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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was tested in two Ames Assays, one mammalian Mutation Assay and one mammalian Cell Micronucleus Assay according to Guidelines (OECD 471, OECD 490 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

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 2021 - January 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: study sponsor, Batch No 201902029
- Purity, including information on contaminants, isomers, etc.: 98.42 %

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item was stored in a closed vessel in the fridge (2 - 8 °C).
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: assumed stable for purposes of the test
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: assumed stable for purposes of the test
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: The solubility of the test item was determined in a non-GLP pre-test in culture medium (RPMI 1640) at a concentration of 20 mg/mL and dimethyl sulfoxide (DMSO), ethanol as well as acetone at a concentration of 400 mg/mL. The test item was completely insoluble in RPMI 1640, DMSO and ethanol but completely dissolved in acetone.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): assumed unreactive for purposes of the test

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
On the 1st day of the pre-test, a stock solution containing 400 mg/mL (nominal) of the test item in acetone was prepared. This stock solution was afterwards used to prepare the ge-ometric series (factor 2) of the resulting test item concentrations. The concentrations of the stock solutions for experiment I and II were 50 mg/mL (experiment I +S9) and 25 mg/mL (experiment I -S9 and experiment II).

FORM AS APPLIED IN THE TEST (if different from that of starting material)
same as starting material

INFORMATION ON NANOMATERIALS
no Nanoform

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
no biocide

OTHER SPECIFICS
none stated

Target gene:

Thymidine Kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: purchased at ATCC (Wesel, Germany) as L5178Y TK+/- clone (3.7.2C) [TK+/- (clone 3.7.2C)] (ATCC® CRL-9518™)
- Suitability of cells: please refer to OECD Guideline
- Normal cell cycle time (negative control): 10-12 h in stock cultures

For cell lines:
- Absence of Mycoplasma contamination: yes, checked
- Number of passages if applicable: not stated
- Methods for maintenance in cell culture: stored in liquid nitrogen in the cell bank of LAUS GmbH
- Cell cycle length, doubling time or proliferation index : 10-12 h in stock cultures
- Modal number of chromosomes: 40 +- 2
- Periodically checked for karyotype stability: no
- Periodically ‘cleansed’ of spontaneous mutants: yes

For lymphocytes:
not applicable

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 complete culture medium with 10 % HS, at 37.0 ± 1.0 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Metabolic activation:

with and without

Metabolic activation system:

S9 (liver enzyme mixture used for the test with metabolic activation)

Test concentrations with justification for top dose:

Experiment I +S9: 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781 mg/mL
Experiment I -S9, Experiment II: 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391 mg/mL


According to the OECD guideline 490 the highest concentration should be 0.01 M or 2 mg/mL or 2 µL/mL (whichever is lowest), unless limited by the solubility or toxicity of the test item. RCE values below 20 % are considered toxic. In case of toxic effects, the high-est test item concentration of the main experiment should reduce the RSG value to 10 - 20 %. For poorly soluble test items that are not cytotoxic at concentrations below the low-est insoluble concentrations, the highest concentration analysed should produce turbidity or a precipitate visible by eye or with the aid of an inverted microscope at the end of the treatment with the test item.
In reference to the results of the pre-test (precipitation was taken into account), 7 concen-trations were chosen for experiment I and II

Vehicle / solvent:

acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

RPMI 1640 medium is used as solvent control for the positive control MMS in a final con-centration of 0.5 % (positive control) during treatment.
0.9 % NaCl is used as solvent control for the positive control CPAm in a final concentra-tion of 0.5 % during

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 4*10^3
- Test substance added in medium;

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: none
- Exposure duration/duration of treatment: 4h/24h
- Harvest time after the end of treatment (sampling/recovery times): 48 h


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 h (= harvest time)
- Selection time (if incubation with a selective agent): 12 d
- Fixation time (start of exposure up to fixation or harvest of cells): no fixation, 48 h (=harvest time)
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. TFT, during selection time
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 4*10^3, counted manually
- Criteria for small (slow growing) and large (fast growing) colonies:
Colonies were counted manually under a binocular magnifying glass. In accordance with their size, the colonies were classified into two groups:
Less than 25 % of the well’s diameter = small colony
More than 25 % of the well’s diameter = large colony


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative cloning efficiency (RCE) and relative total growth (RTG

Rationale for test conditions:

according to Guideline

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if:
• the induced mutation frequency reproducibly exceeds a threshold of 126 colo-nies per 106 cells above the corresponding solvent control.
• the relative increase of the mutation frequency shows a dose relationship.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if:
• the induced mutation frequency does not exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.
• the relative increase of the mutation frequency does not show a dose relationship.

A mutagenic response is considered to be reproducible if it occurs in both parallel cul-tures.

Statistics:

A linear regression (least squares) of the test item concentrations was performed to as-sess a possible dose dependent increase of mutant frequencies.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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

Additional information on results:

two individual experiments performed leading to same results.

The results of the mutagenicity assay with metabolic activation are presented in the following tables.

Mutagenicity Experiment I with Metabolic Activation

Culture	Content	Cells seeded per well	No. of empty wells		Wells with small colonies		Wells with large colonies
			plate 1	plate 2	plate 1	plate 2	plate 1	plate 2
A	Solvent Control Test Item (Acetone 0.5%)	71	60	23	31	9	10	71
B	Solvent Control Test Item (Acetone 0.5%)	66	69	23	19	11	9	66
A	Solvent Control CPAm (NaCl 0.9%)	66	64	23	24	13	13	66
B	Solvent Control CPAm (NaCl 0.9%)	77	75	20	10	3	11	77
A	Positive Control CPAm	49	48	56	58	19	14	49
B	Positive Control CPAm	47	48	63	56	14	19	47
A	Test Item 0.25 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.25 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a	n/a
A	Test Item 0.13 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.13 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a	n/a
A	Test Item 0.06 mg/mL	66	55	34	34	6	15	66
B	Test Item 0.06 mg/mL	70	59	29	40	6	9	70
A	Test Item 0.03 mg/mL	64	66	35	20	8	11	64
B	Test Item 0.03 mg/mL	60	58	37	31	16	16	60
A	Test Item 0.016 mg/mL	70	70	24	24	8	9	70
B	Test Item 0.016 mg/mL	63	55	34	43	7	9	63
A	Test Item 0.008 mg/mL	67	65	29	31	7	6	67
B	Test Item 0.008 mg/mL	57	68	38	26	11	6	57
A	Test Item 0.004 mg/mL	n/e	n/e	n/e	n/e	n/e	n/e	n/e
B	Test Item 0.004 mg/mL	n/e	n/e	n/e	n/e	n/e	n/e	n/e
Additional untreated control (medium control)
A	Solvent control (RPMI)	73	62	24	21	7	17	73
B	Solvent control (RPMI)	67	64	24	26	11	14	67
A	Acetone 0.5 %	71	60	23	31	9	10	71
B	Acetone 0.5 %	66	69	23	19	11	9	66

n/a = not analysed for mutagenicity because of cytotoxicity

n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations

 

