphoma cells.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Only 4 strains instead of the 5 required by current guidelines were tested.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Conducted according to guideline in effect at time of study conduct
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver (S-9 mix)
Test concentrations with justification for top dose:
50 to 5000 µg
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Details on test system and experimental conditions:
An aliquot (0.1 mL) of each concentration of test substance was placed in a sterile tube. Molten, histidine-deficient top-agar (2 mL) and bacterial suspension (0.1 mL), maintained at 45°C, was then added. The tubes were mixed by inversion and 0.5 mL rat liver microsomal preparation (S-9 mix) was added where appropriate. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (20 mL). Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S-9 mix and the test material. A control series of plates was prepared to confirm the inability of DMSO (0.1 ML) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates. Aliquots (0.1 mL) of a 10E-6 dilution of culture were spread on the surface of plates of complete medium to measure the viability and cell density of each culture. All plates were prepared in triplicate, allowed to solidify and incubated at 37°C for 2 days. After incubation, numbers of revertant colonies were counted, either manually or with a Biotran II automatic colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the background lawn of non-revertant cells on minimal plates was verified. Results obtained with all strains were confirmed in a second, independent experiment.
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A solution of the test substance was prepared in DMSO at 25 mg/mL, and an aliquot of the solution (0.2 mL) was transferred to a sterile tube containing molten histidine-deficient top-agar (2.0 mL) maintained at 45°C. An additional aliquot (0.1 mL) of the test material solution was similarly transferred to another tube of molten top-agar (2.0 mL). Three serial 10-fold dilutions in molten top-agar were prepared from each of the above preparations, giving a series of 8 different concentrations of the test material from 2.5 µg to 5 mg per plate. All tubes were inoculated with an overnight culture of strain TA98 (0.1 mL) and overlaid onto minimal medium plates. Control plates were prepared containing top-agar and culture alone, top-agar, DMSP (0.2 mL) and culture, and top-agar and test material (0.1 mL) without bacterial culture. The plates were incubated for 37°C for 2 days and were then examined for the presence of a background lawn of non-revertant colonies; toxicity of the test material was shown by absence or thinning of the background lawn. No visible thinning of the background lawn of non-revertant cells was obtained. A top exposure level of 5 mg/plate was therefore selected for use in the main assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
It was concluded that the test item was devoid of mutagenic activity under the conditions of the test.
Executive summary:

The test substance was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, using pour-plate assays. Results obtained with all strains were confirmed in a second, independent experiment. The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of the test substance from 50 to 5000µg per plate, selected following a toxicity test, and included solvent (dimethyl suiphoxide) controls with and without S-9 mix. No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the levels tested, either in the presence or absence of S-9 mix. It was concluded that the test substance was devoid of mutagenic activity under the conditions of the test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1993
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Short abstract from the 20th Annual meeting of the Japanese Society of Toxicological Sciences. No study details provided.
Principles of method if other than guideline:
The reverse mutation assay was conducted with Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA in the absence and presence of metabolic activation system (S9 mix).
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Remarks:
There were negative controls based on summary: did not increase the number of revertants compared with negative control values
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
other: see Remarks
Remarks:
from summary: 2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
other: see Remarks
Remarks:
2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
other: see Remarks
Remarks:
2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
other: see Remarks
Remarks:
2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
Cytotoxicity / choice of top concentrations:
not specified
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
other: see Remarks
Remarks:
2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.
Cytotoxicity / choice of top concentrations:
not specified
Conclusions:
2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or
without metabolic activation.
Executive summary:

The reverse mutation assay was conducted with Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA in the absence and presence of metabolic activation system (S9 mix).

2,2'-azobis(2-methylbutyronitrile) did not increase the number of revertants compared with negative control values in any strains with or without metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Supporting in vivo micronucleus study on 13472-08-7 (supporting due to lack of reliability due to abstract only) is negative. Further testing not required as all in vitro endpoints are negative.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic and germ cell study: gene mutation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test substance was examined for mutagenic activity in Salmonella typhimurium, strains TA98, TA100, TA1535 and TA1537, using pour-plate assays. Results obtained with all strains were confirmed in a second, independent experiment. The studies, which were conducted in the absence and presence of an activating system employed a range of levels of the test substance from 50 to 5000 µg per plate with and without a metabolic activation system. No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the levels tested. It was concluded that the test substance was devoid of mutagenic activity under the conditions of the test.

An Ames assay, chromosome aberration test, and mouse lymphoma assay with 2,2’-azobis(isobutyronitrile) were used as a read-across to fulfill the data gap for the test substance. The underlying hypothesis for the read-across between the test substance and 2,2’-azobis(isobutyronitrile) is that the two substances share a common alerting group of azo which has the potential to drive outcomes in a battery of genotoxicity tests and as evidenced by other azo and azoxy compounds.Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

2,2’-Azobis(isobutyronitrile) was tested in an Ames assay with Salmonella typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA, with and without a metabolic activation system, the S9 mix, and Salmonella typhimurium TA97 without metabolic activation. Different dose levels of the test material were tested on each strain of Salmonella. 2,2’-Azobis(isobutyronitrile) did not induce any significant increase in the number of revertants, in the Salmonella strains used and no toxicity was observed.In the chromosome aberration test with Chinese hamster lung cells, 2,2’-azobis(isobutyronitrile) did not induce any significant increase in the number of cells with chromosome aberrations, in the number of polyploid cells or in the number of cells with endoreduplicated chromosomes in the presence or absence of the metabolic activation system, and no toxicity was observed. The mouse lymphoma assay showed that 2,2’-azobis(isobutyronitrile) did not induce any significant increase in the mutation frequency and no toxicity was observed in the presence or absence of the metabolic activation system. Taken together the weight of evidence of these studies support the conclusion that the test substance is not considered genetically active.


Justification for selection of genetic toxicity endpoint
In addition to the Ames assay with the test substance, multiple OECG guideline, GLP studies with the read-across chemical, 2,2’-azobis(isobutyronitrile) have been identified for this endpoint. In addition the study selected above, Ames assay, in vitro chromosome aberration assay, and mouse lymphoma assay with the read-across chemical, 2,2’-azobis(isobutyronitrile) are pertinent to the hazard conclusion for this endpoint.

Short description of key information:
The test substance was negative in the Ames assay. In addition, the read-across chemical, 2,2’-azobis(isobutyronitrile), was negative in the Ames assay, in vitro chromosome aberration assay, and mouse lymphoma assay.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance was negative in the Ames assay. In addition, the read-across chemical, 2,2’-azobis(isobutyronitrile), was negative in Ames assay, in vitro chromosome aberration assay, and mouse lymphoma assay. Based on the weight of evidence provided, the test substance does not need to be classified for genetic toxicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.