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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 473)

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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'-dimethyl-2,2'-azodipropiononitrile

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 / cell type:

mammalian cell line, 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

Controls

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

Results and discussion

Test results

Species / strain:

mammalian cell line, 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'.

Any other information on results incl. tables

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     -

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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. According to the criteria laid down in Council Directive 67/548/EEC (and subsequent adaptations), the test item 2,2'-AZOBIS(ISOBUTYRONITRILE) is considered as non-mutagenic and is not classified.