Results Mutation frequency Experiment I with Metabolic Activation

Culture	Content	Small mutant colonies / 10^6cells	Large mutant colonies / 10^6cells	Mutant colonies / 10^6cells	Small mutant colonies / 10^6cells (Mean both cultures)		Mutant colonies / 10^6cells (Mean both cultures)
A	Solvent Control Test Item (Acetone 0.5%)	72.9	23.0	84.4	71	27	91
B	Solvent Control Test Item (Acetone 0.5%)	68.8	30.7	98.2
A	Solvent Control CPAm (NaCl 0.9%)	82.5	42.8	114.7	62	31	86
B	Solvent Control CPAm (NaCl 0.9%)	41.7	18.6	57.3
A	Positive Control CPAm	499.6	104.6	378.7	598	120	443
B	Positive Control CPAm	696.2	135.8	506.6
A	Test Item 0.25 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.25 mg/mL	n/a	n/a	n/a
A	Test Item 0.13 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.13 mg/mL	n/a	n/a	n/a
A	Test Item 0.06 mg/mL	109.3	29.0	115.4	108	24	106
B	Test Item 0.06 mg/mL	107.2	19.6	95.7
A	Test Item 0.03 mg/mL	77.9	24.1	90.0	110	42	124
B	Test Item 0.03 mg/mL	141.8	59.1	157.8
A	Test Item 0.016 mg/mL	74.7	24.1	82.0	110	24	110
B	Test Item 0.016 mg/mL	145.7	24.7	138.4
A	Test Item 0.008 mg/mL	99.0	18.5	99.0	109	23	112
B	Test Item 0.008 mg/mL	119.0	27.2	125.9
A	Test Item 0.004 mg/mL	n/e	n/e	n/e	n/e	n/e	n/e
B	Test Item 0.004 mg/mL	n/e	n/e	n/e
Additional untreated control (medium control)
A	Solvent control (RPMI)	71.8	35.9	94.7	79	38	102
B	Solvent control (RPMI)	85.8	39.7	108.7
A	Acetone 0.5 %	72.9	23.0	84.4	71	27	91
B	Acetone 0.5 %	68.8	30.7	98.2

n/a = not analysed for mutagenicity because of cytotoxicity

n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations

Mutagenicity Experiment I without Metabolic Activation

Culture	Content	Cells seeded per well	No. of empty wells		Wells with small colonies		Wells with large colonies
			plate 1	plate 2	plate 1	plate 2	plate 1	plate 2
A	Solvent Control Test Item (Acetone 0.5%)	67	72	25	26	11	7	67
B	Solvent Control Test Item (Acetone 0.5%)	59	68	37	19	7	13	59
A	Solvent Control MMS (RPMI 1640)	71	69	19	20	9	12	71
B	Solvent Control MMS (RPMI 1640)	66	62	17	24	16	12	66
A	Positive Control MMS	45	44	64	55	15	11	45
B	Positive Control MMS	49	50	64	57	12	12	49
A	Test Item 0.13 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.13 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a	n/a
A	Test Item 0.06 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.06 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a	n/a
A	Test Item 0.03 mg/mL	61	65	38	33	8	7	61
B	Test Item 0.03 mg/mL	50	65	54	37	11	9	50
A	Test Item 0.016 mg/mL	63	57	30	35	11	10	63
B	Test Item 0.016 mg/mL	55	59	37	41	9	9	55
A	Test Item 0.008 mg/mL	65	65	23	26	13	11	65
B	Test Item 0.008 mg/mL	73	62	17	31	9	11	73
A	Test Item 0.004 mg/mL	67	64	20	21	12	14	67
B	Test Item 0.004 mg/mL	65	68	33	15	11	17	65
A	Test Item 0.002 g/mL	n/e	n/e	n/e	n/e	n/e	n/e	n/e
B	Test Item 0.002 g/mL	n/e	n/e	n/e	n/e	n/e	n/e	n/e
Additional untreated control (medium control)
A	Solvent control (RPMI)	59	67	32	23	15	10	59
B	Solvent control (RPMI)	67	67	21	24	13	8	67
A	Acetone 0.5 %	67	72	25	26	11	7	67
B	Acetone 0.5 %	59	68	37	19	7	13	59

n/a = not analysed for mutagenicity because of cytotoxicity

n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations

Results Mutation frequency Experiment I without Metabolic Activation

Culture	Content	Small mutant colonies / 10^6cells	Large mutant colonies / 10^6cells	Mutant colonies / 10^6cells	Small mutant colonies / 10^6cells (Mean both cultures)	Large mutant colonies / 10^6cells (Mean both cultures)	Mutant colonies / 10^6cells (Mean both cultures)
A	Solvent Control Test Item (Acetone 0.5%)	80.3	25.6	84.0	82	26	93
B	Solvent Control Test Item (Acetone 0.5%)	84.6	27.0	101.4
A	Solvent Control MMS (RPMI 1640)	55.7	28.4	77.5	59	35	91
B	Solvent Control MMS (RPMI 1640)	62.5	41.0	105.4
A	Positive Control MMS	601.8	90.6	478.5	573	82	421
B	Positive Control MMS	544.8	73.1	362.8
A	Test Item 0.13 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.13 mg/mL	n/a	n/a	n/a
A	Test Item 0.06 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.06 mg/mL	n/a	n/a	n/a
A	Test Item 0.03 mg/mL	126.7	22.3	115.6	148	26	126
B	Test Item 0.03 mg/mL	169.9	29.1	135.6
A	Test Item 0.016 mg/mL	83.1	23.3	94.5	112	25	118
B	Test Item 0.016 mg/mL	140.7	26.6	140.7
A	Test Item 0.008 mg/mL	79.5	36.0	105.2	76	32	97
B	Test Item 0.008 mg/mL	71.9	27.5	88.0
A	Test Item 0.004 mg/mL	61.3	37.1	97.5	72	41	102
B	Test Item 0.004 mg/mL	82.9	45.4	105.8
A	Test Item 0.002 g/mL	n/e	n/e	n/e	n/e	n/e	n/e
B	Test Item 0.002 g/mL	n/e	n/e	n/e
Additional untreated control (medium control)
A	Solvent control (RPMI)	89.3	36.9	111.5	80	34	103
B	Solvent control (RPMI)	70.7	30.7	95.2
A	Acetone 0.5 %	80.3	25.6	84.0	82	26	93
B	Acetone 0.5 %	84.6	27.0	101.4

n/a = not analysed for mutagenicity because of cytotoxicity

n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations

 

Mutagenicity Experiment II without Metabolic Activation

Culture	Content	Cells seeded per well	No. of empty wells		Wells with small colonies		Wells with large colonies
			plate 1	plate 2	plate 1	plate 2	plate 1	plate 2
A	Solvent Control Test Item (Acetone 0.5%)	3997	61	65	11	11	28	30
B	Solvent Control Test Item (Acetone 0.5%)	4001	69	68	8	8	25	27
A	Solvent Control MMS (RPMI 1640)	3999	61	65	9	8	30	27
B	Solvent Control MMS (RPMI 1640)	4001	71	81	7	5	22	12
A	Positive Control MMS	3995	36	34	54	64	44	50
B	Positive Control MMS	4000	45	41	40	45	36	40
A	Test Item 0.13 mg/mL	4001	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.13 mg/mL	3995	n/a	n/a	n/a	n/a	n/a	n/a
A	Test Item 0.06 mg/mL	4003	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.06 mg/mL	3996	n/a	n/a	n/a	n/a	n/a	n/a
A	Test Item 0.03 mg/mL	4003	71	70	11	6	22	22
B	Test Item 0.03 mg/mL	4000	70	78	6	8	21	12
A	Test Item 0.016 mg/mL	3998	60	63	7	10	32	28
B	Test Item 0.016 mg/mL	3998	65	64	7	10	29	27
A	Test Item 0.008 mg/mL	4003	77	76	4	8	17	15
B	Test Item 0.008 mg/mL	4000	69	65	6	10	24	26
A	Test Item 0.004 mg/mL	4002	75	62	9	9	19	33
B	Test Item 0.004 mg/mL	3999	67	71	9	9	26	21
A	Test Item 0.002 g/mL	4000	n/e	n/e	n/e	n/e	n/e	n/e
B	Test Item 0.002 g/mL	4003	n/e	n/e	n/e	n/e	n/e	n/e
Additional untreated control (medium control)
A	Solvent control (RPMI)	3999	61	65	9	8	30	27
B	Solvent control (RPMI)	4001	71	81	7	5	22	12
A	Acetone 0.5 %	3997	61	65	11	11	28	30
B	Acetone 0.5 %	4001	69	68	8	8	25	27

n/a = not analysed for mutagenicity because of cytotoxicity

n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations

 

Results Mutation frequency Experiment II without Metabolic Activation

Culture	Content	Small mutant colonies / 10^6cells	Large mutant colonies / 10^6cells	Mutant colonies / 10^6cells	Small mutant colonies / 10^6cells (Mean both cultures)	Large mutant colonies / 10^6cells (Mean both cultures)	Mutant colonies / 10^6cells (Mean both cultures)
A	Solvent Control Test Item (Acetone 0.5%)	29.9	88.3	103.4	27	87	97
B	Solvent Control Test Item (Acetone 0.5%)	23.5	85.1	91.0
A	Solvent Control MMS (RPMI 1640)	27.2	103.4	123.6	23	81	97
B	Solvent Control MMS (RPMI 1640)	19.3	58.2	69.7
A	Positive Control MMS	844.0	595.3	893.1	734	566	875
B	Positive Control MMS	623.2	537.1	856.0
A	Test Item 0.13 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.13 mg/mL	n/a	n/a	n/a
A	Test Item 0.06 mg/mL	n/a	n/a	n/a	n/a	n/a	n/a
B	Test Item 0.06 mg/mL	n/a	n/a	n/a
A	Test Item 0.03 mg/mL	26.7	75.1	89.1	25	67	85
B	Test Item 0.03 mg/mL	23.4	58.3	80.4
A	Test Item 0.016 mg/mL	25.9	104.6	124.3	23	89	104
B	Test Item 0.016 mg/mL	19.6	72.9	84.0
A	Test Item 0.008 mg/mL	15.8	44.7	55.7	21	67	82
B	Test Item 0.008 mg/mL	26.0	90.2	107.5
A	Test Item 0.004 mg/mL	28.4	91.1	97.4	30	90	100
B	Test Item 0.004 mg/mL	30.9	88.1	103.6
A	Test Item 0.002 g/mL	n/e	n/e	n/e	n/e	n/e	n/e
B	Test Item 0.002 g/mL	n/e	n/e	n/e
Additional untreated control (medium control)
A	Solvent control (RPMI)	27.2	103.4	123.6	23	81	97
B	Solvent control (RPMI)	19.3	58.2	69.7
A	Acetone 0.5 %	29.9	88.3	103.4	27	87	97
B	Acetone 0.5 %	23.5	85.1	91.0

n/a = not analysed for mutagenicity because of cytotoxicity

n/e = not evaluated because the OECD 490 guideline requires only 4 concentrations

 

Statistical results

A linear regression (least squares) of the test item concentrations was performed to assess a possible dose dependent increase of mutant frequencies. With the assessment of this regression, it can be evaluated whether mutations increase with increasing dose of the test item. A p-value of 0.05 or lower (significance level 95%) is considered as critical.

The following results were obtained:

Table       Regression Parameters of Test Item

Treatment	Correlation coefficient |r|	F krit
Exp. I with metabolic activation	0.321	0.679
Exp. I without metabolic activation	0.919	0.081
Exp. II without metabolic activation	0.300	0.700

 

The positive controls were tested at one concentration only. Therefore, no dose-dependency could be evaluated, although the positive controls showed considerable increases in mutants. In the following table, the statistical significance values are presented. The chi-square test was used.

Statistically significant increase in mutants is considered as given if p is below 0.01.

Table      Statistical Significance

Substance	Concentration in µg/mL	p-Values 1^st and 2^nd Experiment
		without S9	with S9
Positive Control MMS	19.5 (Experiment I) 12.5 (Experiment II)	< 0.001	--
Positive Control CPAm	5.5	--	< 0.001

 

Conclusions:

under the experimental conditions reported the test item did not induce gene mutations at the thymidine kinase locus (Tk1) in heterozygous mouse lymphoma L5178Y Tk+/- cells.
Therefore, the test item 2,2'-Azobis(2,4-dimethylvaleronitrile) is considered to be “not mu-tagenic under the conditions of the mouse lymphoma assay”.

Executive summary:

The study was performed to investigate the potential of the test item 2,2'-Azobis(2,4-dimethylvaleronitrile) to induce mutations at the thymidine kinase locus (Tk1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y Tk ^+/- cells.

 

The assay was performed in a pre-test and two independent and valid experiments, using two parallel cultures each (replicates).

In experiment I, 7 concentrations of the test item were tested with and without metabolic activation and a treatment period of 4 h. Afterwards, 4 concentrations were evaluated for mutant frequency. In experiment II, 7 concentrations of the test item were tested without metabolic activation and a treatment period of 24 h. Afterwards, 4 concentrations were evaluated for mutant frequency.

MMS (19.5 µg/mL in experiment I and 12.5 µg/mL in experiment II) and CPA_m (5.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and exceeded the number of mutant colonies of more than 300 in comparison to the corresponding solvent control.

In both experiments of this study (short exposure with and without S9 mix, extended exposure without S9 mix), the range of the spontaneous mutant frequency of the solvent controls was in the range of the historical data.

Since all further acceptability criteria of the assay were also met the study is valid.

 

The following nominal concentrations of the test item were investigated in experiment I:

+S9: 0.25 mg/mL, 0.13 mg/mL, 0.06 mg/mL, 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL, 0.004 mg/mL

-S9: 0.13 mg/mL, 0.06 mg/mL, 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL, 0.004 mg/mL, 0.002 mg/mL

The following nominal concentrations of the test item were investigated in experiment II: 0.13 mg/mL, 0.06 mg/mL, 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL, 0.004 mg/mL, 0.002 mg/mL.

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration.

In experiment I with metabolic activation, precipitation of the test item, visible to the unaided eye, was noted at the concentrations 0.25 mg/mL and 0.13 mg/mL and without metabolic activation at 0.13 mg/mL and 0.06 mg/mL.

In experiment II, precipitation of the test item, visible to the unaided eye, was noted at the concentrations 0.13 mg/mL and 0.06 mg/mL.

No significant reduction of growth was observed with and without S9 after 4 h treatment with the test item (all concentrations) in all experimental parts.

According to the Guideline OECD490, the highest concentrations analysed, showed precipitates, therefor the following 4 test item concentrations could be evaluated for mutagenicity:

+S9: 0.06 mg/mL, 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL

-S9: 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL, 0.004 mg/mL

In experiment II (- S9) a strong reduction of viability was observed in the three highest concentrations, but the RTG values for all concentrations were higher than 20 %. Since the two highest concentrations showed precipitates, the following test item concentrations could be evaluated for mutagenicity: 0.03 mg/mL, 0.016 mg/mL, 0.008 mg/mL, 0.004 mg/mL

In all evaluated concentrations of the test item no substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item. The mutation frequency did not reach or exceed the threshold of 126 above the corresponding solvent control.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2021 - Sep 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Sponsor, Batch Nr. 201902029
- Purity, including information on contaminants, isomers, etc.: 98.42 %

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: fridge (2-8 °C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable under storage conditions
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: no information provided in report
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: no information provided in report
- Reactivity of the test material with the incubation material used (e.g. plastic ware): no information provided in report

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
no special treatment

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: no differences

INFORMATION ON NANOMATERIALS
no nano material

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)
no biocide/pesticide

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: no information available

Target gene:

histidine deficiency

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Rat Liver S9

Test concentrations with justification for top dose:

The following nominal test item concentrations were prepared for experiment 1:
5000, 1500, 500, 150 and 50 µg/plate.

The following nominal test item concentrations were prepared for experiment 2:
5000, 2500, 1250, 625, 313, 156 and 78 µg/plate.

Due to toxicity observed in experiment 2, additional concentrations were tested
The following nominal test item concentrations were prepared for experiment 2b:
TA98 (+S9), TA102 (-S9), TA1535 (-S9):
625, 313, 156, 78, 39, 19.5, 9.8 µg/plate
TA1335 (+S9), TA1537 (+S9):
313, 156, 78, 39, 19.5, 9.8, 4.88 µg/plate
TA98 (-S9), TA1537 (-S9):
78, 39, 19.5, 9.8, 4.88, 2.44, 1.22 µg/plate

justification: according to Guideline

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

mitomycin C

other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene

Remarks:

The negative controls are consisting of solvent or vehicle alone, without the test item or the positive control substances and otherwise treated in the same way as the treatment samples. The control samples were used to provide the corresponding values fo

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) triplicate
- Number of independent experiments 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): no applicable
- Test substance added in agar (plate incorporation); preincubation;

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min
- Incubation time: 48 h


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition;

METHODS FOR MEASUREMENTS OF GENOTOXICIY
visual colony counting

Rationale for test conditions:

according to Guideline

Evaluation criteria:

A result is considered as clearly positive if all following criteria are fulfilled:
- A concentration-related increase, in revertants
- a clear biological relevant increase in at least one concentration compared to the concurrent solvent control
- at least one concentration with an increase above the distribution of historical solvent control data (mean ± 3 SD).

A biologically relevant increase is described as follows:
- if in the bacteria strains TA98, TA100, TA102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2.0)
- if in the bacteria strains TA1535 and TA1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3.0).

Statistics:

no statistical analysis applied, only mean and standard deviation are calculated and stated.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

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

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

1       Data of Experiment 1

1.1      Determination of Titre

Criterion:            The determination of titre should give a number of at least 10⁹ cells/mL, correlating to 100 colonies/plate after dilution.

 

Exact colony counts could not be determined due to the strong growth of the bacteria, but

by visual inspection it was obvious that the values exceed 100 colonies/ plate

 

Tabelle 14.1‑a Titre Values (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.
Repl. 2	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.
Assessment	ok	ok	ok	ok	ok	ok	ok	ok	ok	ok

s.g.= strong growth, too strong for counting of colonies

ok= the criterion was fulfilled

1.2      Sterility Control

Criterion:            No colony per plate may grow.

Tabelle 14.2‑a Sterility (colonies per plate)

	Demin. water	DMSO	Ethanol
Repl. 1	0	0	0
Repl. 2	0	0	0
Repl. 3	0	0	0
Repl. 4	0	0	0
Assessment	ok	ok	ok

ok= the criterion was fulfilled

 

 

 

1.3      Spontaneous Revertants

1.3.1    Demin. Water

Tabelle 14.3‑a Spontaneous Revertants demin. water (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	16	17	71	74	140	148	9	9	4	5
Repl. 2	16	19	70	70	148	152	9	9	4	5
Repl. 3	18	20	62	69	160	156	11	10	5	6
Mean	17	19	68	71	149	152	10	9	4	5
sd	1.2	1.5	4.9	2.6	10.1	4.0	1.2	0.6	0.6	0.6

sd = standard deviation ±

 

1.3.2    DMSO

Tabelle 14.3‑b Spontaneous Revertants DMSO (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	15	19	65	74	140	144	8	9	5	5
Repl. 2	17	20	62	72	140	152	10	9	6	5
Repl. 3	17	21	58	60	152	160	11	10	6	7
Mean	16	20	62	69	144	152	10	9	6	6
sd	1.2	1.0	3.5	7.6	6.9	8.0	1.5	0.6	0.6	1.2

sd = standard deviation ±

 

1.3.3    Ethanol

Tabelle 14.3‑c Spontaneous Revertants Ethanol (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	14	17	70	74	132	144	8	9	5	4
Repl. 2	15	17	61	70	144	152	9	10	6	4
Repl. 3	15	20	58	66	156	160	11	12	6	6
Mean	15	18	63	70	144	152	9	10	6	5
sd	0.6	1.7	6.2	4.0	12.0	8.0	1.5	1.5	0.6	1.2

sd = standard deviation ±

 

 

1.4      Positive Controls

 

• Without metabolic activation:
  4-Nitro-1,2-phenylene diamine (NPD) in DMSO
  Sodium azide (Na-azide) in demin. water
  Mitomycin C (MMC) in demin. water

 

• With metabolic activation:
  2-Amino anthracene (2-AA) in DMSO
  Benzo-a-pyrene (BaP) in DMSO

 

For used concentrations see chapter 6.2.4, page 14.

 

Tabelle 14.4‑a Diagnostic Mutagens (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Substance	NPD	BaP	Na-azide	2-AA	MMC	2-AA	Na-azide	2-AA	NPD	2-AA
Repl. 1	688	156	720	1536	880	672	328	188	76	43
Repl. 2	704	168	704	1496	896	688	344	200	81	45
Repl. 3	752	184	664	1472	928	728	344	212	87	49
Mean	715	169	696	1501	901	696	339	200	81	46
sd	33.3	14.0	28.8	32.3	24.4	28.8	9.2	12.0	5.5	3.1
f(I)	44.69	8.45	10.24	21.75	6.05	4.58	33.90	22.22	13.50	7.67
Rev. abs.	699	149	628	1432	752	544	329	191	75	40

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

 

 

 

1.5      Mutagenicity Test Test Item 2,2'-Azobis(2,4-dimethylvaleronitrile)

Tabelle 14.5‑a Concentration 5000 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	14	12	45	55	136	144	9	9	3	3
Repl. 2	12	11	41	53	148	148	9	10	3	4
Repl. 3	11	9	37	42	160	156	9	10	4	5
Mean	12	11	41	50	148	149	9	10	3	4
sd	1.5	1.5	4.0	7.0	12.0	6.1	0.0	0.6	0.6	1.0
f(I)	0.80	0.61	0.65	0.71	1.03	0.98	1.00	1.00	0.50	0.80
Rev. abs.	-3	-7	-22	-20	4	-3	0	0	-3	-1

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

Tabelle 14.5‑b Concentration 1500 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	10	17	60	54	140	144	8	8	3	3
Repl. 2	9	16	52	47	148	152	9	9	3	3
Repl. 3	9	15	48	48	152	160	10	9	4	5
Mean	9	16	53	50	147	152	9	9	3	4
sd	0.6	1.0	6.1	3.8	6.1	8.0	1.0	0.6	0.6	1.2
f(I)	0.60	0.89	0.84	0.71	1.02	1.00	1.00	0.90	0.50	0.80
Rev. abs.	-6	-2	-10	-20	3	0	0	-1	-3	-1

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

 

Tabelle 14.5‑c Concentration 500 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	17	14	54	59	152	140	9	8	3	3
Repl. 2	11	14	45	54	164	152	10	8	3	5
Repl. 3	11	13	43	49	168	164	10	9	4	5
Mean	13	14	47	54	161	152	10	8	3	4
sd	3.5	0.6	5.9	5.0	8.3	12.0	0.6	0.6	0.6	1.2
f(I)	0.87	0.78	0.75	0.77	1.12	1.00	1.11	0.80	0.50	0.80
Rev. abs.	-2	-4	-16	-16	17	0	1	-2	-3	-1

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

Tabelle 14.5‑d Concentration 150 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	19	15	56	60	136	144	9	9	3	3
Repl. 2	16	14	53	53	140	152	9	10	3	4
Repl. 3	12	9	46	51	156	160	11	12	4	5
Mean	16	13	52	55	144	152	10	10	3	4
sd	3.5	3.2	5.1	4.7	10.6	8.0	1.2	1.5	0.6	1.0
f(I)	1.07	0.72	0.83	0.79	1.00	1.00	1.11	1.00	0.50	0.80
Rev. abs.	1	-5	-11	-15	0	0	1	0	-3	-1

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

Tabelle 14.5‑e Concentration 50 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	16	19	65	66	144	152	9	9	3	4
Repl. 2	15	18	56	63	164	168	10	11	4	4
Repl. 3	13	16	54	58	168	172	10	11	4	5
Mean	15	18	58	62	159	164	10	10	4	4
sd	1.5	1.5	5.9	4.0	12.9	10.6	0.6	1.2	0.6	0.6
f(I)	1.00	1.00	0.92	0.89	1.10	1.08	1.11	1.00	0.67	0.80
Rev. abs.	0	0	-5	-8	15	12	1	0	-2	-1

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

 

 

2       Data of Experiment 2

2.1      Determination of Titre

Criterion:            The determination of titre should give a number of at least 10⁹ cells/mL, correlating to 100 colonies/plate after dilution.

 

Exact colony counts could not be determined due to the strong growth of the bacteria, but

by visual inspection it was obvious that the values exceed 100 colonies/ plate

 

Tabelle 15.1‑a Titre Values (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.
Repl. 2	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.	s.g.
Assessment	ok	ok	ok	ok	ok	ok	ok	ok	ok	ok

s.g.= strong growth, too strong for counting of colonies

ok= the criterion was fulfilled

2.2      Sterility Control

Criterion:            No colony per plate may grow.

Tabelle 15.2‑a Sterility (colonies per plate)

	Demin. water	DMSO	Ethanol
Repl. 1	0	0	0
Repl. 2	0	0	0
Repl. 3	0	0	0
Repl. 4	0	0	0
Assessment	ok	ok	ok

ok= the criterion was fulfilled

 

 

 

2.3      Spontaneous Revertants

2.3.1    Demin. Water

Tabelle 15.3‑a Spontaneous Revertants demin. water (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	19	20	69	53	160	156	8	8	4	4
Repl. 2	20	22	80	61	164	160	9	9	6	6
Repl. 3	21	25	103	64	172	168	10	9	8	7
Mean	20	22	84	59	165	161	9	9	6	6
sd	1.0	2.5	17.3	5.7	6.1	6.1	1.0	0.6	2.0	1.5

sd = standard deviation ±

 

2.3.2    DMSO

Tabelle 15.3‑b Spontaneous Revertants DMSO (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	19	20	59	55	156	160	9	8	4	5
Repl. 2	19	22	60	65	164	164	10	9	6	5
Repl. 3	20	24	72	90	208	168	12	10	7	8
Mean	19	22	64	70	176	164	10	9	6	6
sd	0.6	2.0	7.2	18.0	28.0	4.0	1.5	1.0	1.5	1.7

sd = standard deviation ±

 

2.3.3    Ethanol

Tabelle 15.3‑c Spontaneous Revertants Ethanol (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	19	21	68	88	156	160	8	8	4	6
Repl. 2	20	21	72	96	164	168	9	9	5	7
Repl. 3	21	25	75	114	176	176	9	9	7	10
Mean	20	22	72	99	165	168	9	9	5	8
sd	1.0	2.3	3.5	13.3	10.1	8.0	0.6	0.6	1.5	2.1

sd = standard deviation ±

 

 

2.4      Positive Controls

 

• Without metabolic activation:
  4-Nitro-1,2-phenylene diamine (NPD) in DMSO
  Sodium azide (Na-azide) in demin. water
  Mitomycin C (MMC) in demin. water

 

 

• With metabolic activation:
  2-Amino anthracene (2-AA) in DMSO
  Benzo-a-pyrene (BaP) in DMSO

 

For used concentrations see chapter 6.2.4, page 14.

 

Tabelle 15.4‑a Diagnostic Mutagens (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Substance	NPD	BaP	Na-azide	2-AA	MMC	2-AA	Na-azide	2-AA	NPD	2-AA
Repl. 1	560	132	656	1112	968	616	240	104	112	184
Repl. 2	616	136	688	1152	976	632	280	108	136	184
Repl. 3	680	148	696	1208	1008	648	288	124	140	200
Mean	619	139	680	1157	984	632	269	112	129	189
sd	60.0	8.3	21.2	48.2	21.2	16.0	25.7	10.6	15.1	9.2
f(I)	32.58	6.32	8.10	16.53	5.96	3.85	29.89	12.44	21.50	31.50
Rev. abs.	600	117	596	1087	819	468	260	103	123	183

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

 

 

 

2.5      Mutagenicity Test Test Item 2,2'-Azobis(2,4-dimethylvaleronitrile)

Tabelle 15.5‑a Concentration 5000 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	2	4	7	60	14	140	0	3	0	0
Repl. 2	0	4	5	65	23	156	0	3	0	0
Repl. 3	1	5	10	63	30	168	0	3	0	0
Mean	1	4	7	63	22	155	0	3	0	0
sd	1.0	0.6	2.5	2.5	8.0	14.0	0.0	0.0	0.0	0.0
f(I)	0.05	0.18	0.10	0.64	0.13	0.92	0.00	0.33	0.00	0.00
Rev. abs.	-19	-18	-65	-36	-143	-13	-9	-6	-5	-8

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

Tabelle 15.5‑b Concentration 2500 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	2	3	52	80	26	144	0	4	0	0
Repl. 2	2	5	46	67	31	148	0	6	0	0
Repl. 3	1	6	67	73	35	156	0	8	0	0
Mean	2	5	55	73	31	149	0	6	0	0
sd	0.6	1.5	10.8	6.5	4.5	6.1	0.0	2.0	0.0	0.0
f(I)	0.10	0.23	0.76	0.74	0.19	0.89	0.00	0.67	0.00	0.00
Rev. abs.	-18	-17	-17	-26	-134	-19	-9	-3	-5	-8

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

 

Tabelle 15.5‑c Concentration 1250 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	5	3	80	70	35	140	0	7	0	0
Repl. 2	3	7	75	71	41	144	3	4	0	0
Repl. 3	5	11	81	71	49	152	5	4	0	0
Mean	4	7	79	71	42	145	3	5	0	0
sd	1.2	4.0	3.2	0.6	7.0	6.1	2.5	1.7	0.0	0.0
f(I)	0.20	0.32	1.10	0.72	0.25	0.86	0.33	0.56	0.00	0.00
Rev. abs.	-16	-15	7	-28	-123	-23	-6	-4	-5	-8

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

Tabelle 15.5‑d Concentration 625 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	5	10	85	85	160	152	4	3	0	0
Repl. 2	7	8	75	73	168	160	2	6	0	0
Repl. 3	13	7	78	50	180	164	1	4	0	0
Mean	8	8	79	69	169	159	2	4	0	0
sd	4.2	1.5	5.1	17.8	10.1	6.1	1.5	1.5	0.0	0.0
f(I)	0.40	0.36	1.10	0.70	1.02	0.95	0.22	0.44	0.00	0.00
Rev. abs.	-12	-14	7	-30	4	-9	-7	-5	-5	-8

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

Tabelle 15.5‑e Concentration 313 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	6	16	65	81	148	144	3	2	1	2
Repl. 2	11	13	75	81	152	148	3	3	4	5
Repl. 3	2	9	54	52	164	160	5	5	1	6
Mean	6	13	65	71	155	151	4	3	2	4
sd	4.5	3.5	10.5	16.7	8.3	8.3	1.2	1.5	1.7	2.1
f(I)	0.30	0.59	0.90	0.72	0.94	0.90	0.44	0.33	0.40	0.50
Rev. abs.	-14	-9	-7	-28	-10	-17	-5	-6	-3	-4

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

 

Tabelle 15.5‑f   Concentration 156 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	9	12	55	80	140	144	9	5	4	6
Repl. 2	7	8	74	60	156	148	2	4	1	3
Repl. 3	6	15	74	72	160	164	4	4	2	6
Mean	7	12	68	71	152	152	5	4	2	5
sd	1.5	3.5	11.0	10.1	10.6	10.6	3.6	0.6	1.5	1.7
f(I)	0.35	0.55	0.94	0.72	0.92	0.90	0.56	0.44	0.40	0.63
Rev. abs.	-13	-10	-4	-28	-13	-16	-4	-5	-3	-3

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

Tabelle 15.5‑g Concentration 78 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	6	13	43	100	156	156	5	8	3	2
Repl. 2	6	16	56	97	156	160	2	6	1	6
Repl. 3	10	14	60	87	172	164	3	5	3	7
Mean	7	14	53	95	161	160	3	6	2	5
sd	2.3	1.5	8.9	6.8	9.2	4.0	1.5	1.5	1.2	2.6
f(I)	0.35	0.64	0.74	0.96	0.98	0.95	0.33	0.67	0.40	0.63
Rev. abs.	-13	-8	-19	-4	-4	-8	-6	-3	-3	-3

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

 

 

3      Data of Experiment 2b

3.1      Determination of Titre

Criterion:            The determination of titre should give a number of at least 10⁹ cells/mL, correlating to 100 colonies/plate after dilution.

 

Exact colony counts could not be determined due to the strong growth of the bacteria, but

by visual inspection it was obvious that the values exceed 100 colonies/ plate

 

Tabelle 16.1‑a Titre Values (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	s.g.	s.g.	--	--	s.g.	--	s.g.	s.g.	s.g.	s.g.
Repl. 2	s.g.	s.g.	--	--	s.g.	--	s.g.	s.g.	s.g.	s.g.
Assessment	ok	ok	--	--	ok	--	ok	ok	ok	ok

s.g.= strong growth, too strong for counting of colonies

ok= the criterion was fulfilled

-- = not tested

3.2      Sterility Control

Criterion:            No colony per plate may grow.

Tabelle 16.2‑a Sterility (colonies per plate)

	Demin. water	DMSO	Ethanol
Repl. 1	0	0	0
Repl. 2	0	0	0
Repl. 3	0	0	0
Repl. 4	0	0	0
Assessment	ok	ok	ok

ok= the criterion was fulfilled

 

 

 

3.3      Spontaneous Revertants

3.3.1    Demin. Water

Tabelle 16.3‑a Spontaneous Revertants demin. water (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	15	15	--	--	164	--	5	6	4	4
Repl. 2	17	17	--	--	168	--	7	6	4	5
Repl. 3	17	20	--	--	172	--	8	7	4	5
Mean	16	17	--	--	168	--	7	6	4	5
sd	1.2	2.5	--	--	4.0	--	1.5	0.6	0.0	0.6

sd = standard deviation ±

-- = not tested

 

3.3.2    DMSO

Tabelle 16.3‑b Spontaneous Revertants DMSO (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	15	15	--	--	168	--	7	5	3	3
Repl. 2	18	18	--	--	176	--	7	6	4	4
Repl. 3	19	18	--	--	176	--	7	11	5	4
Mean	17	17	--	--	173	--	7	7	4	4
sd	2.1	1.7	--	--	4.6	--	0.0	3.2	1.0	0.6

sd = standard deviation ±

-- = not tested

 

3.3.3    Ethanol

Tabelle 16.3‑c Spontaneous Revertants Ethanol (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	16	17	--	--	152	--	3	4	3	4
Repl. 2	17	18	--	--	160	--	6	6	3	5
Repl. 3	18	19	--	--	164	--	7	6	4	5
Mean	17	18	--	--	159	--	5	5	3	5
sd	1.0	1.0	--	--	6.1	--	2.1	1.2	0.6	0.6

sd = standard deviation ±

-- = not tested

 

 

3.4      Positive Controls

 

• Without metabolic activation:
  4-Nitro-1,2-phenylene diamine (NPD) in DMSO
  Sodium azide (Na-azide) in demin. water
  Mitomycin C (MMC) in demin. water

 

 

• With metabolic activation:
  2-Amino anthracene (2-AA) in DMSO
  Benzo-a-pyrene (BaP) in DMSO

 

For used concentrations see chapter 6.2.4, page 14.

 

Tabelle 16.4‑a Diagnostic Mutagens (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Substance	NPD	BaP	Na-azide	2-AA	MMC	2-AA	Na-azide	2-AA	NPD	2-AA
Repl. 1	720	76	--	--	816	--	296	248	72	33
Repl. 2	776	88	--	--	848	--	328	256	80	36
Repl. 3	816	100	--	--	888	--	344	264	92	37
Mean	771	88	--	--	851	--	323	256	81	35
sd	48.2	12.0	--	--	36.1	--	24.4	8.0	10.1	2.1
f(I)	45.35	5.18	--	--	5.07	--	46.14	36.57	20.25	8.75
Rev. abs.	754	71	--	--	683	--	316	249	77	31

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

 

 

3.5      Mutagenicity Test Test Item 2,2'-Azobis(2,4-dimethylvaleronitrile)

Tabelle 16.5‑a Concentration 625 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	--	11	--	--	84	--	5	--	--	--
Repl. 2	--	11	--	--	92	--	6	--	--	--
Repl. 3	--	12	--	--	96	--	8	--	--	--
Mean	--	11	--	--	91	--	6	--	--	--
sd	--	0.6	--	--	6.1	--	1.5	--	--	--
f(I)	--	0.61	--	--	0.57	--	1.20	--	--	--
Rev. abs.	--	-7	--	--	-68	--	1	--	--	--

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

Tabelle 16.5‑b Concentration 313 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	--	15	--	--	156	--	3	4	--	4
Repl. 2	--	16	--	--	160	--	6	5	--	5
Repl. 3	--	17	--	--	176	--	7	6	--	5
Mean	--	16	--	--	164	--	5	5	--	5
sd	--	1.0	--	--	10.6	--	2.1	1.0	--	0.6
f(I)	--	0.89	--	--	1.03	--	1.00	1.00	--	1.00
Rev. abs.	--	-2	--	--	5	--	0	0	--	0

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

 

Tabelle 16.5‑c Concentration 156 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	--	16	--	--	156	--	4	5	--	3
Repl. 2	--	17	--	--	160	--	4	8	--	4
Repl. 3	--	17	--	--	164	--	4	11	--	5
Mean	--	17	--	--	160	--	4	8	--	4
sd	--	0.6	--	--	4.0	--	0.0	3.0	--	1.0
f(I)	--	0.94	--	--	1.01	--	0.80	1.60	--	0.80
Rev. abs.	--	-1	--	--	1	--	-1	3	--	-1

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

Tabelle 16.5‑d Concentration 78 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	11	18	--	--	152	--	3	5	3	4
Repl. 2	11	18	--	--	160	--	5	9	4	5
Repl. 3	12	18	--	--	168	--	6	9	4	5
Mean	11	18	--	--	160	--	5	8	4	5
sd	0.6	0.0	--	--	8.0	--	1.5	2.3	0.6	0.6
f(I)	0.65	1.00	--	--	1.01	--	1.00	1.60	1.33	1.00
Rev. abs.	-6	0	--	--	1	--	0	3	1	0

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

Tabelle 16.5‑e Concentration 39 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	16	17	--	--	152	--	3	4	3	3
Repl. 2	17	19	--	--	152	--	6	4	4	3
Repl. 3	17	20	--	--	156	--	7	6	5	5
Mean	17	19	--	--	153	--	5	5	4	4
sd	0.6	1.5	--	--	2.3	--	2.1	1.2	1.0	1.2
f(I)	1.00	1.06	--	--	0.96	--	1.00	1.00	1.33	0.80
Rev. abs.	0	1	--	--	-6	--	0	0	1	-1

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

 

Tabelle 16.5‑f   Concentration 19.5 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	17	17	--	--	160	--	4	5	3	3
Repl. 2	18	18	--	--	164	--	4	6	3	5
Repl. 3	18	19	--	--	176	--	5	9	5	5
Mean	18	18	--	--	167	--	4	7	4	4
sd	0.6	1.0	--	--	8.3	--	0.6	2.1	1.2	1.2
f(I)	1.06	1.00	--	--	1.05	--	0.80	1.40	1.33	0.80
Rev. abs.	1	0	--	--	8	--	-1	2	1	-1

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

Tabelle 16.5‑g Concentration 9.8 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	17	19	--	--	152	--	4	6	3	3
Repl. 2	18	19	--	--	156	--	5	7	3	4
Repl. 3	18	19	--	--	168	--	8	10	5	4
Mean	18	19	--	--	159	--	6	8	4	4
sd	0.6	0.0	--	--	8.3	--	2.1	2.1	1.2	0.6
f(I)	1.06	1.06	--	--	1.00	--	1.20	1.60	1.33	0.80
Rev. abs.	1	1	--	--	0	--	1	3	1	-1

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

Tabelle 16.5‑h Concentration 4.88 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	16	--	--	--	--	--	--	4	3	5
Repl. 2	16	--	--	--	--	--	--	4	4	5
Repl. 3	18	--	--	--	--	--	--	4	5	5
Mean	17	--	--	--	--	--	--	4	4	5
sd	1.2	--	--	--	--	--	--	0.0	1.0	0.0
f(I)	1.00	--	--	--	--	--	--	0.80	1.33	1.00
Rev. abs.	0	--	--	--	--	--	--	-1	1	0

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

 

Tabelle 16.5‑i   Concentration 2.44 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	17	--	--	--	--	--	--	--	3	--
Repl. 2	17	--	--	--	--	--	--	--	4	--
Repl. 3	19	--	--	--	--	--	--	--	4	--
Mean	18	--	--	--	--	--	--	--	4	--
sd	1.2	--	--	--	--	--	--	--	0.6	--
f(I)	1.06	--	--	--	--	--	--	--	1.33	--
Rev. abs.	1	--	--	--	--	--	--	--	1	--

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

Tabelle 16.5‑j   Concentration 1.22 µg/plate (colonies per plate)

Strain	TA98		TA100		TA102		TA1535		TA1537
Induction	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Repl. 1	15	--	--	--	--	--	--	--	3	--
Repl. 2	16	--	--	--	--	--	--	--	4	--
Repl. 3	19	--	--	--	--	--	--	--	5	--
Mean	17	--	--	--	--	--	--	--	4	--
sd	2.1	--	--	--	--	--	--	--	1.0	--
f(I)	1.00	--	--	--	--	--	--	--	1.33	--
Rev. abs.	0	--	--	--	--	--	--	--	1	--

sd = standard deviation ±

f(I) = increase factor, calculation see chapter 7.4, page 21

Rev.abs. = absolute revertants, calculation see chapter 7.4, page 21

-- = not tested

 

 

 

Conclusions:

Based on the results of this study it is concluded that 2,2'-Azobis(2,4-
dimethylvaleronitrile) is not mutagenic in the Salmonella typhimurium strains TA98,
TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activa-
tion under the experimental conditions in this study.

Executive summary:

Findings and Results:
The study procedures described in this report were based on the most recent Guideline
OECD 471 (2020) and EU Method B.13/14 (2008).
The test item 2,2'-Azobis(2,4-dimethylvaleronitrile) was tested in the Bacterial reverse mu-
tation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and
TA1537).
The test was performed in three valid experiments in the presence and absence of metabolic
activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing
for absence of metabolic activation.
Exp. 2 had to be repeated (exp. 2b) due to an insufficient number of analyzable non-toxic
concentrations.

 

Experiment 1:
In the first experiment, the test item (dissolved in ethanol) was tested up to concentrations
of 5000 µg/plate in the absence and presence of S9 mix in the strains TA98, TA100, TA102,
TA1535 and TA1537 using the plate incorporation method.
The test item showed a few precipitates on the plates with TA98, TA100, TA1535 and
TA1537 at 5000 µg/plate (for details see chapter 8.1.2, page 22).
No signs of toxicity towards the bacteria strains were observed.
The results of this experiment showed that none of the tested concentrations induced a
relevant or dose-related increase in the number of revertants in all tested strains, in the
presence and the absence of metabolic activation.

 

Experiment 2:
Based on the results of the first experiment, the test item was tested up to concentrations of
5000 µg/plate in the presence and absence of S9 mix in all bacteria strains using the pre-
incubation method.
The test item showed a few precipitates on the plates with TA98, TA100, TA102, TA1535
and TA1537 at 5000, 2500 and 1250 µg/plate.
Signs of toxicity towards the bacteria strains TA98 (+/-S9), TA100 (-S9), TA102 (-S9),
TA1535 (+/-S9), TA1537 (+/-S9) were observed (for details see chapter 8.2.2, page 24).
Only for the strains TA100 (+/-S9) and TA102 (+S9), sufficient (at least five) non-toxic con-
centrations could be evaluated in this experiment. In these strains, none of the tested con-
centrations induced a relevant or dose-related increase in the number of revertants.
For the other strains, a repetition was performed in exp. 2b.

Experiment 2b:
Based on the results of exp. 2, the test item was tested up to individual concentrations per
strain (see chapter 7.2, page 18) in the presence and absence of S9 mix in all bacteria
strains using the pre-incubation method.
The test item showed no precipitates on the plates at any of the concentrations and no signs
of toxicity could be observed. Therefore, sufficient concentrations (at least five) could be
evaluated for mutagenicity.
The results of this experiment showed that none of the tested concentrations induced a
relevant or dose-related increase in the number of revertants in all tested strains, in the
presence and the absence of metabolic activation

Reference 3

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

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Qualifier:

according to guideline

Guideline:

other: EU Method B.49

GLP compliance:

yes (incl. QA statement)

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 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.

Justification for classification or non-classification

The available information is conclusive but not sufficient for classification.