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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was tested in an Ames Assay and Mammalian Cell Micronucleus Assay according to Guidelines (OECD 471 and OECD 487 respectively)

Information on Gene Mutation in Mammalian Cells is provided by Predictions obtained with OECD QSAR Toolbox. Predictions are provided from two categories formed by application of

the profiles "organic functional groups: Azo" and "organic functional groups: Nitrile". This approach was followed as no category of substances was available having both (or all*) profiles of the substance.

*The substance has the profile "organic functional groups: Azo, Nitrile, Azonitrile, Isopropyl and Alkane, branched with tertiary carbon"

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25. October 2017 - 16. February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Qualifier:
according to
Guideline:
other: EU Method B.49
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
no single target gene, assay aims for whole genom
Species / strain / cell type:
primary culture, other: peripheral lymphocytes
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy donors who neither smoke nor receive medication.
Cytokinesis block (if used):
cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
S9 (liver enzyme mixture used for the test with metabolic activation) was obtained from
Trinova Biochem GmbH, Gießen, and stored at – 80 ± 5°C.
Test concentrations with justification for top dose:
200 / 100 / 50 / 25 / 12.5 mg/mL

The solubility of the test item was determined in a non-GLP pre-test using cell culture medium,
dimethyl sulfoxide (DMSO) and ethanol absolute. The test item was insoluble in medium,
DMSO and ethanol at the required concentrations (20 mg/mL in medium and
400 mg/mL in DMSO and ethanol) but completely soluble in DMSO at the next lower concentration
(200 mg/mL).
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
7.2.1 Cell Cultivation
The blood cultures were set up in defined time intervals within 24 h after collection in sterile
culture vessels, each containing 1 part of heparinised blood and 9 parts of complete
culture medium RPMI 1640 for cell proliferation. The cultures were incubated for 72 h at
37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
7.2.2 Cell Treatment
After the initial cell cultivation, duplicate cultures were prepared for each test group. After
centrifugation (10 min, 500 * g), the cells were resuspended in serum free RPMI 1640 and
solvent control, positive control or the single test item concentrations were added.
In the case of metabolic activation, 50 μL S9 mix per mL medium were used. The cell cultures
were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 4 h
(exposure period)
After the exposure time of 4 h, the cells were spun down by gentle centrifugation for 5 min
(500 * g). The supernatant was discarded, the cells were re-suspended in 5 mL Saline G
and centrifuged again. The washing procedure was repeated once as described.
After washing, the cells were resuspended in complete culture medium RPMI 1640, cytochalasin
B (final concentration 5 μg/ml) was added and the cells were incubated at
37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 19 h until preparation.
In experiment II without metabolic activation, 72 h after seeding, the blood cultures were
centrifuged (10 min, 500 * g). The cell pellet was re-suspended in complete culture medium
RPMI 1640, cytoB (final concentration 5 μg/mL) and solvent control, positive control or
the test item concentrations were added. The exposure duration was 23 h.
7.2.3 Harvesting Procedure
Each cell culture was harvested and processed separately. The cells were spun down by
gentle centrifugation (10 min, 500 * g). The supernatant was discarded and the cells were
re-suspended in 12 mL hypotonic KCl solution. The cell suspension was allowed to stand
for 10 min at room temperature (20 ± 5°C). After removal of the hypotonic solution by centrifugation
(10 min, 500 * g), the cell pellet was fixed with a mixture of methanol and glacial
acetic acid (3:1). After fixation at 2 – 8 °C for minimum 30 min, the cell suspension was
spun down by gentle centrifugation (10 min, 500 * g), the supernatant was discarded and
the cell pellet was re-suspended in fixative again. The washing procedures were repeated
until the cell pellet was white.
7.2.4 Preparation of Slides
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.
7.2.5 Determination of the Cytokinesis-Block Proliferation Index
In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per culture)
was determined in order to assess the cytotoxicity of the test item. From these determinations,
the test item concentrations which were evaluated for scoring of micronuclei
were defined.
7.2.6 Determination of Binucleated Cells with Micronuclei
At least 1000 binucleated cells per culture were scored for micronuclei. Only cells with sufficiently
distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included
in the analysis.
Rationale for test conditions:
according to Guideline
Evaluation criteria:
The test item is considered to have no genotoxic effects if:
 Neither a statistically significant nor a concentration-related increase of the number of
micronucleate cells in the evaluated test concentrations is observed.
 The obtained results lie within the range of the historical laboratory control data for solvent
controls, considering also e.g. 95.5 % control limits where appropriate.
The test item is considered to have genotoxic effects if all of the following conditions are
met:
 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 using trend analysis.
 Any of the results lies outside the range of the historical laboratory control data for solvent
controls, considering also e.g. 95.5 % control limits where appropriate.
Statistics:
The number of binucleated 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 binucleated cells with micronuclei, the chi-squaretest was used
Key result
Species / strain:
primary culture, other: human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test item 2,2'-Azobis(2,4-dimethylvaleronitrile) is considered as “not genotoxic” under
the conditions of the test.
Executive summary:

This study was performed to assess the genotoxic potential of 2,2'-Azobis(2,4-

dimethylvaleronitrile) to induce formation of micronuclei in human lymphocytes cultured in

vitro in absence and presence of an exogenous metabolic activation system (liver S9 mix

from male rats, treated with Aroclor 1254).

The test item was dissolved in DMSO to prepare a stock solution with a concentration of

200 mg/mL, corresponding to the highest concentration (1000 μg/mL) in the test. In addition,

a geometric series of 4 dilutions was prepared from the stock solution.

Two valid experiments were performed.

Human peripheral blood lymphocytes, on whole blood culture, were stimulated to divide by

addition of phytohaemagglutinin and exposed to solvent control, test item and positive control.

After the culture harvest time, the cells were harvested and slides were prepared. Then,

the proportion of cells containing micronuclei was determined.

Two independent experiments were performed. In each experiment, 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 (CBPI) was calculated for all cultures

treated with solvent control, positive control and test item. On the basis of the data of the

cytokinesis-block proliferation index, the appropriate concentrations were selected for micronuclei

scoring.

None of the test item concentrations induced a cytotoxic effect in experiment I and II.

In experiment I without metabolic activation, precipitates were observed at the two highest

test item concentrations (1000 μg/mL and 500 μg/mL) at the end of treatment. In the approach

with metabolic activation, precipitates were clearly visible at the concentrations

1000 μg/mL, 500 μg/mL and 250 μg/mL. According to the OECD 487, 250 μg/mL was

chosen as highest analysable concentration in the approach with metabolic activation and

500 μg/mL in the approach without metabolic activation. In experiment II without metabolic

activation, no precipitates were observed up to the maximum test item concentration.

Therefore, the three highest test item concentrations were evaluated for micronuclei.

Neither a statistically significant nor a biologically relevant increase in the number of binucleated

cells containing micronuclei at the evaluated concentrations was observed.

All positive control compounds caused large, statistically significant increases in the proportion

of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported, 2,2'-Azobis(2,4-

dimethylvaleronitrile) does not induce the formation of micronuclei in human lymphocytes

in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
Jan. 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: results derived from OECD QSAR Toolbox, with adequate documentation
Justification for type of information:
In General: The OECD QSAR Toolbox is a software providing databases and tools to characterise a given substance by structure and/or activity ("profiling").
In consequence the databases are screened for analogous with same profilers and categories are formed having same structural or activity profilers.
Therefore the applied approach can be interpreted as Structure-Activity-Relationship.

1. SOFTWARE
OECD QSAR Toolbox 4.2

2. MODEL (incl. version number)

no specific model used.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL

CC(C)CC(C)(N=NC(C)(CC(C)C)C#N)C#N

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
The software is released by OECD and used by ECHA.

5. APPLICABILITY DOMAIN
during category formation and subcategorisation, a "domain" of analogous is build having same profils as the target.

6. ADEQUACY OF THE RESULT
The predicted endpoint "in vitro Mammalian Cell Gene Mutation" is adequate to fulfill the information request "Mammalian Cell Gene Mutation"

For further information, please refer to the attached documentation.
Qualifier:
no guideline followed
Principles of method if other than guideline:
OECD QSAR Toolbox is used as a standard program for Read Across
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
not applicable for Read Across with OECD QSAR Toolbox
Cytokinesis block (if used):
not applicable for Read Across with OECD QSAR Toolbox
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver S9
Test concentrations with justification for top dose:
not applicable for Read Across with OECD QSAR Toolbox
Vehicle / solvent:
not applicable for Read Across with OECD QSAR Toolbox
Details on test system and experimental conditions:
not applicable for Read Across with OECD QSAR Toolbox
Rationale for test conditions:
not applicable for Read Across with OECD QSAR Toolbox
Evaluation criteria:
not applicable for Read Across with OECD QSAR Toolbox
Statistics:
not applicable for Read Across with OECD QSAR Toolbox
Key result
Species / strain:
not specified
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
other: not applicable
Untreated negative controls validity:
other: not applicable
True negative controls validity:
other: not applicable
Positive controls validity:
other: not applicable
Key result
Species / strain:
not specified
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
other: not applicable
Untreated negative controls validity:
other: not applicable
True negative controls validity:
other: not applicable
Positive controls validity:
other: not applicable
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
The substance is predicted to be negative for in vitro Mammalian Cell Gene Mutation.
Executive summary:

The genotoxic potential of the substance 2,2'-Azobis(2,4-dimethylvaleronitrile)

, CAS 4419-11-8, has been assessed by using Read Across to analogeous substances.

