3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(3-chloro-o-tolyl)benzamide]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

adapted for nanomaterials

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 Jul 2016

Deviations:

yes

Remarks:

additional characterization of particles in cell culture medium; no 4h exposure (too short for insoluble particles)

Principles of method if other than guideline:

Adapations (test material preparation, particle characterization) NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018

GLP compliance:

yes (incl. QA statement)

Remarks:

The particle characterization in cell culture medium was not performed under GLP.

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(3-chloro-o-tolyl)benzamide]

EC Number:

226-970-7

EC Name:

3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(3-chloro-o-tolyl)benzamide]

Cas Number:

5580-57-4

Molecular formula:

C43H35Cl5N8O6

IUPAC Name:

3,3'-{(2-chloro-5-methyl-1,4-phenylene)bis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}bis[4-chloro-N-(3-chloro-2-methylphenyl)benzamide]

Test material form:

solid: nanoform

Details on test material:

Physical state/ appearance: yellow powder

Specific details on test material used for the study:

Batch no.: 0004460012
Purity: 99.3 wt%
Date of production: 18 Jan 2011
Expiry/Retest date: 18 Jan 2031
Appearance: Yellow powder
Storage conditions: ambient (RT)

Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance preparations

Storage stability: The stability of the test substance was guaranteed until 18 Jan 2031 as indicated by the sponsor, and the sponsor holds this responsibility.

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: primary human lymphocytes (buffy coat cells) Fresh Blood was collected from a single donor for each experiment.
Only healthy, non-smoking donors and not receiving medication were used. In this study, in the main experiment a 28 year old male donor was used.
The lymphocytes of each donor have previously been shown to respond well to stimulation of proliferation with phytohemagglutinin (PHA) and to the used positive control substances.

Cytokinesis block (if used):

Cytochalasin B (6 µg/mL)

Metabolic activation:

without

Test concentrations with justification for top dose:

The test material fulfills the criteria of an insoluble nanomaterial and shall be present in the cell culture medium as a homogenous stable nanoparticle dispersion.
In a pretest, inhomogeneous suspensions (aggregation) of the test substance in the vehicle 0.05% w/v BSA in water were obtained in concentrations of 5.0, 10.0 and 20.0 mg/mL. The highest concentration, which could be homogenously formulated in 0.05% w/v BSA-water was 2.56 mg/mL corresponding to 256 µg/mL in culture medium (1:10 dilution).

Vehicle / solvent:

In accordance to the “SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2,
dated 6 May 2018, 0.05% w/v bovine serum albumin water (BSA-water) was used as vehicle.
The final concentration of the vehicle 0.05% w/v BSA-water in culture medium was 10% (v/v).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

mitomycin C

other: Tungsten Carbide-Cobalt (nanomaterial positive control)

Details on test system and experimental conditions:

TIME SCHEDULE:
Day 1: Activation of the cells with Phytohemagglutinin
Day 3: Test substance incubation (approx. 48 hours after activation)
Day 4: Removal of test substance by intense washing; treatment with Cyt B
Day 5: Preparation of the slides

Since a pulse treatment (as described in OECD 487) does not allow enough time for the nanoparticles to enter the cell, only continuous treatment protocol was used.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate (two flasks with two slides prepared for each)
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48 h (stimulating with phytohemagglutinin)
- Exposure duration/duration of treatment: 20 h
- Harvest time after the end of treatment: 20 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
cytokinesis blocking: 6 µg/mL Cytochalasin B (Cyt B), 20 h
- Methods of slide preparation and staining technique: The cells were centrifuged (900 g, 5 min, 4°C) and suspended in fresh fixative and incubated for 20 min at 4°C. The fixation step was repeated twice. After the last fixation step, the cells were centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fresh fixative and spread on slides. The slides were dipped in deionized water, the cells were pipetted on the slide and fixed by passing through a flame. The cells were stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2, 10 min) and mounted.
- Number of cells spread and analysed per concentration: At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.
- Criteria for scoring micronucleated cells: The analysis of micronuclei was carried out according to the following criteria of Countryman and Heddle (1976).
- The diameter of the micronucleus was less than 1/3 of the main nucleus
- The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
- Only binucleated cells were scored.
Slides were coded randomly before microscopic analysis with an appropriate computer program. Cultures with few isolated cells were analyzed for micronuclei.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it was determined in 500 cells per culture (1000 cells per test group). This value indicates the average number of cell cycles per cell during the period of exposure to the actin polymerization inhibitor Cyt B.

CBPI = ((No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)) / (Total number of cells)

The CBPI was used to calculate the % cytostasis (relative inhibition of cell growth compared to the respective vehicle control group) - a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

% Cytostasis = 100 - 100 {(CBPIT - 1) / (CBPIC - 1)}

T = test substance treated culture
C = vehicle control culture

pH value:
At the beginning of the treatment period, the pH was measured at least for the top concentration and for the vehicle control, each.

Evaluation criteria:

Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells in the control groups (vehicle/positive) and in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged
acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.
Assessment criteria
A test substance is considered to be clearly positive if all 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.
• The number of micronucleated cells exceeded both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

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).
In addition, a statistical trend test (SAS procedure REG (16)) was performed to assess a possible dose-related increase of micronucleated cells. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report (17).
The dependent variable was the number of micronucleated cells and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: pH values were not relevantly influenced by test substance treatment.
- Data on osmolality: not determined
- Possibility of evaporation from medium: not applicable
- Water solubility: insoluble
- Precipitation and time of the determination: The test material was tested as a stable dispersion of particles in the nano-size range.

RANGE-FINDING/SCREENING STUDIES (if applicable):
Non-GLP experiments on the stability of the test material dispersion and the particle size distribution were carried out to determine the adequate concentrations.

STUDY RESULTS
In the Main experiment all evaluated values of the test substance (0.3 – 0.7% micronucleated cells) were within the range of the 95% control limit of the historical negative control data (0.2 – 0.9% micronucleated cells). A statistical significance compared to the concurrent vehicle control value (0.7% micronucleated cells) or dose dependency was not observed.
The positive control substances MMC (0.04 μg/mL) and Colchicine (0.05 μg/mL) induced statistically significantly increased micronucleus frequencies. In this study, the frequencies of micronucleated cells (7.9% and 3.7% micronucleated cells (MMC and Col, respectively) were compatible to the historical positive control data range.
The particular positive control substance Tungsten Carbide-Cobalt (WC-Co) induced an increased micronucleus frequency at 60 μg/mL (1.2%) and exceeded the 95% upper control limit of the historical negative data range (0.2 – 0.9% micronucleated cells). This increase was, however, not statistically significant.

Results from cytotoxicity measurements: No cytotoxicity observed for the test material (CBPI)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
See: Any other information (Tables 5 and 6)

Any other information on results incl. tables

Table 1: Summary Table:

Exp.	Exposure/ Recovery/ Preparation interval [h]	Test groups [µg/mL]	Micro- nucleated cells [%]	Cytotoxicity Proliferation index cytostasis [%]
1	20/0/20	Vehicle control^1	0.7	0.0
		1.0	n.d.	1.6
		3.0	n.d.	-1.3
		10.0	0.7	-11.7
		30.0	0.3	-1.6
		60.0	n.d.	-1.1
		100.0	0.3	-7.1
		256.0	0.3	-4.2
		WC-Co 30	0.7	51.8
		WC-Co 60	1.2	58.5
		WC-Co 100	n.d.	65.2
		Positive control^2	7.9^s	6.5
		Positive control^3	3.7^s	19.0

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

^S         Frequency statistically significantly higher than corresponding control values

n.d.    Not determined

n.s.    Not scorable due to strong cytotoxicity

¹         0.05% BSA-water (w/v)         ²        MMC 0.04 µg/mL                   ³        Col 0.05 µg/mL

 

