2,2'-azobis[2-methylbutyronitrile]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 01 June 2022 to 28 September 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

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:

2,2'-azobis[2-methylbutyronitrile]

EC Number:

236-740-8

EC Name:

2,2'-azobis[2-methylbutyronitrile]

Cas Number:

13472-08-7

Molecular formula:

C10H16N4

IUPAC Name:

2,2'-diazene-1,2-diylbis(2-methylbutanenitrile)

Test material form:

solid: granular

Details on test material:

Name: SAMPLE PERKADOX AMBN-GRANULES EUG
Chemical name: 2,2’-Azobis(2-methylbutyronitrile)
appearance: White granules,
Purity info 99.9%
expiry date: 22 Feb 2023 (1 year after production/analysis)
Storage; In refrigerator (2-8°C)
SADT: 45°C
Molecular Weight: 192.27
Handling: No specific handling conditions required
Contact with the following incompatible materials will result in hazardous decomposition:
Acids and bases
Iron
Copper
Reducing agents
Heavy metals
Rust
Do not mix with peroxide accelerators, unless under controlled processing.
Use only stainless steel 316, PP, polyethylene or glass-lined equipment.

Method

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Rat S9 homogenate prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
(CoA S9-fraction included)

Test concentrations with justification for top dose:

At a concentration of 1000 µg/mL, the test material precipitated in the culture medium.
Without and with S9-mix: 250, 500 and 1000 µg/mL culture medium (3 hours exposure time, 27 hours harvest time). Highest level based on solubility.
Without S9-mix: 20, 40, 60, 80, 100, 125, 150, 175, 200 and 250 µg/mL culture medium (24 hours exposure time, 24 hours harvest time). Highest level based on cytotoxicity.

Vehicle / solvent:

DMSO 1.0% (v/v).

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 ± 2 h
- Exposure duration/duration of treatment: 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma; 5 µg/mL) was added to the cells simultaneously with the test material at the 24 hours exposure time.
- Harvest time after the end of treatment: After 3 hours exposure to test material, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B and incubated for another 24 hours (1.5 times normal cell cycle).

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: Cytochalasine B (5 µg/mL), for 24 hrs.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
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 6.7% (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 and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
All slides were randomly coded before examination of micronuclei and scored. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not performed.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
The following criteria for scoring of binucleated cells were used:
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
• Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.

METHODS FOR MEASUREMENT OF CYTOTOXICITY:
cytokinesis-block proliferation index: A minimum of 500 cells (with a maximum deviation of 5%) per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells). The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).

Rationale for test conditions:

3 hours exposure time, 27 hours harvest time: Highest concentration based on solubility
24 hours exposure time, 24 hours harvest time: Highest concentration based on cytotoxicity (

Evaluation criteria:

An in vitro micronucleus 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 materials MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).

A test material is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
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 in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test material is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None 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) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Fisher’s exact test, one-sided, p < 0.05: Comparison number of micronuclei to control.
Cochran Armitage trend test for dose-related increase

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

For 3 hours exposure time, 27 hours harvest time both with and without S9-mix

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

3 hours exposure time, 27 hours harvest time

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

24 hours exposure time, 24 hours harvest time

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table 1           
Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with 2,2’-azobis[2-methylbutyronitrile] in the Dose-range Finding Test

3 hours exposure time, 27 hours harvest time

Without metabolic activation (-S9-mix)

Concentration µg/mL	Number of cells with ….nuclei			CBPI	% cytostasis
	1	2	3 or more
0	57	297	154	2.19	0
31.3	71	271	179	2.21	-1
62.5	57	314	142	2.17	2
125	59	294	167	2.21	-1
250	61	301	160	2.19	0
500	56	314	133	2.15	3
1000^1)	142	338	31	1.78	34

With metabolic activation (+S9-mix)

Concentration µg/mL	Number of cells with ….nuclei			CBPI	% cytostasis
	1	2	3 or more
0	43	295	173	2.25	0
31.3	50	256	210	2.31	-4
62.5	61	277	185	2.24	1
125	57	298	163	2.2	4
250	59	305	152	2.18	6
500	67	306	134	2.13	10
1000^1)	72	314	138	2.13	10

24 hours exposure time, 24 hours harvest time
Without metabolic activation (-S9-mix)

Concentration µg/mL	Number of cells with ….nuclei			CBPI	% cytostasis
	1	2	3 or more
0	81	351	185	2.17	0
62.5	91	378	106	2.03	12
125	175	317	14	1.68	42
250	399	125	1	1.24	79
500	486	27	0	1.05	95
1000^1)	^2)	^2)	^2)	^2)	^2)
1923^1)	^2)	^2)	^2)	^2)	^2)

Note: All calculations were performed without rounding off.
1) The test material precipitated in the culture medium
2) Cell lysis

Cytokinesis-Block Proliferation Index of Human Lymphocytes Cultures Treated with 2,2’-azobis[2-methylbutyronitrile] in the First Cytogenetic Assay

