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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

two in vitro studies according to resp. Guideline available:

in vitro reverse mutation testing in bacteria

in vitro Micronucelus Test in mammalian cells.

Further in vitro tests are not performed due to clear results in available studies.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

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

Target gene:

whole genom is targeted

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.

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04.04.2003 - 23.04.2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine and/or trypthophane

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:

rat liver S9 mix

Test concentrations with justification for top dose:

1.2, 4.9, 20, 78, 313, 1250 and 50001.1 g/plate
In the dose-finding test, the growth inhibition by the test substance was observed at 5000 µg/plate in
all strains both with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the information from the sponsor, because the test substance was insoluble in water and
dissolved in DMSO and acetone at over 0.5g/g, DMSO was used as the solvent for the test substance.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

treated as negative controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide, 2-Aminoanthracene, 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine*2HCl

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation;
- Cell density at seeding (if applicable): not applicable

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): not applicable

STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable

NUMBER OF CELLS EVALUATED: not applicable

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition expressed by colony numbers

OTHER EXAMINATIONS:
not applicable

Rationale for test conditions:

standard test conditions

Evaluation criteria:

In the dose-finding test and the main test, if the number of revertant colonies on the test plates
increased significantly in comparison with that on the control plates (based on twice as many as that of
the negative control), and dose-response and reproducibility were also observed, the test substance was
judged positive.

Statistics:

Statistical analysis was not done

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity was observed at the highest dose level.

Conclusions:

From the results described above, it is concluded that Dimethyl-2,2' -azobis(2-methylpropionate) is
mutagenic in the bacterial reverse mutation assay carried out under the experimental conditions.

Executive summary:

Mutagenicity potential of Dimethyl=2,2’- azobis(2-methylpropionate) was assessed with Salmonella

typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA.

In this study, dose-related increase in the number of revertant colonies more than twice in comparison

with that of the negative control and reproducibility were observed in E. coli WP2 uvrA with metabolic

activation.

From the above, it is judged that Dimethyl=2,2'-azobis(2-methylpropionate) has mutagenicity forward to

bacteria under the described study conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

no in vivo tests available. further information needed.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Mode of Action Analysis / Human Relevance Framework

based on the available results, no clear mode of action can be identified.

The substance induces mutations in the bacterial strain E. Coli with metabolic activation

and induces micronuclei in mammalian cells with and without metabolic activation

Justification for classification or non-classification

The available information is conclusive and sufficient for classification as Mutagenic cat. 2.

However further information has to be generated to conclude on classification or non-classification as Mutagenic cat.1