4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jul 09, 2021 - Dec 20, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

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:

nanoparticle: as largely insoluble, selection of concentration based on cytotoxicity or homogeneity; no metabolic activation; no pulse treatment, but for a period corresponding to 1 cell cycle; Tungsten-Carbide-Cobalt as positive control

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU method B.49

Version / remarks:

Commission Regulation (EC) No 735/2017 of 14 Feb 2017 amending, for the purpose of its
adaptation to technical progress, Regulation (EC) No 640/2012 of 06 Jul 2012 laying down
test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of
the Council on the Registration, Evaluation, Authorization and Restriction of Chemicals
(REACH): In vitro Mammalian Cell Micronucleus Test, No B.49; No L 193

Deviations:

yes

Remarks:

modifications of the protocol for nanomaterial testing according to NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt - Rheinland Pfalz

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

EC Number:

221-423-9

EC Name:

4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Cas Number:

3089-16-5

Molecular formula:

C20H10Cl2N2O2

IUPAC Name:

4,11-dichloro-5,12-dihydroquino[2,3-b]acridine-7,14-dione

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- Batch 200389
- State of aggregation: solid, powder
- The number-based concentration of particles with sizes below 100 nm (smallest feret diameter) is 99%
- Particle size distribution (TEM): 43.3 nm (D50)
- Shape of particles: multimodal/orthorhombic
- Coating: no
- Surface properties: not applicable
- Density: 1689 kg/m³ at 20°C
- Moisture content: refer to IUCLID chapter 1
- Residual solvent: refer to IUCLID chapter 1
- Activation: not applicable
- Stabilisation: not applicable

Test material purity is 100% pigment minus extractables and volatiles

Specific details on test material used for the study:

TEST MATERIAL
- Batch number: 200389
- Purity: 94.11 area-% (HPLC, 300 nm), 94.82 area-% (HPLC, 335 nm)
- Content w(pigment) = 98.4 g/100g
- Physical state/ appearance: solid/ red

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition: ambient (RT)
- Stability: under storage conditions guaranteed until 2030 as indicated by thes ponsor, and the sponsor holds this responsibility
- Homogeneity: was ensured by mixing before preparation of the test substance preparations

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test substance preparation was performed 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 06 May 2018.
- 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.
- Two stock dispersions were prepared (2.56 mg/mL and 21.0 mg/mL) in the 1st Experiment.
- The stock dispersions of 21.0 mg/mL in the 1st Experiment and 3.0 mg/mL, 9.0 mg/mL and 15.0 mg/mL in the 2nd Experiment were handled separately. In the 4th and 5th Experiment stock dispersions of 9.0 mg/mL and 15.0 mg/mL were handled separately.
- For further dilutions the stock dispersions of 2.56 mg/mL were used in Experiment 1, 3, 4 and 5.
- 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.
- All test substance concentrations below 256 μg/mL were serially diluted from the stock solution (2.56 mg/mL) with 0.05% w/v BSA-water to a 10 times higher concentration of the planned doses.
- All concentrations above 256 μg/mL were individually prepared.
- All 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.

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Fresh Blood was collected from a single donor for each experiment.
- Only healthy, non-smoking donors and not receiving medication were used (1st Experiment 31 year-old male, 2nd Experiment 28 year-old male, 3rd Experiment 30 year-old female, 5th Experiment 32 year-old male).
- The lymphocytes of each donor have previously shown to respond well to stimulation of proliferation with phytohemagglutinin (PHA) and to the used positive control substances.
- Buffy coat cells were isolated from whole blood and cultures thereof were treated with the test substance.

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; 1st, 2nd and 5th Experiment), 0.5% [v/v] Phytohemagglutinin (PHA, stock solution 0.6 mg/mL, final concentration 3 μg/mL; 3rd and 4th Experiment)
- Culture medium: RPMI 1640 medium containing stable glutamine supplemented with 20% [v/v] FCS

Cytokinesis block (if used):

30 μL Cytochalasin B (Cyt B, stock solution: 2 mg/mL in DMSO, final concentration: 6 μg/mL)

Metabolic activation:

without

Metabolic activation system:

Nanoparticles do not generally require metabolic activation (Elespuru 2018). Therefore, parallel cultures using S9 mix were not carried out.

Test concentrations with justification for top dose:

The test substance is a nanomaterial and is largely insoluble. Therefore, the selection of the concentration was based on the homogeneity of the dispersion in the vehicle (stable dispersion, avoiding agglomeration to large (non-nano)particles). 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 that did not change significantly during the genotoxicity testing. Across all doses tested, the median diameter D50, D10 and D90 percentiles showed a trend with dose. More than 50% of the dispersed particles showed a diameter < 100 nm. As there was no reference spectrum available, a calculation of the dissolved content could not be carried out.
In a pretest, homogeneous suspensions of the test substance in the vehicle 0.05% w/v BSAwater were obtained in concentrations of 5.0, 11.7 and 20.0 mg/mL. Thus, the highest tested concentration in the 1st Experiment was 2100.0 μg/mL in culture based on the pretest and the purity of the test substance. 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. In case a relevant cytotoxicity is not observed 2100 μg/mL should be used as the top concentration. At least three concentrations were evaluated to detect a possible dose-response relationship. At least 2 cultures were prepared per test group, and at least 1000 cells per culture were evaluated for the occurrence of micronucleated cells.

1st Experiment: 1; 3; 10; 30; 60; 100 and 2100 µg/mL
2nd Experiment: 300; 900; 1500 µg/mL
3rd Experiment: 1; 3; 10; 30; 60 and 100 µg/mL
4th Experiment: 10; 30; 60; 100; 900 and 1500 µg/mL
5th Experiment: 10; 30; 60; 100; 900 and 1500 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

Remarks:

Additionally Nanomaterial positive control (100, 60 and 30 μg/mL 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

STIMULATION OF THE CELLS
Buffy coat cells were isolated from whole blood using the density centrifugation method. Briefly, the blood was diluted in a 1:2 ratio with culture medium and carefully layered on top of Ficoll- PaqueTM PLUS (GE Healthcare Bio-Sciences AB) in a 50 mL tube (20 mL of diluted on top of 25 mL Ficoll). The tubes were centrifuged at 1055 g for 20 minutes (without a break function in the deceleration phase). The buffy coat (mid layer) from each tube was collected and centrifuged for 15 minutes at 491 g (using the break function in the deceleration phase). The cells were washed twice with cell culture medium and centrifuged at 900 g for 5 minutes. The final washed cell pellets were resuspended in a volume of cell culture medium containing PHA corresponding to five times of the original blood volume. The cell suspension was cultured for 48 h in 175 cm2 flasks using 50 mL cell suspension per flask.

DEFINITION OF TEST CULTURES
A test group consists of two separately treated flasks. From each flask least two slides were prepared.

PREPARATION AND TREATMENT OF TEST CULTURES
The activated cell cultures were pooled and centrifugated in 10 mL aliquotes at 900 g for 5 minutes. After centrifugation the supernatant (culture medium) was removed and the cells suspended in 9 mL test substance dilution in culture medium. A separate aliquote of the test substance dilution was incubated with the cultures to determine test substance precipitation at the end of exposure. At the end of the exposure period, the cells were transferred in tubes, centrifuged for 5 minutes at 900 g and resuspended in HBSS (Hanks Balanced Salt Solution). Washing of the cells was repeated at least once. Then the cells were centrifuged at (900 g, 5 min) and resuspended in RPMI 1640 medium with 20% [v/v] FCS and transferred into 25 cm² cell culture flasks. Cyt B was added to the cultures and incubated under agitation at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity for 20 hours.

CELL HARVEST AND PREPARATION OF SLIDES
The cells were transferred into tubes, centrifugated at 900 g for 5 min and washed with HBSS. After washing, the cells were centrifuged (900 g, 5 min) and suspended in 0.65% KCl (37°C), incubation for 10 minutes at 37°C. After the hypotonic treatment, the cells were fixed by adding of fixative (19 parts methanol and 1 part acetic acid). 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 will be repeated twice. After the last fixation step, the cells were centrifugated directly (900 g, 5 min, 4°C), suspended in 0.5-2 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, 20 min) and mounted.

MICRONUCELUS ANALYSIS
The slides were scored microscopically. As a rule, at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei. The analysis of micronuclei was carried out according to the following criteria of Countryman and Heddle.
i) The diameter of the micronucleus was less than 1/3 of the main nucleus
ii) The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
iii) 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.

PROLIFERATION INDEX (CBPI)
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. The number of mononucleated, binucleated and multinucleated cells were recorded and the CBPI was calculated using the following formula:

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

Evaluation criteria:

ACCEPTANCE CRITERIA
The in vitro micronucleus assay is considered valid if the following criteria are met:
i) 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.
ii) Sufficient cell proliferation was demonstrated in the vehicle control.
iii) 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”.
iv) The positive controls 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:
i) A statistically significant increase in the number of micronucleated cells was obtained.
ii) A dose-related increase in the number of cells containing micronuclei was observed.
iii) 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:
i) Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
ii) 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, BASF SE). If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05. 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. 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:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity, stability of dispersion as nanoform

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TREATMENT CONDITIONS
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.

