N,N'-(2-chloro-1,4-phenylene)bis[4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

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:

N,N'-(2-chloro-1,4-phenylene)bis[4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

EC Number:

252-772-5

EC Name:

N,N'-(2-chloro-1,4-phenylene)bis[4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Cas Number:

35869-64-8

Molecular formula:

C40H23Cl3N8O8

IUPAC Name:

N,N'-(2-chloro-1,4-phenylene)bis{4-[(4-chloro-2-nitrophenyl)diazenyl]-3-hydroxy-2-naphthamide}

Test material form:

solid: nanoform

Specific details on test material used for the study:

Batch identification: 0004501436
Content: 99.4 %
physical state: solid; brown
storage at room temperature

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.

- Suitability of cells:
- Normal cell cycle time (negative control):

Cytokinesis block (if used):

Cytochalasin B (6 µg/mL)

Metabolic activation:

not applicable

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 was tested. 1 mg/L was the highest concentration which remained in a homogenous state as determined by an
Analytical Ultracentrifugation (AUC) method.

1, 3, 10, 30, 60, 100, 1000 mg/L

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

Remarks:

WC was used as a particle control for nanomaterials

Details on test system and experimental conditions:

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

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

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 1000 per culture (2000 per concentration)
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): The analysis of micronuclei was carried out according to the following criteria of Countryman and Heddle (14).
- 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.

- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification):
- Determination of polyploidy:
- Determination of endoreplication:


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: In case of toxicity, the top concentration should produce 55 ± 5% cytotoxicity: reduction of the
proliferation index (CBPI) to 45 ± 5% of the concurrent vehicle control. The CBPI was determined in 500 cells per culture (1000 cells per test group).

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:

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:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: homogeneous dispersion of nanomaterial

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

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: 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
- Concurrent vehicle negative and positive control data : yes

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o 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)
- Positive historical control data: see table 4
- Negative (solvent/vehicle) historical control data: see table 5

Any other information on results incl. tables

Table 1: Summary of results

Exposure/Recovery/ Preparation interval	Test groups [μg/mL]	Micro- nucleated cells** [%]	Cytotoxicity Proliferation index cytostasis [%]
20/0/20	Vehicle control (0.05% BSA-water (w/v))	0.7	0.0
	1.0	n.d.	-2.0
	3.0	n.d.	-8.2
	10.0	0.8	-0.9
	30.0	0.5	6.3
	60.0	n.d.	-0.4
	100.0	0.5	2.4
	1000.0	0.5	-3.9
	WC-Co 30	0.7	51.8
	WC-Co 60	1.2^S	58.5
	WC-Co 100	n.d.	65.2
	Positive control (MMC 0.04 μg/mL)	7.9^S	6.5
	Positive control (Col 0.05 μg/mL)	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

Table 2: Proliferation Index (CBPI)

Test group [µg/mL]	Culture	Mononucleated cells	Binucleated cells	Multinucleated cells	CBPI absolute	CBPI cyto- stasis [%]
BSA	A	90	399	11	1.79	0.0
	B	153	327	20
1.0	A	113	371	16	1.80	-2.0
	B	124	351	25
3.0	A	84	386	30	1.85	-8.2
	B	114	365	21
10.0	A	102	375	23	1.80	-0.9
	B	145	336	19
30.0	A	153	339	8	1.74	6.3
	B	126	365	9
60.0	A	114	371	15	1.79	-0.4
	B	131	348	21
100.0	A	109	368	23	1.77	2.4
	B	153	339	8
1000.0	A	119	363	18	1.82	-3.9
	B	98	384	18
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

Table 3: Analysis of micronuclei

Test group   Culture [µg/mL]		No. of evaluated cells	Cells containing Micronuclei
BSA	A	1000	7	14	0.7
	B	1000	7
1.0	A	n.d.
	B
3.0	A
	B
10.0	A	1000	9	15	0.8
	B	1000	6
30.0	A	1000	4	9	0.5
	B	1000	5
60.0	A	n.d.
	B
100.0	A	1000	7	10	0.5
	B	1000	3
1000.0	A	1000	5	9	0.5
	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	7.9S
	B	1000	76
Col 0.05	A	1000	42	74	3.7S
	B	1000	32

S Frequency statistically significantly higher than corresponding control values

Table 4 Historical control data (positive control)

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

Table 5: historical control data (vehicle)

	Micronucleated cells [%]
Exposure period of 20h
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