2,2'-azobis[4-methoxy-2,4-dimethylvaleronitrile]

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:

19. May 2016 - 15 Nov. 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-azobis[4-methoxy-2,4-dimethylvaleronitrile]

EC Number:

239-593-8

EC Name:

2,2'-azobis[4-methoxy-2,4-dimethylvaleronitrile]

Cas Number:

15545-97-8

Molecular formula:

C16H28N4O2

IUPAC Name:

2,2'-azobis[4-methoxy-2,4-dimethylvaleronitrile]

impurity 1

Reference substance name:

IUPAC-name not available, the reference substance may consist of one or more impurities

Molecular formula:

not applicable

IUPAC Name:

IUPAC-name not available, the reference substance may consist of one or more impurities

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TY2031
- Expiration date of the lot/batch: 17 February 2017
- Purity test date: not stated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in freezer (=< -15 °C) protected from light
- Stability under test conditions: not stated
- Solubility and stability of the test substance in the solvent/vehicle: not stated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not stated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
A solubility test was performed. Batch TY2031 was soluble in dimethyl
sulfoxide of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany) at
concentrations of 16.4 mg/ml and below but formed a suspension at concentrations of 51.2
mg/ml and above in the dose range finding study and first cytogenetic assay. In the second
cytogenetic assay, the test item was dissolved at concentrations of 12.5 mg/ml and below but
formed a suspension at concentrations of 15.0 mg/ml and above. The stock solution was
treated with ultrasonic waves to obtain a homogeneous suspension. To protect the test item
from light, amber coloured glassware or aluminium-foil was used.
Test item concentrations were used within 2.5 hours after preparation.

Method

Target gene:

no target gene according to protocol

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking volunteers
- Suitability of cells: Cultured peripheral human lymphocytes were used as test system. Peripheral human
lymphocytes are recommended in international guidelines (e.g. OECD, EC).
- Cell cycle length, doubling time or proliferation index: AGT = 12.7 h - 13.8 h
- Sex, age and number of blood donors if applicable: age 30 - 35
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Number of passages if applicable: 0
- Methods for maintenance in cell culture if applicable: not stated
- Modal number of chromosomes: 46 +-2
- Normal (negative control) cell cycle time: not stated

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with
20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Life technologies), L-glutamine
(2 mM) (Life technologies), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively)
(Life technologies) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).
5.0 ± 0.5% CO2
- Properly maintained: not stated
- Periodically checked for Mycoplasma contamination: not stated
- Periodically checked for karyotype stability: not stated
- Periodically 'cleansed' against high spontaneous background: not stated

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 homogenate

Test concentrations with justification for top dose:

Dose-range finding: 17, 52, 164, 512, 1000 µg/ml
first experiment: 52, 164, 512 µg/ml
second experiment: 52, 164, 512 µg/ml (24 h exposure)
10, 25, 50, 75, 100, 125, 150 µg/ml (48 h exposure)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: based on solubility test and well-known vehicle

Controls

Untreated negative controls:

no

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): not determined

DURATION
- Preincubation period: 48 +-2 h
- Exposure duration: 3 h
- 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): 24 h

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of
96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked
with the Charles River Den Bosch study identification number and group number. At least
two slides were prepared per culture. Slides were allowed to dry and thereafter stained for
10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter
slides were rinsed in water and allowed to dry. The dry slides were automatically embedded
in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Histolab,
Gothenburg, Sweden) and mounted with a coverslip in an automated cover slipper (Leica
Microsystems B.V., Rijswijk, The Netherlands).

NUMBER OF CELLS EVALUATED: at least 1000 for mitotic index

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 150 per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Rationale for test conditions:

according to Guideline

Evaluation criteria:

Acceptability of the assay
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).

Evaluation
A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, onesided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat
Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis
of the data.
Fisher’s exact test, one-sided, p < 0.05

In case the Fisher’s exact test shows that there are statistically significant differences between
one or more of the test item groups and the vehicle control group a Cochran Armitage trend
test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: cultured peripheral human

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: cultured peripheral human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not stated
- Effects of osmolality: not stated
- Evaporation from medium: not stated
- Water solubility: not stated
- Precipitation: At a concentration of 512 μg/ml the test item precipitated in the culture medium.
- Definition of acceptable cells for analysis: not stated
- Other confounding effects: not stated

RANGE-FINDING/SCREENING STUDIES:
see section "any other information on results incl. tables"


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see section "any other information on results incl. tables"

- Negative (solvent/vehicle) historical control data: see section "any other information on results incl. tables"


ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: based on number of metaphases and calculation of mitotic index

Any other information on results incl. tables

Results of Range finding study

1000 cells scored per concentration

Test item concentration (µg/ml)	Number of metaphases	Percentage of control
without metabolic activation (-S9 -mix)
3 h exposure time, 24 h fixation time
control	56	100
17	65	116
52	78	131
164	64	114
512	85	152
1000	69	123
24 h exposure time, 24 h fixation time
control	37	100
17	42	114
52	45	124
164	32	86
512	21	57
1000	23	62
48 h exposure time, 48 h fixation time
control	55	100
17	52	96
52	32	59
164	15	28
512	11	20
1000	9	16
with metabolic activation (+S9 -mix)
control	67	100
17	69	102
52	57	85
164	79	118
512	64	96
1000	66	99

Positive historical control data

	3 hours exposure time				24 hours exposure time		48 hours exposure time
	Gaps included		Gaps excluded		Gaps included	Gaps excluded	Gaps included	Gaps excluded
	+ S9-Mix	-S9-Mix	+ S9-Mix	-S9-Mix	-S9-Mix	-S9-Mix	-S9-Mix	-S9-Mix
Mean number of aberrant cells per 100 cells	33.08	29.34	32.44	28.90	30.61	29.71	35.57	34.54
SD	12.90	13.35	12.92	13.42	13.45	13.77	15.00	15.11
N	254	254	254	254	250	250	248	248
Upper control limit (95 % control limits)	58.67	53.21	57.86	52.60	57.38	57.09	64.87	63.82
Lowercontrol limit (95 % control limits)	7.48	5.47	7.01	5.19	3.84	2.34	6.26	5.26

Negative historical control data

	3 hours exposure time				24 hours exposure time		48 hours exposure time
	Gaps included		Gaps excluded		Gaps included	Gaps excluded	Gaps included	Gaps excluded
	+ S9-Mix	-S9-Mix	+ S9-Mix	-S9-Mix	-S9-Mix	-S9-Mix	-S9-Mix	-S9-Mix
Mean number of aberrant cells per 100 cells	0.79	0.80	0.73	0.77	0.78	0.63	0.99	0.74
SD	1.03	1.11	1.03	1.12	1.08	1.02	1.24	1.13
N	256	254	256	254	250	250	248	248
Upper control limit (95 % control limits)	3.32	3.21	3.21	3.10	3.20	2.75	4.06	3.12
Lowercontrol limit (95 % control limits)	-1.73	-1.62	-1.75	-1.57	-1.64	-1.48	-2.08	-1.64

Applicant's summary and conclusion

Conclusions:

2,2’-Azobis (4-methoxy-2,4-dimethyl valeronitrile) is clastogenic in human lymphocytes under the experimental conditions described in this report. The clastogenic activity is confined only to incubations in the absence of S9-mix at the 24 h exposure time.

Executive summary:

Evaluation of the ability of 2,2’-Azobis (4-methoxy-2,4-dimethyl valeronitrile) to induce

chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of 2,2’-Azobis (4-methoxy-2,4-dimethyl valeronitrile) on the

number of chromosome aberrations in cultured peripheral human lymphocytes in the

presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone

induced rat liver S9-mix). The possible clastogenicity of the test item was tested in two

independent experiments.

The study procedures described in this report are in compliance with the most recent OECD

guideline.

Batch TY2031 of the test item was a white powder. Batch TY2031 was soluble in dimethyl

sulfoxide at concentrations of 16.4 mg/ml and below but formed a suspension at

concentrations of 51.2 mg/ml and above.

In the first cytogenetic assay, the test item was tested up to 512 μg/ml for a 3 h exposure time

with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. The test item

precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test item was tested up to 512 μg/ml for a 24 h

continuous exposure time with a 24 h fixation time and up to 100 μg/ml for a 48 h continuous

exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was

reached at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was

within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically

significant increase in the incidence of cells with chromosome aberrations. In addition, the

number of cells with chromosome aberrations found in the positive control cultures was

within the 95% control limits of the distribution of the historical positive control database. It

was therefore concluded that the test conditions were adequate and that the metabolic

activation system (S9-mix) functioned properly.

In the first cytogenetic assay, the test item did not induce any statistically significant or

biologically relevant increase in the number of cells with chromosome aberrations in the

absence and presence of S9-mix.

At the 24 hours continuous exposure time, the test item induced a statistically significant

increase in the number of cells with chromosome aberrations at the highest dose level. The

number of cells with chromosome aberrations was outside the 95% control limits of the

distribution of the historical negative control database. In addition, a statistical significant

dose related trend was observed. These results indicate that Batch TY2031 is positive in the

in vitro chromosome aberration study and might be considered a clastogenic compound.

At the 48 h continuous exposure time, the test item did not induce any statistically significant

or biologically relevant increase in the number of cells with chromosome aberrations.

No biologically relevant effects of the test item on the number of polyploid cells and cells

with endoreduplicated chromosomes were observed both in the absence and presence of S9-

mix. Therefore it can be concluded that the test item does not disturb mitotic processes and

cell cycle progression and does not induce numerical chromosome aberrations under the

experimental conditions described in this report.