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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 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

Test material

Test material information

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

This reference substance refers to from one to several impurities with potentially differing molecular formulas.

IUPAC Name:

Unknown impurities

Constituent 1

Reference substance name:

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

EC Number:

224-583-8

EC Name:

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

Cas Number:

4419-11-8

Molecular formula:

C14H24N4

IUPAC Name:

2-[(E)-2-(1-cyano-1,3-dimethylbutyl)diazen-1-yl]-2,4-dimethylpentanenitrile

Test material form:

solid: crystalline

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

Method

Target gene:

Thymidine Kinase locus

Species / strain

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

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.