4,4'-azobis[4-cyanovaleric] acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March 2017 - 08 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Methode B.49 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-azobis[4-cyanovaleric] acid

EC Number:

220-135-0

EC Name:

4,4'-azobis[4-cyanovaleric] acid

Cas Number:

2638-94-0

Molecular formula:

C12H16N4O4

IUPAC Name:

4,4'-azobis[4-cyanovaleric] acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: 150726 (Test item No.: 16/0252-1)
- Expiration date of the lot/batch: 26 July 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator

Method

Species / strain

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The TK6 cell line had shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of CytB.
- Doubling time: about 12 - 14 hours
- Methods for maintenance in cell culture: Deep-frozen cell stocks were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5 mL RPMI 1640 medium (containing a L-glutamine source and antibiotics) supplemented with 10% (v/v) FCS. Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity and subcultured twice weekly.

MEDIA USED
- Type and identity of media: RPMI 1640 containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL) and 1% (v/v) amphotericine B (250 μg/mL).
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

Test concentrations: 0; 78.1; 156.3; 312.5; 625.0; 1250.0; 2500.0 μg/mL
Justification for top dose: 2500 μg/mL (correspond to 2038 μg/mL of the main ingredient) were selected as top dose based on current OECD guidline

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to insolubility of the test substance in water, DMSO was selected as vehicle, which has been demonstrated to be suitable in the in vitro cytogenetic assay and for which historical control data are available.

Controls

Untreated negative controls:

yes

Remarks:

medium only

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4 h (experiment I, II and II with S9 mix, experiment I without S9 mix), 24 h (experiment III without S9 mix)
- Recovery time: 20 hours (experiment I and II with S9 mix, experiment I without s9 mix), 40 hours (Experiment III with S9 mix)
- Harvest time: 24 hours (experiment I and II with S9 mix, experiment I and I without S9 mix), 44 hours (experiment II with S9 mix

STAIN: mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) and propidium iodide

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Single cell suspensions were prepared from each test group by resuspending. Then, the cell number per flask of each cell suspension was determined using a cell counter. Subsequently, 5x10^4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides using a Cytospin centrifuge. At least two slides per flask were prepared. In the case of strongly reduced cell numbers no slides were prepared. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes. Before scoring, the slides were stained with a mixture of DAPI and propidium iodide.

NUMBER OF CELLS EVALUATED: at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells were scored.

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)

Evaluation criteria:

A test substance is considered to be clearly positive if the following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
A test substance is considered to be clearly negative if the following criterion is met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.

Statistics:

The statistical evaluation of the data was carried out using an appropriate statistical analysis. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test). Furthermore, a statistical trend test (SAS; Proc Reg) was performed to assess a possible doserelated increase of micronucleated cells.

Results and discussion

Test results

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

only in the 2nd Experiment with S9 mix at one concentration level (625.0 μg/mL) reduced cell counts (below 50% of the concurrent vehicle control value) was observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH values were not influenced by test substance treatment. However, the pH of the stock solutions was prediluted with culture medium and adjusted to a physiological value using small amounts of NaOH.
- Effects of osmolality: Osmolality was not influenced by test substance treatment.
- Water solubility: The test substance was insoluble in water.
- Precipitation: No precipitation of the test substance in culture medium was observed.

RANGE-FINDING/SCREENING STUDIES: yes, Pretests for choice of vehicle and dose selection

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: at least 1000 binucleated cells per culture (in total at least 2000 binucleated cells per test group)
- Indication whether binucleate or mononucleate where appropriate: Only binucleated cells were scored.

HISTORICAL CONTROL DATA
- Please refer to the field “Any other information on results incl. tables’”

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

OTHER:
- The 1st experiment (with S9 mix) was invalid and therefore repeated (2nd experiment).

Any other information on results incl. tables

Table 1: Summary table - experimental parts without S9 mix

Exp.	Exposure/ Preparation interval	Test groups [µg/mL]	S9 mix	Prec.*	Micro-nucleated cells** [%]	Cytotoxicity
						Proliferation index cytostasis (CBPI)	Cell count [%]
1	4/24	Vehicle control^1	-	n.d.	0.7	0.0	100.0
		78.1	-	-	n.d.	n.d.	94.4
		156.3	-	-	n.d.	n.d.	93.8
		312.5	-	-	n.d.	n.d.	83.9
		625.0	-	-	0.7	3.6	75.1
		1250.0	-	-	1.3	9.1	81.2
		2500.0	-	-	1.0	-0.4	87.7
		Positive control^2	-	n.d.	2.1^S	27.7	82.7
		Positive control^3	-	n.d.	4.2^S	51.2	83.0
3	24/24	Vehicle control^1	-	n.d.	1.1	0.0	100.0
		Negative control	-	n.d.	1.3	-13.6	102.9
		78.1	-	-	n.d.	n.d.	103.3
		156.3	-	-	n.d.	n.d.	105.3
		312.5	-	-	n.d.	n.d.	100.5
		625.0	-	-	1.1	-7.4	95.1
		1250.0	-	-	1.7	-5.2	105.7
		2500.0	-	-	1.0	9.4	101.2
		Positive control^4	-	n.d.	2.4^S	17.4	81.3
		Positive control^5	-	n.d.	n.d.	n.d.	88.7

*       Precipitation in culture medium at the end of exposure period (macroscopic)

**      Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

^S       Frequency statistically significant higher than corresponding control values

n.d.   Not determined

n.p.   No cytospin slides prepared due to strong cytotoxicity

¹        DMSO 1% (v/v)

²        MMC 0.125μg/mL

³        MMC 0.250μg/mL

⁴        MMC 0.015μg/mL

⁵        MMC 0.030μg/mL

 

Table 2: Summary table - experimental parts with S9 mix

Exp.	Exposure/ Preparation interval	Test groups [µg/mL]	S9 mix	Prec.*	Micro-nucleated cells** [%]	Cytotoxicity
						Proliferation index cytostasis (CBPI)	Cell count [%]
2	4/24	Vehicle control^1	+	n.d.	1.0	0.0	100.0
		156.3	+	-	n.d.	n.d.	75.1
		312.5	+	-	n.d.	n.d.	65.4
		625.0	+	-	0.7	-12.3	41.5
		1250.0	+	-	1.4	-16.3	64.9
		2500.0	+	-	1.1	-20.0	80.5
		Positive control^6	+	n.d.	2.5^S	-2.4	87.3
		Positive control^7	+	n.d.	n.d.	n.d.	89.3
3	4/44	Vehicle control^1	+	n.d.	1.0	0.0	100.0
		156.3	+	-	n.d.	n.d.	101.8
		312.5	+	-	n.d.	n.d.	102.3
		625.0	+	-	1.1	-4.3	97.7
		1250.0	+	-	1.4	-4.2	95.0
		2500.0	+	-	1.3	-11.5	113.8
		Positive control^6	+	n.d.	2.9^S	1.0	126.6
		Positive control^7	+	n.d.	n.d.	n.d.	119.7

*       Precipitation in culture medium at the end of exposure period (macroscopic)

**      Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

^S       Frequency statistically significant higher than corresponding control values

n.d.   Not determined

n.p.   No cytospin slides prepared due to strong cytotoxicity

¹        DMSO 1% (v/v)

⁶        CPP 0.5 μg/mL

⁷        CPP 1.0 μg/mL

 

Table 3: Historical negative control data

Cytochalasin B Method, Period: June 2015 – December 2016

Without and with S9 mix – all vehicles*
Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h, 24 h / 24 h
Mean	0.7 %
Minimum	0.3 %
Maximum	1.4 %
Standard Deviation	0.28 %
95% Lower Control Limit	0.1 %
95% Upper Control Limit	1.3 %
No. of experiments	43

* = culture medium, DMSO 1% (v/v)

 

Table 4: Historical negative control data - without S9 mix

Cytochalasin B Method, Period: June 2015 - December 2016

Without S9 mix – all vehicles*
Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h & 24 h / 24 h
Mean	0.7 %
Minimum	0.3 %
Maximum	1.4 %
Standard Deviation	0.29 %
95% Lower Control Limit	0.1 %
95% Upper Control Limit	1.3 %
No. of experiments	38

* = culture medium, DMSO 1% (v/v)

 

Table 5: Historical negative control data - with S9 mix

Cytochalasin B Method, Period: June 2015 - December 2016

With S9 mix – all vehicles*
Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h
Mean	0.9 %
Minimum	0.6 %
Maximum	1.3 %
Standard Deviation	0.25 %
95% Lower Control Limit	0.1 %
95% Upper Control Limit	1.6 %
No. of experiments	5

* = culture medium, DMSO 1% (v/v)

 

Table 6: Historical positive control data - without S9 mix

Cytochalasin B Method, Period: June 2015 - December 2016

Without S9 mix Mitomycin C (MMC) 0.125 – 0.250 μg/mL
Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h
Mean	4.3 %
Minimum	3.0 %
Maximum	6.5 %
Standard Deviation	1.68 %
95% Lower Control Limit	0.0 %
95% Upper Control Limit	10.3 %
No. of experiments	4

 

Table 7: Historical positive control data - with S9 mix

Cytochalasin B Method, Period: June 2015 - December 2016

With S9 mix Cyclophosphamide (CPP) 0.625 – 1.250 μg/mL
Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h
Mean	2.3 %
Minimum	2.0 %
Maximum	2.4 %
Standard Deviation	0.22 %
95% Lower Control Limit	1.2 %
95% Upper Control Limit	3.3 %
No. of experiments	3

 

 

95% Control limit (= Prediction interval)

For the control mean the 95% control limit was calculated according to the formulae

Mean ± SD * Sqrt (1/n + 1) * t_n-1;0.975

 

Mean    = Mean of the control means

SD        = Standard deviation of the control means

n           = Number of controls

t_n-1;0.975    = t-Quantile with n-1 degrees of freedom

Sqrt      = Square root (√)