Table 2: Proliferation index (CBPI) - Main experiment

Test group [µg/mL]	Culture	Mononucleated cells	Binucleated cells	Multinucleated cells	CBPI absolute	CBPI cyto- stasis [%]
Vehicle control*	A	90	399	11	1.79	0.0
	B	153	327	20
1.0	A	110	385	5	1.78	1.6
	B	141	338	21
3.0	A	141	341	18	1.80	-1.3
	B	114	351	35
10.0	A	134	354	12	1.88	-11.7
	B	87	324	89
30.0	A	135	357	8	1.80	-1.6
	B	101	370	29
60.0	A	115	380	5	1.80	-1.1
	B	115	363	22
100.0	A	112	373	15	1.84	-7.1
	B	97	365	38
256.0	A	117	368	15	1.82	-4.2
	B	102	373	25
WC-Co 30	A	348	142	10	1.38	51.8
	B	284	214	2
WC-Co 60	A	354	139	7	1.33	58.5
	B	326	174	0
WC-Co 100	A	362	130	8	1.27	65.2
	B	373	126	1
MMC 0.04	A	159	322	19	1.74	6.5
	B	141	341	18
Col 0.05	A	255	230	15	1.64	19.0
	B	134	354	12

* 0.05% w/v BSA-water

 

Table 3: Analysis of micronuclei - Main experiment

Test group [µg/mL]	Culture	No. of evaluated cells	Cells containing Micronuclei
			[n]	[n]	[%]
Vehicle control*	A	1000	7	14	0.7
	B	1000	7
1.0	A	n.d.
	B
3.0	A
	B
10.0	A	1000	6	13	0.7
	B	1000	7
30.0	A	1000	4	6	0.3
	B	1000	2
60.0	A	n.d.
	B
100.0	A	1000	5	6	0.3
	B	1000	1
256.0	A	1000	2	6	0.3
	B	1000	4
WC-Co 30	A	1000	5	14	0.7
	B	1000	9
WC-Co 60	A	1000	4	23	1.2
	B	1000	19
WC-Co 100	A	n.d.
	B
MMC 0.04	A	1000	81	157	7.9^s
	B	1000	76
Col 0.05	A	1000	42	74	3.7^s
	B	1000	32

* 0.05% w/v BSA-water

^S Frequency statistically significantly higher than corresponding control values

 

Table 4: Linear trend test

Linear trend-test	Slope	One-sided p-value*
Main experiment	-0.00124	0.94854

*     The linear trend-test testing for an increased number of micronucleated cells is significant (significance level of 5%), if the one-sided p-value is lower than 0.05.

 

Table 5: Hitorcial negative control data (2018 - 2020)

Micronucleated cells [%]
Exposure period	20 hrs
Mean	0.5
Minimum	0.2
Maximum	1.2
Standard Deviation	0.17
95% Lower Control Limit	0.2
95% Upper Control Limit	0.9
No. of Experiments	54

 

Table 6: Historical positice control data (2018-2020)

Micronucleated cells [%]
Exposure period	20 hrs	20 hrs
Substance and concentration	MMC 0.04 µg/mL	Col 0.05 µg/mL
Mean	4.1	4.0
Minimum	2.1	2.4
Maximum	7.1	7.2
Standard Deviation	0.93	1.05
No. of Experiments	48	45

Applicant's summary and conclusion

Conclusions:

Thus, under the experimental conditions chosen here, the conclusion is drawn that the test item has no potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence of metabolic activation.

Executive summary:

The test substance was tested for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity) in this study according OECD 487 and in complince with GLP. One experiment was carried out, incubating the cells for 20 h (20 h harvest time) with the test substance at concentrations in the range of 1.0 to 256 µg/mL. A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. In this study, 0.05% w/v BSA-water was selected as vehicle. The characterization of the nanomaterial in cell culture medium showed, that the particles were successfully dispersed into a stable suspension with partial agglomeration, that did not change significantly during the treatment period. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicine (Col) and the nanomaterial positive control Tungsten Carbide-Cobalt (WC-Co), led to the expected increase in the number of cells containing micronuclei.

The test substance was formulated in the given vehicle according to the NANOGENOTOXProject (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018.

In this study, no cytotoxicity indicated by reduced proliferation index (CBPI) was observed up to the highest applied test substance concentration. On the basis of the results of the present study, the test substance did not cause any biologically relevant in the number of cells containing micronuclei.
Thus, under the experimental conditions described, test material is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes.