2,2'-azobis[2-methylbutyronitrile]

Genetic toxicity: in vitro

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

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1999

Reference 2

Reference Type:

other: english study summary

Title:

Unnamed

Year:

1999

Reference 3

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-dimethyl-2,2'-azodipropiononitrile

EC Number:

201-132-3

EC Name:

2,2'-dimethyl-2,2'-azodipropiononitrile

Cas Number:

78-67-1

Molecular formula:

C8H12N4

IUPAC Name:

2,2'-diazene-1,2-diylbis(2-methylpropanenitrile)

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 / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 97

Additional strain / cell type characteristics:

not applicable

Species / strain 2

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 results

Test results 1

Species / strain:

S. typhimurium, 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

Test results 2

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

Test results 3

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.