3 hours exposure time, 27 hours harvest time

Without metabolic activation (-S9-mix)

Concentration µg/mL	CBPI			Mean CBPI	% cytostatis
0	2.01	-	2.04	2.02	0
250	1.98	-	1.99	1.99	4
500	1.95	-	2.01	1.98	4
1000	1.83	-	1.93	1.88	14
0.20 MMC-C	1.73	-	1.8	1.76	26
0.25 MMC-C	1.67	-	1.76	1.72	30
0.05 Colch	1.68	-	1.84	1.76	26
0.1 Colch	1.31	-	1.37	1.34	67

With metabolic activation (+S9-mix)

Concentration µg/mL	CBPI			Mean CBPI	% cytostatis
0	2.06	-	2.13	2.09	0
250	2.11	-	2.12	2.12	-2
500	1.98	-	2	1.99	9
1000	1.84	-	1.92	1.88	19
7.5 CP	1.7	-	1.72	1.71	35
10 CP	1.55	-	1.6	1.58	47

Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with 2,2’-azobis[2-methylbutyronitrile] in the First Cytogenetic Assay

Without metabolic activation (-S9-mix)

Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei ^1)
		1000	1000	2000
		A	B	A+B
0	0	3	4	7
250	4	4	6	10
500	4	3	1	4
1000	14	3	3	6
0.20 MMC-C	26	21	22	43^****
0.05 Colch	26	10	6	16^*
0.1 Colch	67	18	21	39^****

With metabolic activation (+S9-mix)

Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei ^1)
		1000	1000	2000
		A	B	A+B
0	0	3	2	5
250	-2	2	4	6
500	9	5	1	6
1000	19	2	4	6
7.5 CP	35	16	12	18^****

*)  Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.
¹⁾ 1000-1005 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

 

Cytokinesis-Block Proliferation Index of Human Lymphocyte Cultures Treated with 2,2’-azobis[2-methylbutyronitrile] in the Cytogenetic Assay 2A

24 hours exposure time, 24 hours harvest time
Without metabolic activation (-S9-mix)

Concentration µg/mL	CBPI			Mean CBPI	% cytostatis
0	1.83	-	1.86	1.84	0
20	1.8	-	1.85	1.82	2
40	1.66	-	1.82	1.74	12
60	1.65	-	1.68	1.67	21
80	1.53	-	1.6	1.56	33
100	1.57	-	1.63	1.6	29
125	1.48	-	1.53	1.5	40
150	1.42	-	1.52	1.47	44
175	1.33	-	1.39	1.36	57
200	1.3	-	1.35	1.32	61
250	1.19	-	1.2	1.2	77
0.125 MMC-C	1.61	-	1.74	1.68	19
0.15 MMC-C	1.62	-	1.65	1.63	25
0.01 Colch	1.67	-	1.73	1.7	17
0.05 Colch	1.03	-	1.03	1.03	97

Number of Binucleated Cells with Micronuclei of Human Lymphocyte Cultures Treated with 2,2’-azobis[2-methylbutyronitrile] in the Second Cytogenetic Assay

24 hours exposure time, 24 hours harvest time
Without metabolic activation (-S9-mix)

Concentration (µg/mL)	Cytostasis (%)	Number of binucleated cells with micronuclei ^1)
		1000	1000	2000
		A	B	A+B
0	0	4	3	7
20	2	3	2	6
80	33	2	1	3
175	57	3	1	4
0.125 MMC-C	19	15	12	27^***
0.01 Colch	17	2	2	4
0.05 Colch	97	6 ^2)	3 ^2)	9^***

*) Significantly different from control group (Fisher’s exact test), * P < 0.05, ** P < 0.01, *** P < 0.001 or **** P < 0.0001.

¹⁾ 1000 binucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.

²⁾ 238 and 125 binucleated cells were scored for the presence of micronuclei, respectively.

Applicant's summary and conclusion

Conclusions:

2,2’-azobis[2-methylbutyronitrile] is not clastogenic or aneugenic in human lymphocytes.

Executive summary:

2,2’-azobis[2-methylbutyronitrile] was evaluated for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of the test material was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline (OECD 487, July 2016). The vehicle of the test material was dimethyl sulfoxide.

In the first cytogenetic assay, the test material was tested up to 1000 µg/mL for a 3-hour exposure time with a 27-hour harvest time in the absence and presence of S9-fraction. The test material precipitated in the culture medium at this dose level.

In the second cytogenetic assay, the test material was tested up to 175 µg/mL for a 24-hour exposure time with a 24-hour harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of binucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C, colchicine and cyclophosphamide all produced a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of binucleated cells with micronuclei 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.

The test material did not induce a statistically significant or biologically relevant increase in the number of binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments.

In conclusion, this test is valid and 2,2’-azobis[2-methylbutyronitrile] is not clastogenic or aneugenic in human lymphocytes under the experimental conditions of this study.