The Read Across was performed with the well established programm OECD QSAR Toolbox.

A category was formed based on the profile "organic functional groups" where members fulfill the criteria for "Azo".

Predictions for the Endpoints "Genotoxicity, in vitro, Mammalian Cell Gene Mutation, with S9",

"Genotoxicity, in vitro, Mammalian Cell Gene Mutation, with",

"Genotoxicity, in vitro, Mammalian Cell Gene Mutation, without" and

"Genotoxicity, in vitro, Mammalian Cell Gene Mutation, with and without"

was made.

(As data is not entered uniformly in databases implemented in OECD QSAR Toolbox, Information on metabolic activation is expressed for example as "with S9" or "with", having the same meaning)

The substance is predicted to be negative for in vitro Mammalian Cell Gene Mutation in.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
Jan. 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: results derived from OECD QSAR Toolbox, with adequate documentation
Justification for type of information:
In General: The OECD QSAR Toolbox is a software providing databases and tools to characterise a given substance by structure and/or activity ("profiling").
In consequence the databases are screened for analogous with same profilers and categories are formed having same structural or activity profilers.
Therefore the applied approach can be interpreted as Structure-Activity-Relationship.

1. SOFTWARE
OECD QSAR Toolbox 4.2

2. MODEL (incl. version number)

no specific model used.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL

CC(C)CC(C)(N=NC(C)(CC(C)C)C#N)C#N

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
The software is released by OECD and used by ECHA.

5. APPLICABILITY DOMAIN
during category formation and subcategorisation, a "domain" of analogous is build having same profils as the target.

6. ADEQUACY OF THE RESULT
The predicted endpoint "in vitro Mammalian Cell Gene Mutation" is adequate to fulfill the information request "Mammalian Cell Gene Mutation"

For further information, please refer to the attached documentation.
Qualifier:
no guideline followed
Principles of method if other than guideline:
OECD QSAR Toolbox is used as a standard program for Read Across
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
not applicable for Read Across with OECD QSAR Toolbox
Cytokinesis block (if used):
not applicable for Read Across with OECD QSAR Toolbox
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver S9
Test concentrations with justification for top dose:
not applicable for Read Across with OECD QSAR Toolbox
Vehicle / solvent:
not applicable for Read Across with OECD QSAR Toolbox
Details on test system and experimental conditions:
not applicable for Read Across with OECD QSAR Toolbox
Rationale for test conditions:
not applicable for Read Across with OECD QSAR Toolbox
Evaluation criteria:
not applicable for Read Across with OECD QSAR Toolbox
Statistics:
not applicable for Read Across with OECD QSAR Toolbox
Key result
Species / strain:
not specified
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
other: not applicable
Untreated negative controls validity:
other: not applicable
True negative controls validity:
other: not applicable
Positive controls validity:
other: not applicable
Key result
Species / strain:
not specified
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
other: not applicable
Untreated negative controls validity:
other: not applicable
True negative controls validity:
other: not applicable
Positive controls validity:
other: not applicable
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
The substance is predicted to be negative for in vitro Mammalian Cell Gene Mutation.
Executive summary:

The genotoxic potential of the substance 2,2'-Azobis(2,4-dimethylvaleronitrile)

, CAS 4419-11-8, has been assessed by using Read Across to analogeous substances.

The Read Across was performed with the well established programm OECD QSAR Toolbox.

A category was formed based on the profile "organic functional groups" where members fulfill the criteria for "Nitrile".

A prediction for the Endpoints "Genotoxicity, in vitro, Mammalian Cell Gene Mutation, with S9", "Genotoxicity, in vitro, Mammalian Cell Gene Mutation, without S9" and

"Genotoxicity, in vitro, Mammalian Cell Gene Mutation, with and without"

was made.

(As data is not entered uniformly in databases implemented in OECD QSAR Toolbox, Information on metabolic activation is expressed for example as "with S9" or "with", having the same meaning)

The substance is predicted to be negative for in vitro Mammalian Cell Gene Mutation.

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

Genetic toxicity in vivo

Description of key information

as no genotoxic effects are present for the substance, no in vivo studies are needed.

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

as no genotoxic effects are present, no mode of action can be identified.

Additional information

Justification for classification or non-classification

The available information is conclusive but not sufficient for classification.