2,2'-azobis[2-methylbutyronitrile]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All in vitro studies on 2,2'-Azodi(2-methylbutyronitrile) (AMBN) (two Ames, MN in HL), and on read across substance AIBN (Ames, MLA, Chrome ab) are negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

other justification

Justification for data waiving:

other:

Reference 2

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

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

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

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

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

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.

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.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 May 2022 - 19 May 2022

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 June 2020

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: male Sprague Dawley rats injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 mL Milli-Q water; 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution ; 1 mL 0.33 M KCl solution. The above solution was filter (0.22 μm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

- volume of S9 mix and S9 in the final culture medium: 0.5 mL

- quality controls of S9: Each S9 batch is characterized with the mutagens benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Test concentrations with justification for top dose:

DOSE-RANGE FINDING TEST
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of the test material used in the subsequent mutation assays was 5000 μg/plate.
At least five different doses (increasing with approximately half-log steps) of the test material were tested in triplicate in each strain in the absence and presence of S9-mix.

FIRST EXPERIMENT: DIRECT PLATE ASSAY
The above mentioned dose-range finding study with two tester strains is reported as a part of the direct plate assay. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the remaining strains
- TA1535, TA1537 and TA98: 52, 164, 512, 1600 and 5000 μg/plate (with and without metabolic activation, 5% v/v S9 fraction)

SECOND EXPERIMENT: PRE-INCUBATION ASSAY
Based on the results of the first mutation assay, the test material was tested up to the dose level of 5000 μg/plate in all tester strains:
- TA1535, TA1537, TA98, TA100 and WP2uvrA: 52, 164, 512, 1600 and 5000 μg/plate (with and without metabolic activation, 5% v/v S9 fraction)


ADDITIONAL EXPERIMENT: PRE-INCUBATION ASSAY
To verify the result obtained in tester strain TA1535 in the absence of S9-mix (second experiment), an additional experiment was performed. In the additional experiment, the test material was tested up to concentrations of 5000 μg/plate in the absence of S9-mix:
-TA1535: 52, 164, 512, 1600 and 5000 μg/plate (without metabolic activation)

Vehicle / solvent:

- Vehicle/solvent used: dimethyl sulfoxide (DMSO)

- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test material formed a clear colourless solution in DMSO. Test material concentrations were used within 3.5 hours after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 ; 2-aminoanthracene (2AA)

Remarks:

See Table 1 and 2 under "Any other information on materials and methods incl. tables" for details on positive control substances

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: three

METHOD OF TREATMENT/ EXPOSURE:
- Cell density: 10^9 cells/mL
- Test substance added in agar (plate incorporation) - Experiment 1; pre-incubation - Experiment 2, Additional experiment

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: Pre-incubation assay (Second + additional experiment): 30 ± 2 minutes
- Exposure duration/duration of treatment: 48 ± 4 h
- Temperature: 37.0 ± 1.0 °C (actual range 34.7 – 40.5°C).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The revertant colonies were counted automatically with the colony counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.

Rationale for test conditions:

Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. The test item was soluble in the selected vehicle. The number of cultures and the strains selected are in agreement with the OECD TG 471.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.

b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Exp. 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Exp. 2

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Exp. 2

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

In the first experiment a positive response. As scientifically unlikely, the most likely cause is a slight cross-contamination with the positive control. In repeat response was negative (See additional comments)

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Additional experiment

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Exp. 1 + 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Exp. 1 + 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Exp. 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Exp. 2

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Exp. 2

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest tested concentration, evidenced by a decrease in the number of revertants

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Exp. 1 + 2

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle 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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
First Experiment (dose-range finding study is reported as part of the first experiment) and and additional experiment:
No precipitation of the test material on the plates was observed in any tester strain.
Second experiment:
Precipitation of the test material on the plates was observed at concentrations of 5000 μg/plate in tester strain WP2uvrA in the absence of S9-mix. Precipitation of the test material on the plates was not observed in any other tester strains.


RANGE-FINDING/SCREENING STUDIES:
The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly..

- Signs of toxicity
First Experiment (dose-range finding study is reported as part of the first experiment) and and additional experiment: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
Second Experiment: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the absence of S9-mix at the highest tested concentration.

- Mean number of revertant colonies per plate:
First Experiment (dose-range finding study is reported as part of the first experiment) and and additional experiment: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested. See Table 3 and 4 in section "Any other information on results incl. tables"

Second Experiment: In the pre-incubation test, an increase in the number of revertants was observed upon treatment in tester strain TA1535 in absence of S9-mix. The highest increase observed was 4.5-fold the concurrent solvent control, in the absence of S9-mix. In any other tester strains, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested. See Table 5 in section "Any other information on results incl. tables"

Additional experiment: To verify the result obtained in tester strain TA1535 in the absence of S9-mix, an additional experiment was performed. In this pre-incubation test, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested. See Table 6 in section "Any other information on results incl. tables"


HISTORICAL CONTROL DATA
- Negative vehicle historical control data: Valid; Please see table 7 in "Any other information on results incl. tables" section.
- Positive historical control data: Valid; Please see table 8 in "Any other information on results incl. tables" section.

Table 3 - Dose-Range Finding Test: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Dose (µg/plate)		TA100				WP2uvrA
Without S9-mix
Positive control		990	±	65		1460	±	106
Solvent control		12	±	9		20	±	5
1.7		119	±	14		26	±	4
5.4		103	±	21		12	±	3
17		125	±	4		19	±	1
52		107	±	15		19	±	4
164		113	±	11		16	±	1
512		112	±	12		22	±	8
1600		97	±	15		15	±	6
5000		130	±	9	^n NP	21	±	4	^n NP
With S9-mix
Positive control		1865	±	54		377	±	8
Solvent control		95	±	8		18	±	6
1.7		98	±	12		29	±	6
5.4		90	±	9		24	±	4
17		110	±	13		20	±	8
52		101	±	14		24	±	7
164		104	±	2		17	±	2
512		95	±	12		29	±	2
1600		87	±	15		28	±	1
5000		91	±	10	^n NP	23	±	7	^n NP
Mean number of revertant colonies (3 replicate plates) ± SD is reported
NP	No precipitate
n	Normal bacterial background lawn

 

Table 4 - Experiment 1: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay

Dose (µg/plate)			TA1535				TA1537				TA98
Without S9-mix
Positive control		208		±	28		652	±	47		962	±	87
Solvent control		5		±	1		6	±	1		5	±	5
52		4		±	3		1	±	2		8	±	4
164		9		±	5		1	±	2		10	±	2
512		10		±	1		1	±	1		7	±	2
1600		5		±	2		1	±	1		5	±	4
5000		5		±	4	^n NP	1	±	1	^n NP	6	±	2	^n NP
With S9-mix
Positive control		499		±	70		321	±	25		1638	±	186
Solvent control		9		±	3		3	±	2		10	±	4
52		5		±	2		2	±	2		17	±	3
164		8		±	3		1	±	0		12	±	7
512		9		±	2		2	±	2		8	±	4
1600		4		±	1		2	±	1		8	±	3
5000		7		±	4	^n NP	1	±	1	^n NP	11	±	4	^n NP
Mean number of revertant colonies (3 replicate plates) ± SD is reported
NP	No precipitate
n	Normal bacterial background lawn

 

Table 5 - Experiment 2: Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Dose (µg/plate)	TA1535				TA1537				TA98				TA100				WP2uvrA
Without S9-mix
Positive control	633	±	40		71	±	17		910	±	122		627	±	94		1278	±	132
Solvent control	11	±	1		3	±	1		10	±	6		100	±	9		15	±	2
52	4	±	4		4	±	1		16	±	10		124	±	13		20	±	2
164	11	±	8		4	±	1	^i	11	±	4		122	±	9		15	±	2
512	12	±	12		3	±	2		9	±	3		127	±	13		12	±	3
1600	50	±	21		2	±	1		7	±	0		93	±	12		15	±	1
5000	45	±	38	^n NP	3	±	2	^n NP	4	±	1	^n NP	35	±	14	^n NP	12	±	3	^n NP
S9-mix
Positive control	162	±	75		275	±	74		963	±	429		1515	±	45		448	±	64
Solvent control	8	±	4	^i	4	±	1		18	±	6		86	±	12		17	±	4
52	12	±	7		1	±	2		20	±	2		115	±	21		17	±	8
164	8	±	2		4	±	1		13	±	8		100	±	17		18	±	5
512	8	±	8		4	±	4		13	±	2		79	±	11		15	±	5
1600	6	±	3		6	±	1		14	±	4		82	±	19		16	±	6	^NP
5000	5	±	3	^n NP	3	±	2	^n NP	14	±	3	^n NP	47	±	20	^n NP	20	±	5	^n SP
Mean number of revertant colonies (3 replicate plates) ± SD is reported
NP	No precipitate
SP	Slight Precipitate
i	Plate infected: Mean of two plates
n	Normal bacterial background lawn

 

Table 6 - Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay

Dose (µg/plate)	TA1535
Without S9-mix
Positive control	677	±	39
Solvent control	8	±	5
52	7	±	3
164	10	±	5
512	6	±	4
1600	4	±	4
5000	4	±	3	^n NP
Mean number of revertant colonies/3 replicate plates (± S.D.)
NP	No precipitate
n	Normal bacterial background lawn

 

Table 7 – Historical Control Data of the the Solvent Control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	2-26	3-23	1-24	1-20	3-43	5-62	58-188	39-169	9-61	9-68
Mean	9	10	5	5	12	17	100	90	21	25
SD	3	3	2	2	4	5	16	21	8	10
Total number of plates	2110	2080	2135	2118	2200	2221	2226	2156	2156	2068
95% upper limit	14	15	9	10	20	27	131	131	131	44
95% lower limit	4	5	0.7	0.5	4	7	69	49	49	6
SD = Standard deviation
Historical control data from experiments performed between Dec 2018 and Dec 2021.

 

Table 8 – Historical Control Data of the Positive Control Materials

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	95-1373	78-1195	71-1587	48-1989	379-2141	251-3369	173-1852	166-2666	89-2027	109-1942
Mean	897	272	855	265	1371	951	800	1339	1312	424
SD	169	110	350	167	304	405	192	428	369	189
Total number of plates	1924	1911	1520	1955	2064	2001	2007	1984	1910	1879
95% upper limit	1229	488	1541	593	1966	1745	1177	2177	2035	795
95% lower limit	565	56	169	-63	776	157	423	501	589	53
SD = Standard deviation
Historical control data from experiments performed between Dec 2018 and Dec 2021.

 

Conclusions:

The results of an AMES test, performed according to OECD TG 471 and in accordance with GLP principles, showed that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In an Ames test, performed according to OECD TG 471 and in accordance with GLP principles, the test item was assessed for its potential to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and at the tryptophan locus of one Escherichia coli strain (WP₂uvrA).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. In addition, an additional experiment was performed to investigate a possible mutagenic response.

In the dose-range finding study, the test material was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP₂uvrA in the direct plate assay. The test material did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.
In the first mutation experiment, the test material was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. The test material did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
In the second mutation experiment, the test material was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP₂uvrA in the pre-incubation assay. Precipitation of the test material on the plates was observed at a concentration of 5000 μg/plate in tester strain WP₂uvrA in the absence of S9-mix. Precipitation of the test material on the plates was not observed in any other tester strains. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the absence of S9-mix at the highest tested concentration.
In tester strain TA1535 increases in the number of revertants were observed in the absence of S9-mix. The highest increase observed was 4.5-fold the concurrent solvent control, in the absence of S9-mix. The most likely cause is a possible slight cross-contamination with the positive control (See additional comments). To verify the results obtained in tester strain TA1535 in the second mutation experiment, an additional experiment was performed. In the additional experiment the test item was tested up to concentrations of 5000 μg/plate in tester strain TA1535 in the absence of S9-mix in the pre-incubation assay. The test item did not precipitate on the plates at this dose level. No cytotoxicity and no increases in the number of revertants were observed. The test material did not induce a biologically relevant, dose-related increase in the number of revertant (His⁺) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp⁺) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a followup experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that 2,2’-azobis[2-methylbutyronitrile] is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Additional comments on the elevated revertant number in TA1535 without S9 in Experiment 2:

• TA-1535 and TA-100 are derived from same strain, but TA100 is made more sensitive. It is very uncommon that TA1535 show more absolute increase of revertants than TA100.


• Often when a strain shows positive effects without S9, also an increase is often observed with S9 (at it takes time to metabolise)


• A specific effect in pre-incubation method only but not in direct plate for a non-toxic substance is not obvious.


• Although the whole dose ranges showed lack of toxicity, there is a remarkable lack of increase in revertants between 1600 and 5000 µg/plate (from 50 to 45) indicating a lack of consistent dose-response.


• The variability between the three plates at the same dose levels is increased at 1600 and 5000 µg/plate at a response of 45-50 revertants becomes with ± 38 very close to ± 40 seen for the positive control with around 600 revertants in each plate.
  (Also positive HCD for this strain show a SD less than 20% (897 ± 169) which is less than at 1600  & 5000 µg in this study with SD about 50% of the number of revertants (50±21 resp. 45±38))

Considering this, the most likely cause of the increase in revertants above 20 in the concerned plates with TA1535 was from a possible slight cross-contamination with the positive control.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Supporting in vivo micronucleus study on 13472-08-7 (supporting due to lack of reliability due to abstract only) is negative. Further testing not required as all in vitro endpoints are negative.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The test substance was examined for mutagenic activity in Salmonella typhimurium, strains TA98, TA100, TA1535 and TA1537, using pour-plate assays. Results obtained with all strains were confirmed in a second, independent experiment. The studies, which were conducted in the absence and presence of an activating system employed a range of levels of the test substance from 50 to 5000 µg per plate with and without a metabolic activation system. No increases in reversion to prototrophy were obtained with any of the four bacterial strains at the levels tested. It was concluded that the test substance was devoid of mutagenic activity under the conditions of the test.

 

In an additional Ames test, the test item was assessed for its potential to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and at the tryptophan locus of one Escherichia coli strain (WP₂uvrA). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. Also this study concluded that 2,2’-azobis[2-methylbutyronitrile] is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

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 (OECD 487). 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 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 addition to the Ames assay and in vitro micronucleus study with the test substance, multiple OECD guideline, GLP studies with the read-across chemical, 2,2’-azobis(isobutyronitrile) (AIBN) have been performed, involving Ames assay, in vitro chromosome aberration assay, and mouse lymphoma assay. None of these studies have indicated a potential for genotoxic properties.

Justification for classification or non-classification

Available studies have not identified a concern for genotoxicity.

Therefore, the substance need not be classified for genetic toxicity according EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.