GENOTOXICITY _ MICRONUCLEUS ANALYSIS
In the 1st Experiment the highest dose group (2100.0 μg/mL) was not scorable due to excessive test substance precipitation. The next lower tested dose group was 100.0 μg/mL at which 1.0% micronucleated cells were found. At 10.0 μg/mL and 30.0 μg/mL 0.8% or 1.0% cells containing micronuclei, respectively were observed. The values at 30.0 and 100.0 μg/mL were above the 95% control limit of our historical vehicle control data range (0.2 - 0.9% micronucleated cells). The respective vehicle control had 0.9% micronucleated cells. A statistical significance compared to the concurrent vehicle control value was not observed.
In the 2nd Experiment the highest dose group (1500.0 μg/mL) was not scorable due to excessive test substance precipitation. The values obtained at 300.0 μg/mL and 900.0 μg/mL (0.5 and 0.6% micronucleated cells, respectively) were within the 95% upper control limit of the historical negative data range (0.2 - 0.9% micronucleated cells; see Appendix 5). The respective vehicle control had 0.4% binucleated cells containing micronuclei. A statistical significance compared to the concurrent vehicle control value was not observed.
In the 3rd Experiment the values of the test groups after test substance treatment ranged between 0.4 - 0.9% binucleated cells containing micronuclei. All values were within the 95% upper control limit of the historical negative data range (0.2 - 0.9% micronucleated cells). The respective vehicle control had 0.6%. No statistically increased significance was observed.
After incomplete evaluation of Experiment 4 (all cultures of part A) the test groups of the particular positive control Tungsten Carbide-Cobalt were not scorable for micronuclei due to insufficient number of binuclear cells. The test group treated with 1500 μg/mL was also not scorable. Therefore, this experiment was not valid and was repeated as Experiment 5.
In the 5th Expermiment the values ranged between 0.3 - 0.8% binucleated cells containing micronuclei were within the 95% control limit of the historical negative data range (0.2 - 0.9% micronucleated cells). The respective vehicle control had 0.5% binucleated
cells containing micronuclei. A statistical significance compared to the concurrent vehicle control value was not observed.
A positive dose response as assessed by a trend analysis was not observed in any of the experiments scored for micronuclei.
The positive control substances MMC (0.04 μg/mL) and Colchicine (0.05 μg/mL) induced statistically significantly increased micronucleus frequencies. In this study, increased frequencies of micronucleated cells (10.3% and 3.0% micronucleated cells in the 1st Experiment; 8.6% and 2.0% in the 2nd Experiment and 7.6% and 4.3% in the 3rd Experiment (MMC and Col, respectively) were scored. In the 5th Experiment 6.2% or 2.1% binucleated cells containg micronuclei (MMC and Col, respectively) were obtained. All values found were compatible to the historical positive control data range (Appendix 6). In the 2nd Experiment the values of the individual cultures treated with colcemide were 11 and 29 micronucleated cells per 1000 cells. In order to confirm the discrepancy between the two cultures a reassessment of the respective slides was performed. In the reassessment the values obtained were 14 and 25 micronucleated cells per 1000 cells. Thus, the values were confirmed.
The particular positive control substance Tungsten Carbide-Cobalt (WC-Co) induced increased micronucleus frequencies in four valid experiments. In this study, the frequencies of micronucleated cells in the 1st Experiment were 1.1% and 1.2% at 30 and 60 μg/mL, respectively. Although the values were not statistically significant, they were above the upper 95% control limit of the historical control data (0.2 – 0.9% micronucleated cells). In the 2nd Experiment the values were 0.9% and 1.3% at 30 and 60 μg/mL, respectively. Both values were statistically significant as compared to the corresponding vehicle control value. The value of the cultures treated with 60 μg/mL was above the upper 95% control limit of the historical control data (0.2 - 0.9% micronucleated cells). In the 3rd Experiment the values were 0.5% , 1.5% and 0.6% at 30, 60 and 100 μg/mL, respectively. Only the cultures treated with 60 μg/ml were statistically significant and above the upper 95% control limit of the historical control data base. In the 5th Experiment the values were 0.5% and 1.3% at 30 and 60 μg/mL, respectively. Only the cultures treated with 60 μg/ml were statistically significant and above the upper 95% control limit of the historical control data base. (0.2 - 0.9% micronucleated cells).

Any other information on results incl. tables

Table 1: Analysis of Microcuclei - 1st Experiment

Test group [µg/mL]	Culture	No. of evaluated cells	Cells containing Micronuclei
			[n]	[n]	[%]
Vehicle control	A	1000	8	17	0.9
	B	1000	9
1.0	A
	B
3.0	A
	B
10.0	A	1000	13	16	0.8
	B	1000	3
30.0	A	1000	6	19	1.0
	B	1000	13
60.0	A	n.d.
	B
100.0	A	1000	7	19	1.0
	B	1000	12
2100.0	A	n.s.
	B
WC-Co 30	A	1000	14	22	1.1
	B	1000	8
WC-Co 60	A	1000	15	23	1.2
	B	1000	8
WC-Co 100	A	n.d.
	B
MMC 0.04	A	1000	138	206	10.3
	B	1000	68
Col 0.05	A	1000	29	60	3.0
	B	1000	31

 

Table 2: Analysis of Microcuclei - 2nd Experiment

Test group [µg/mL]	Culture	No. of evaluated cells	Cells containing Micronuclei
			[n]	[n]	[%]
Vehicle control	A	1000	3	7	0.9
	B	1000	4
300.0	A	1000	8	10	0.5
	B	1000	2
900.0	A	1000	6	12	0.6
	B	1000	6
1500.0	A	n.s.
	B
WC-Co 30	A	1000	8	17	0.9
	B	1000	9
WC-Co 60	A	1000	9	25	1.3
	B	1000	16
WC-Co 100	A	n.s.
	B
MMC 0.04	A	1000	62	172	8.6
	B	1000	110
Col 0.05	A	1000	29	40	2.0
	B	1000	11
	A	1000	25	39	2.0
	B	1000	14

 

Table 3: Analysis of Microcuclei - 3rd Experiment

Test group [µg/mL]	Culture	No. of evaluated cells	Cells containing Micronuclei
			[n]	[n]	[%]
Vehicle control	A	1000	4	11	0.6
	B	1000	7
1.0	A	n.d.
	B
3.0	A
	B
10.0	A	1000	7	13	0.7
	B	1000	6
30.0	A	1000	9	17	0.9
	B	1000	8
60.0	A	1000	5	12	0.6
	B	1000	7
100.0	A	1000	4	7	0.4
	B	1000	3
WC-Co 30	A	1000	3	10	0.5
	B	1000	7
WC-Co 60	A	1000	19	30	1.5
	B	1000	11
WC-Co 100	A	1000	2	12	0.6
	B	1000	10
MMC 0.04	A	1000	87	151	7.6
	B	1000	64
Col 0.05	A	1000	50	86	4.3
	B	1000	36

 

Table 4: Analysis of Microcuclei - 5th Experiment

Test group [µg/mL]	Culture	No. of evaluated cells	Cells containing Micronuclei
			[n]	[n]	[%]
Vehicle control	A	1000	6	10	0.5
	B	1000	4
10.0	A	1000	8	16	0.8
	B	1000	8
30.0	A	1000	7	15	0.8
	B	1000	8
60.0	A	1000	8	16	0.8
	B	1000	8
100.0	A	1000	9	12	0.6
	B	1000	3
900.0	A	1000	4	9	0.5
	B	1000	5
1500.0	A	1000	3	6	0.3
	B	1000	3
WC-Co 30	A	1000	5	9	0.5
	B	1000	4
WC-Co 60	A	1000	13	26	1.3
	B	1000	13
WC-Co 100	A	n.d.
	B
MMC 0.04	A	1000	74	123	6.2
	B	1000	49
Col 0.05	A	1000	19	41	2.1
	B	1000	22

Applicant's summary and conclusion

Conclusions:

not clastogenic, not aneugenic

Executive summary:

In a reliable GLP-conform study according to OECD TG 487, the test substance was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Five independent experiments were carried out, of which 4 are considered valid. The test substance is an insoluble pigment, which fulfills the criteria of a nanomaterial. Thus, in accordance to the OECD 487 guideline the following modifications have been considered for the testing of the nanomaterial:

 

1) Solubility properties: the test substance is a pigment and is largely insoluble. 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 (stable dispersion, avoiding agglomeration to large (non-nano)particles).

2) Metabolic activation: nanoparticles do not generally require metabolic activation. 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.

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.

 

The following concentrations were selected based on a pre-test on homogeneity of the dispersions as well as the purity of the test substance. The highest used concentration was 2100.0 μg/m. Test groups printed in bold type were evaluated for the occurrence of micronuclei:

 

1st Experiment

20 hours exposure

0; 1.0; 3.0; 10.0; 30.0; 60.0; 100.0; 2100.0 μg/mL

 

2nd Experiment

20 hours exposure

0; 300.0; 900.0; 1500.0 μg/mL

 

3rd Experiment

20 hours exposure

0; 1.0; 3.0; 10.0; 30.0; 60.0; 100.0; μg/mL

 

4th Experiment (not valid, data not shown)

20 hours exposure

0; 10.0; 30.0; 60.0; 100.0; 900.0; 1500.0 μg/mL

 

5th Experiment

20 hours exposure

0; 10.0; 30.0; 60.0; 100.0; 900.0; 1500.0 μg/mL

 

A sample of 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The cultures treated with Colcemide in the 2nd Experiment were renalysed in order to confirm the results from the first assessment. 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 the laboratories 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 increase in the number of cells containing micronuclei. Thus, under the experimental conditions described, the test substance is considered not to have chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes.