Diisopropyl 3,3'-[(2,5-dichloro-1,4-phenylene)bis[iminocarbonyl(2-hydroxy-3,1-naphthylene)azo]]bis[4-methylbenzoate]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021- 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 Jul 2016

Deviations:

yes

Remarks:

- concentration selection - no metabolic activation - only continuous treatment protocol was used - additional nanomaterial positive control

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.49 (In Vitro Mammalian Cell Micronucleus Test)

Version / remarks:

14 Feb 2017

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: NANOGENOTOX-Project (Grant Agreement No 2009 21 01)

Version / remarks:

Version 1.2, dated 06 May 2018

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:

Diisopropyl 3,3'-[(2,5-dichloro-1,4-phenylene)bis[iminocarbonyl(2-hydroxy-3,1-naphthylene)azo]]bis[4-methylbenzoate]

EC Number:

275-639-3

EC Name:

Diisopropyl 3,3'-[(2,5-dichloro-1,4-phenylene)bis[iminocarbonyl(2-hydroxy-3,1-naphthylene)azo]]bis[4-methylbenzoate]

Cas Number:

71566-54-6

Molecular formula:

C50 H42 Cl2 N6 O8

IUPAC Name:

diisopropyl 3,3'-[(2,5-dichloro-1,4-phenylene)bis[iminocarbonyl(2-hydroxy-3,1-naphthylene)azo]]bis[4-methylbenzoate]

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- State of aggregation:
- Particle size distribution:
- Mass median aerodynamic diameter (MMAD):
- Geometric standard deviation (GSD):
- Shape of particles:
- Surface area of particles: BET = 29.5 +/- 0.3 m2/g
- Coating: none

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

Batch identification: 0004967613
CAS No.: 71566-54-6
Purity: ≥ 98 < 100 %
Validity: 2023
Physical state, appearance: Solid, red

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage conditions: ambient (RT)
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
Storage stability: The stability of the test substance under storage conditions was guaranteed until 18 Jan 2023 as indicated by the sponsor, and the sponsor holds this responsibility.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
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. Onestock dispersions were prepared (2.56 mg/mL) further dilutions.
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-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.

A physical-chemical characterization of the test substance in the vehicle, including both intrinsic properties (size, shape, specific surface area) and extrinsic properties (agglomeration and solubility in the genotoxicity test medium), was determined analytically.

Method

Species / strain

Species / strain / cell type:

primary culture, other: human lymphocytes (buffy coat cells)

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors: one 24 year old, healthy and non-smoking female
- Whether whole blood or separated lymphocytes were used: Buffy coat cells were isolated from whole blood and cultures thereof were treated with the test substance.
- Mitogen used for lymphocytes: Phytohemagglutinin M form (PHA-M)
- The lymphocytes of each donor have previously shown to respond well to stimulation of proliferation with PHA and to the used positive control substances.

MEDIA USED
All media were supplemented with:
• 1% [v/v] penicillin/streptomycin (final concentration 100 μg/mL)
• 20% [v/v] fetal calf serum (FCS)

For the stimulation the medium was supplemented with:
• 1.5% Phytohemagglutinin M form (PHA-M)

For the Cytochalasin B treatment the medium was supplemented with:
• 30 μL Cytochalasin B (Cyt B, stock solution: 2 mg/mL in DMSO, final concentration: 6 μg/mL)

Culture medium:
RPMI 1640 medium containing stable glutamine supplemented with 20% [v/v] FCS.

In this study all incubations were performed at 37°C with a relative humidity of ≥ 90% in a 5% [v/v] CO2 atmosphere.

Cytokinesis block (if used):

Cytochalasin B (Cyt B)

Metabolic activation:

without

Metabolic activation system:

Metabolic activation: nanoparticles do not generally require metabolic activation (Elspuru, 2018). Therefore, parallel cultures using S9 mix were not carried out.

Test concentrations with justification for top dose:

The selection of the top concentration to be used were based on a pre-test on homogeneity of the test substance dispersion. As a result, 256 μg/m was the highest tested concentration.
Selected doses: 1, 3, 10, 30, 60, 100, 256 µg/mL.

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

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.

Stimulating time: 48 h; Exposure time: 20 h; Harvest time: 20 h

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: at least 2
- Number of independent experiments: 1

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

TREATMENT AND HARVEST SCHEDULE:
- 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.

Rationale for test conditions:

The test substance is a nanomaterial and is largely insoluable.

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:

An appropriate statistical analysis was performed. 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).
If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (s) were printed in the tables.
In addition, a statistical trend test (SAS procedure REG) 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.
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.
However, both, biological and statistical significance were considered together.

Results and discussion

Test results

Species / strain:

primary culture, other: human lymphocytes (buffy coat cells)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity, tested up to homogenous nanoparticle disperspersion

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

CHARACTERIZATION OF THE TEST SUBSTANCE IN VEHICLE
Compared to the size of the constituent particles determined independently by TEM, the particles are successfully dispersed into a stable suspension with partial agglomeration that does not change significantly during the genotoxicity testing. The percentiles of the size distribution (D10, D50, D90) did not show a trend with dose. The dissolved content at the end of the incubation time of 20h is around 0.05%.

TREATMENT CONDITIONS
The pH values were not relevantly influenced by test substance treatment.

CYTOTOXICITY
In this study, no reduced proliferative activity was observed after 20 hours continuous test substance treatment in the test groups scored for cytogenetic damage.

STUDY RESULTS
The values (0.4 – 0.7% micronucleated cells) were within the 95% upper control limit of the historical negative data range (0.2 – 0.9% micronucleated cells; see table). 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 all independently performed experiments. In this study, the frequencies of micronucleated cells (5.3% and 2.9% micronucleated cells (MMC and Col, respectively) were compatible to the historical positive control data range. The nanomaterial positive control substance Tungsten Carbide-Cobalt (WC-Co) induced statistically significantly increased micronucleus frequencies. In this study, the frequencies of micronucleated cells (60 μg/mL: 1.5%) and exceeded the 95% upper control limit of the historical negative data range (0.2 – 0.9% micronucleated cells)

For further details see table 2.

HISTORICAL CONTROL DATA
see table 3 and 4 in section "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1: Linear trend-test 

Slope	One-sided p-value*
0.00037368	0.25058

* 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 2: Summary of results

Exp.	Exposure/ Recovery/ Preparation intervall	Test groups	Micronucleated cells *	Cytotoxicity Proliferation index cytostasis
	[h]	[µg/mL]	[%]	[%]
1	20/0/20	VC^1	0.6	0.0
		1.0	n.d.	8.1
		3.0	n.d.	1.2
		10.0	0.4	4.8
		30.0	0.5	6.8
		60.0	n.d.	5.7
		100.0	0.7	11.8
		256.0	0.6	13.7
		WC-Co 30	n.d	5.7
		WC-Co 60	1.1	8.1
		WC-Co 100	1.0	29.6
		PC^2	5.6^S	10.4
		PC^3	1.6^S	11.2

* 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

VC vehicle control; PC positive control

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

 

Table 3: HISTORICAL NEGATIVE CONTROL DATA

Period: April 2018 - November 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 4: HISTORICAL POSITIVE CONTROL DATA

Period: April 2018 – November 2020

	Micronucleated cells [%]
Substance and concentration	Mitomycin C (0.04 µg/mL)	Colchicin (0.05 µg/mL)
Exposure period	20 hrs	20 hrs
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

 

Fractionating techniques with selective detection (AUC, UIVVis) 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 that did not change significantly during the genotoxicity testing.

The size distribution was polydisperse, ranging from below 20 nm to 500 nm

The percentiles of the size distribution (D10, D50, D90) did not show a trend with dose. The dissolved content at the end of the incubation time of 20h was around 0.05%.

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, the conclusion is drawn that the test material 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). 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 NANOGENOTOX Project (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.