Dimethyl 2,2'-azobis(2-methylpropionate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2015 - June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: B.49 (In Vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl 2,2'-azobis(2-methylpropionate)

EC Number:

219-976-6

EC Name:

Dimethyl 2,2'-azobis(2-methylpropionate)

Cas Number:

2589-57-3

Molecular formula:

C10H18N2O4

IUPAC Name:

dimethyl 2,2'-azobis(2-methylpropionate)

Test material form:

solid: flakes

Method

Target gene:

whole genom is targeted

Species / strain

Species / strain / cell type:

lymphocytes: peripheral

Details on mammalian cell type (if applicable):

The blood sample was obtained from a healthy donor who neither smokes nor receives
medication. The following donor was chosen for the experimental part:
female, 34 years old

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Nominal concentration of test item solution (mg/mL): 400, 200, 100, 50, 25, 12.5, 6.3, 3.2

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item was determined in a non-GLP pre-test. The test item was sufficiently soluble in DMSO.
Therefore, DMSO was chosen as solvent.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 47.5 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 18 h
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 22 h (Exposure duration + expression time)

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin
B

STAIN (for cytogenetic assays): 10 % solution of Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension onto a clean microscope slide.
The cells were then stained with a 10 % solution of Giemsa. All slides were independently
coded before microscopic analysis.

NUMBER OF CELLS EVALUATED: at least 500 cells per culture, cytokinesis-block proliferation index
At least 1000 binucleate cells per culture were scored for micronuclei

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Only cells with sufficiently
distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included
in the analysis.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was calculated as reduction in CBPI compared to the CBPI of the concurrent
solvent control

OTHER EXAMINATIONS:
-none

- OTHER:

Rationale for test conditions:

according to Guideline

Evaluation criteria:

The test item is considered to have genotoxic effects if:
 At least one test concentration shows a statistically significant increase of micronucleate
cells compared to the concurrent solvent control.
 In at least one experimental condition a dose-related increase of micronucleate cells
can be observed.
 Any of the results lies outside the range of the historical laboratory control data for solvent
controls.

Statistics:

The number of binucleate cells with micronuclei in each treatment group was compared
with the solvent control. Statistical significance was tested using Fisher’s exact test at the
five per cent level (p 0.05)
For positive controls with high values of binucleate cells with micronuclei, the chi-squaretest
was used

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: peripheral

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, under the experimental conditions reported, dimethyl-2,2'-
azobisisobutyrate induced the formation of micronuclei in human lymphocytes in
vitro.
The test item dimethyl-2,2'-azobisisobutyrate is considered as “genotoxic under the
conditions of the test”.

Executive summary:

One valid experiment was performed.

This study was performed to assess the genotoxic potential of dimethyl-2,2'-

azobisisobutyrate to induce formation of micronuclei in human lymphocytes cultured in

vitro in the absence and the presence of an exogenous metabolic activation system (liver

S9 mix from male rats, treated with Aroclor 1254).

The test item was dissolved in DMSO. A stock solution with a concentration of 400 mg/mL

(corresponding to a final test concentration of 2 mg/mL) and thereof a geometric series of

dilutions was prepared.

Human lymphocytes, on whole blood culture, were stimulated to divide by addition of phytohaemagglutinin

and exposed to solvent control, test item and positive control.

After exposure period and expression time in growth medium (= culture harvest time), the

cells were harvested and slides were prepared. Then, the proportion of binucleate cells

containing micronuclei was determined.

The following schedule was observed:

Procedure	Exp. I
Metabolic activation	Without S9 mix	With S9 mix
Exposure period	4 h	4 h
Expression time in growth medium	18 h	18 h
Culture harvest time	22 h	22 h
Concentrations selected for scoring of micronuclei	2, 0.25 and 0.03 mg/mL	2, 1 and 0.5 mg/mL

One valid experiment with 2 experimental conditions - without and with metabolic activation

- was performed. All cell cultures were set up in duplicates. In order to assess the toxicity

of the test item to cultivated human lymphocytes, the cytokinesis-block proliferation

index was calculated for all cultures treated with solvent control, positive control and test

item. On the basis of these data, the concentrations indicated in the table above were selected

for micronuclei scoring.