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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro: Ames (Prival): negative; OECD 471
Clastogenicity in human lymphocytes: non clastogenic (OECD 487, nanomaterial protocol)

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

adapted for nanomaterials

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016, specific adaptions for nanomaterials as of - NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Issued by Landesamt für Umwelt, Rheinland-Pfalz, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Lot 0004079400
storage at room temperature
yellow solid
purity 99.4%

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For lymphocytes:
- 34 year old female donor, non-smoking
- Buffy coat cells were isolated from whole blood
- Whether blood from different donors were pooled or not: not applicable; single donor
- Mitogen used for lymphocytes:Cytochalasin B

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Based on the solubility properties of the test substance 256 µg/mLwas used as top concentration. Higher concentrations led to inhomogeneous formulations.
1, 3, 10, 30, 60, 100, 256 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 is 10% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

mitomycin C

other: 100, 60 and 30 µg/mL Tungsten Carbide-Cobalt (Nanostructures and Amorphous Material Inc.; Houston, Texas)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : At least 2 cultures were prepared per test group (referred to
as A and B), and at least 1000 cells per culture were evaluated for the occurrence of micronucleated
cells.
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium;
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 20h
- Harvest time after the end of treatment (sampling/recovery times): see treamtent with cyokinesis
blocking substance
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Identity of cytokinesis blocking substance: cytoB, 20h exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic
assays): The cells are transferred into tubes, centrifugated at 900 g for 5 min and washed with HBSS
. After washing, the cells are centrifuged (900 g, 5 min) and suspended in 0.65% KCl (37°C), incuba
tion for 10 minutes at 37°C. After the hypotonic treatment, the cells are fixed by adding of fixative (19
parts methanol and 1 part acetic acid). The cells are centrifuged (900 g, 5 min, 4°C) and fixed suspe
nded in fresh fixative and incubated for 20 min at 4°C. The fixation step is repeated twice. After the
last fixation step, the cells can be centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fre
sh fixative and spread on slides. The slides are dipped in deionized water, the cells are pipetted on
the slide and fixed by passing through a flame. The cells are stained with May-Grünwald (3 min) and
10% [v/v] Giemsa (in Titrisol, pH 7.2, 20 min).

- Number of cells spread and analysed per concentration (number of replicate cultures and total n
umber of cells scored): at least 1000 binucleated cells per culture, in total at least 2000 binucleated
cells per test group
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identi
fication): criteria of Countryman and Heddle.
- The diameter of the micronucleus is less than 1/3 of the main nucleus
- The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
- Only binucleated cells will be scored.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: 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

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The slides were scored microscopically for micronuclei.

OTHER:
The test substance was weighed, pre-wetted with 0.5 vol% ethanol (pre-wetting is introduced to
enable dispersion of hydrophobic materials in water-based systems) and topped up with the
vehicle 0.05% w/v BSA-water to achieve the required concentration of the stock dispersions.
One stock dispersion was prepared (2.56 mg/mL). A homogeneous test substance preparation in the
vehicle was prepared by using a Branson Sonifier S-550D (Branson Ultrasonics Corp., Danbury, CT,
USA) equipped with a standard 13 mm disruptor horn.
The further concentrations were serially diluted from the stock solution with 0.05% w/v BSA in water
to a 10 times higher concentration of the planned doses. Then the test substance formulations were
diluted 1:10 in culture medium according to the planned doses. All test substance formulations were
prepared immediately before administration.

Rationale for test conditions:

Pre-experiments were performed to characterize the concentration dependent aggllomeration of particles.

Evaluation 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 is obtained.
• A dose-related increase in the number of cells containing micronuclei is observed.
• The number of micronucleated cells exceeds 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 is observed under any experimental condition.
• The number of micronucleated cells in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Species / strain:

lymphocytes: human

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity, tested up to highest stable dispersion

Remarks:

adapted for poorly soluble nanoparticle

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: At the beginning of the treatment period, the pH was measured at least for the top
concentration and for the vehicle control, each. No influence was observed.
- Data on osmolality: not determined
- Possibility of evaporation from medium: none
- Water solubility: Insoluble; see adaption for pooly soluble nanoparticles

RANGE-FINDING/SCREENING STUDIES :
Fractionating techniques with selective detection (AUC, UVVis) were used to characterise the test item preparations in the cell culture medium. Compared to the size of the constituent particles
determined independently by TEM, the particles were successfully dispersed into a stable suspension with partial agglomeration. The re-characterization after 20h showed a change in shape from polydisperse to monodisperse in size distribution. The
percentiles of the size distribution (D10, D50, D90) did show a trend with dose. The dissolved content at the end of the incubation time of 20h was around 1.2%.
Details are shown in the figure.

STUDY RESULTS
- Concurrent vehicle negative and positive control data are shown in table 1
- Cytotoxicity and number of micronucleated cells are shown in table 1
- Historical control data are provided in the attachment.

The values (0.4 – 0.7% micronucleated cells) were were within the 95% upper control limit of the historical negative data range (0.2 – 0.9% micronucleated cells; see Appendix 5). A
statistical significance compared to the concurrent vehicle control value (0.6 % micronucleated cells) was not observed.
The experimental part described above showed no positive dose response as assessed by a trend analysis.
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 (5.6% and 1.6% micronucleated cells (MMC and Col, respectively)) were compatible to the historical positive control data range.
The initial scoring of the WC-Co cutltures gave a value of 1.1% micronucleus frequency after treatment with either 60 or 100 μg/mL. The value was compatible with the historical control
data of this compound. In order to verify the results additional 1000 cells per culture were scored for the WC-Co cultures treated with 60 and 100 μg/mL as well as the
vehicle control cultures. The obtained results showed a micronucleus frequency of 1.1% and 1.0% for cultures treated with 60 and 100 μg/mL WC-Co, respectively. The vehicle control
value remained at 0.6%. The values of WC-Co at both 60 and 100 μg/mL were statistically signinifacnt as compared to the vehicle control value.

Table 1: Summary of results

Test groups [μg/mL]	Micronucleated cells* [%]	Cytotoxicity Proliferation index cytostasis [%]
vehicle (0.05% BSA-water (w/v))	0.6	0.0
1	n.d.	-11.0
3	n.d.	-11.0
10	0.7	-9.6
30	0.6	-7.4
60	n.d.	-7.2
100	0.5	-10.2
256	0.4	-11.3
WC-Co 30	n.d.	5.7
WC-Co 60	1.1	8.1
WC-Co 100	1.1	29.6
Positive control (MMC 0.04 μg/mL)	5.6^S	10.4
Positive control (Col 0.05 μg/mL)	1.6^S	11.2
vehicle control**	0.6	-
WC-Co 60**	1.1^S	-
WC-Co 100**	1.0^S	-

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

** Relative number of binucleated cells with micronuclei per 4000 cells scored per test group

^S Frequency statistically significantly higher than corresponding control values

n.d. Not determined

Conclusions:

No clastogenicity was observed in the in-vitro micronucleus test (OECD 487, GLP) adapted for nanoparticles.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial gene mutation:
Mutagenicity of Pigment Yellow 128 in the bacterial reverse mutation assay (Ames test) was investigated using the Prival modification for azo substances (BASF 2000). Negative results were obtained in all tests with and without metabolic activation. All relevant tester stains were tested and test item concentrations were adequate.


Cytogenicity study in mammalian cells:

Pigment Yellow 128 was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). One experiment was carried out. The test material fulfills the criteria of a nanomaterial according to the EU REACH definition. Thus, in accordance to the OECD 487 guideline the following modifications were made:

1) Solubility properties: the test substance is a poorly soluble nanomaterial. Therefore, the selection of the concentration to be tested and scored is based either on the induced cytotoxicity or the homogeneity of the dispersion in the vehicle.

2) Metabolic activation: nanoparticles do not generally require metabolic activation (Elespuru R. (2018) Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods. Toxicological Sciences 164(2), 391-416). Therefore, parallel cultures using S9 mix were not carried out.

3) The time required for the target cell to take up the nanoparticles differs significantly from that required for testing of soluble chemicals. Therefore, pulse treatment of the cultures is omitted. Cells are treated for a period corresponding to approx. 1 cell cycle (20h) (Elespuru R (2018) Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods. Toxicological Sciences 164(2), 391-416).

4) The compatibility of the used test procedure for the assessment of the putative mutagenic potential of a nanomaterial is confirmed by the additional testing of the nanomaterial positive control Tungsten-Carbide-Cobalt (WC-Co). This compound has been shown to be a suitable nanomaterial positive control (Moche H, Chevalier D, Barois N, Lorge E, Claude N, Neslany F (2014) Tungsten Carbide-Cobalt as a nanoparticulate reference positive control in in vitro genotoxicity).

Previous assessments have shown that isolated buffy coat cells are more suitable for nanomaterial testing as compared to whole blood (Burgum M J, Ulrich C, Partosa N, Evans S J, Gomes C, Beritt-Seifert S, Landsiedel R,

Bogni A, Honarvar N, Doak S. Adapatation of the in vitro micronucleus assay for nanomaterial testing (manuscript in preparation)). Therefore, in this study buffy coat cells isolated from whole blood were used.

Concentrations of 0; 1; 3; 10; 30; 60; 100; 256 µg/mL were selected based on a pre-test on homogeneity of the dispersions. The highest used concentration was 256 µg/mL. Higher concentrations could not be homogenously formulated.

Test groups 0; 10; 30; 100 and 256 µg/mL were evaluated for the occurrence of micronuclei: 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 used as vehicle. 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.

The characterization of the nanomaterial in cell culture medium showed that the particles were successfully dispersed into a stable suspension with partial agglomeration which did not change significantly during the treatment period. The dissolved content at the end of the incubation time of 20h was about 1.2%. The vehicle controls gave frequencies of micronucleated cells within the 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.

In this study, no cytotoxicity as indicated by reduced proliferation index (CBPI) was observed up to the highest applied test substance concentration.

Fractionating techniques with selective detection (AUC, UVVis) were used to characterise the test item preparations in the cell culture medium. Compared to the size of the constituent particles determined independently by TEM, the particles were successfully dispersed into a stable suspension with partial agglomeration. The percentiles of the size distribution (D10, D50, D90) did show a trend with dose. The re-characterization after 20h showed a change in shape from polydisperse to monodisperse in size distribution. The dissolved content at the end of the incubation time of 20h was around 1.2%

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP).