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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test: negative, according to OECD TG 471, GLP, Salmonella typhimurium strains: TA 98, TA 100, TA 1535, TA 1537 and TA 102 with and without metabolic activation, 2000, K1

Mouse lymphoma Assay: negative, according to OECD TG 476, GLP, mouse lymphoma L5178Y cells, with and without metabolic activation, 2000, K1

Micronucleus test: negative, according to OECD TG 487, GLP, human lymphocytes, without metabolic activation, 2022, K1

Link to relevant study records

Reference

Reference 1

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

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

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

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.

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).

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.

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).

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug - Oct 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 6218-N7508-PPR 99-2565
- Purity: approx. 98 %
- Expiration date: August 19, 2005
- Test material form: solid: particulate/powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature in a tightly closed container
- Solubility and stability of the test substance in the solvent/vehicle: stable for at least 4 hours in DMSO

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
On the day of the experiment (immediately before treatment), the test item was suspended in DMSO.
The final concentration of DMSO in the culture medium was 0.5 % (v/v).

FORM AS APPLIED IN THE TEST: suspended in DMSO

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: not specified; Cell stocks were stored in the cell bank of the testing facility.
- Suitability of cells: Each master cell stock was screened for mycoplasm contamination, checked for karyotype stability and cleansed against spontaneous mutants.
- Doubling time: 10 - 12 hours
- Methods for maintenance in cell culture: Thawed stock cultures were propagated in plastic flasks in RPMI 1640 complete culture medium. The cells were subcultured at least three times a week. The cell cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide and 95.5 % air.
- Modal number of chromosomes: 40 ± 2
- Number of passages if applicable: not specified
- Periodically checked for Mycoplasma contamination: yes, before freezing
- Periodically checked for karyotype stability: yes, before freezing
- Periodically 'cleansed' against high spontaneous background: yes (Cells were grown in RPMI 1640-HAT medium supplemented with 1x10^-4 M hypoxanthine, 2x10^-7 M aminopterin, 1.6x10^-5 M thymidine, followed by a 2-day recovery period in RPMI 1640 medium containing 1x10^-4 M hypoxanthine and 1.6x10^-5 M thymidine.)

MEDIA USED
- Type and identity of media:
Complete Culture Medium: RPMI 1640 medium supplemented with 15 % horse serum (HS), 100 U/100 µg/ml Penicillin/Streptomycin, 220 µg/ml Sodium-Pyruvate, and 1.25 U/ml Amphotericin used as antifungal.

Cloning Medium: RPMI 1640 (complete culture medium)

Selective Medium: RPMI 1640 (complete culture medium) by addition of 5 µg/ml Trifluorothymidine (TFT).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: prepared by the testing facility; obtained from the livers of 8 - 12 weeks old male rats (strain Wistar Hanlbm), which received daily applications of 80 mg/kg bw Phenobarbital i.p. and p-Naphthoflavone orally on three subsequent days. The livers were prepared 24 hours after the last treatment.
- method of preparation of S9 mix: After decapitation of the anaesthetised animals, the livers of the animals were removed, washed in 150 mM KCl and homogenized. The homogenate, diluted 1+3 with KCl was centrifuged at 9000 g for 10 minutes (4° C). Aliquots of the supernatant containing the microsomal fraction were frozen and stored in ampoules at -80° C. Small numbers of the ampoules were kept at -20° C for up to one week. The protein content was determined using the analysis kit of Bio-Rad Laboratories. The protein concentration of the S9 preparation was 28.9 mg/ml in the pre-experiment and experiment 1. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to give a final protein concentration of 0.75 mg/ml in the cultures. Cofactors were
added to the S9 mix to reach the following concentrations:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not specified; As the positive control (with S9 mix) showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large colonies, a proper enzymatic activity as well as metabolic capability of the S9 mix is assumed.

Test concentrations with justification for top dose:

Experiment 1:
with and without S9 mix: 2.5*; 5.0; 10.0; 20.0; 100.0; and 320.0 µg/ml

Experiment 2:
without S9 mix: 2.5*; 5.0; 10.0; 20.0; 100.0; and 320.0 µg/ml

* At day 4 the cultures of this concentration were not continued since a minimum of only four concentrations is required by the guidelines.

A pre-test was performed in order to determine the concentration range for the mutagenicity experiments, the pH-value, and the osmolarity. The highest concentration used in the pre-test was chosen with regard to the solubility of the test item. Test item concentrations between 19.5 and 2500 µg/ml (with and without S9 mix) were used to evaluate toxicity. Precipitation visible to the naked eye occurred at 39.1 µg/ml and above with and without metabolic activation. No relevant toxic effects were observed up to the maximal analysable concentration of 312.5 µg/ml. Higher concentrations led to heavy precipitation interfering with the cell counting of the suspension cell cultures.
In the main experiments the concentration range covered 4 concentrations in the soluble range up to the limit of solubility. Additionally two widely spaced concentrations in the precipitating concentration range were included to detect possible toxic and mutagenic effects under precipitation.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at treatment: 1×10^7 cells/flask

DURATION
- Exposure duration: 4 h; 24 h (without S9 mix)
- Expression time (cells in growth medium): 72 h, 24 h in experiment 2
- Selection time (if incubation with a selection agent): 10 - 15 days

SELECTION AGENT (mutation assays): 5 µg/ml Trifluorothymidine (TFT)

DETERMINATION OF CYTOTOXICITY
- cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
- The test item is classified as mutagenic if it reproducibly induces with at least one of the concentrations a mutation frequency that is two times higher than the mean spontaneous mutation frequency in the experiment.
- The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such evaluation may be considered independently of an enhancement factor for induced mutants.

Statistics:

No statistical evaluation was performed.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Cytotoxicity in one culture at the maximal concentration in the first experiment with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No relevant and reproducible increase in mutant colony numbers was observed in both experiments in the absence and presence of metabolic activation. In the first experiment, the threshold of twice the colony count of the corresponding solvent control was reached in the second culture with metabolic activation at the maximal concentration under heavy precipitation. Since no comparable effect occurred in the parallel culture under identical conditions, this increase was judged as an artefact based upon precipitation of the test item.

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: no (solvent control - pH 7.3; 2500 µg test substance/ml - pH 7.2)
- Effects of osmolality: no (solvent control - 390 mOsm; 2500 µg test substance/ml - 348 mOsm)
- Precipitation: Precipitation of the test item visible to the unaided eye was observed at 20.0 µg/ml and above in both main experiments.

HISTORICAL CONTROL DATA:
- Positive historical control data: see table 2
- Negative (solvent/vehicle) historical control data: see table 2

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant toxic effects occurred in both main experiments with the exception of culture II at the maximal concentration in the first experiment with metabolic activation.

Table 1: Summary of results

 

	conc., µg per ml	S9 mix	relative cloning efficiency 1	relative total growth	mutant colonies/ 10^6cells	induction factor	relative cloning efficiency 1	relative total growth	mutant colonies/ 10^6cells	induction factor
Experiment I			Culture I				Culture II
Neg. contr. with medium Neg. contr. with DMSO Pos. control with MMS Test item Test item Test item Test item Test item Test item	13.0 2.5 5.0 10.0 20.0 100.0 320.0	-	100.0 100.0 58.1 101.6 116.2 105.0 108.5 88.4 55.8	100.0 100.0 40.6 # 92.8 81.4 79.6 56.5 71.6	129 109 505 # 161 175 188 194 154	1.0 3.9 # 1.5 1.6 1.7 1.8 1.4	100.0 100.0 89.8 100.0 91.1 103.3 68.4 95.4 67.1	100.0 100.0 51.6 # 97.3 118.5 98.4 105.1 92.5	89 89 367 # 98 95 93 103 81	1.0 4.1 # 1.1 1.1 1.0 1.2 0.9
Neg. contr. with medium Neg. contr. with DMSO Pos. control with MMS Test item Test item Test item Test item Test item Test item	13.0 2.5 5.0 10.0 20.0 100.0 320.0	+	100.0 100.0 88.3 88.3 103.3 101.6 101.6 80.8 73.0	100.0 100.0 64.1 # 112.1 99.4 103.9 136.8 83.1	101 113 274 # 127 96 116 118 129	1.0 2.4 # 1.1 0.8 1.0 1.0 1.1	100.0 100.0 68.0 85.0 101.7 109.3 105.4 77.6 26.2	100.0 100.0 65.0 # 83.8 112.1 131.5 94.6 20.2	100 100 328 # 169 104 126 99 202	1.0 3.3 # 1.7 1.0 1.2 1.0 2.0
Experiment II			Culture I				Culture II
Neg. contr. with medium Neg. contr. with DMSO Pos. control with MMS Test item Test item Test item Test item Test item Test item	13.0 2.5 5.0 10.0 20.0 100.0 320.0	-	100.0 100.0 41.6 96.6 101.8 118.8 100.0 101.8 87.4	100.0 100.0 21.0 # 113.3 105.9 114.6 75.8 99.5	127 98 551 # 61 103 64 114 90	1.0 5.6 # 0.6 1.0 0.6 1.2 0.9	100.0 100.0 66.6 88.4 92.4 89.2 97.1 69.7 68.5	100.0 100.0 26.5 # 125.9 108.3 87.7 121.6 91.0	123 112 704 # 73 79 109 55 90	1.0 6.3 # 0.7 0.7 1.0 0.5 0.8

# culture was not continued since a minimum of four concentrations is required by the guidelines

 

Table 2: Historical Data from 1998 -1999

 

Controls	mutant colonies per 10^6 cells
	4 h treatment		24 h treatment
	without S9 mix	with S9 mix	without S9 mix
Negative Controls	24 -135	25 - 135	27 -148
Solvent controls	15 -132	24 -153	27 - 149
Positive Controls (MMS)	151 - 760	-	168 - 1944
Positive Controls (3-MC)	-	155 - 595	-
Positive Controls (DMNA)	-	115 - 345	-

Conclusions:

In the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation. Therefore, the test substance is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of the test substance to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours.

The test item was evaluated at the following concentrations: 5.0; 10.0; 20.0; 100.0; and 320.0 µg/ml.

According to the pre-experiment on toxicity and the solubility of the test item the concentration range was selected. In the pre-experiment precipitation visible to the unaided eye occurred already at 39.1 µg/ml and above with and without metabolic activation during pulse and continuous treatment. No relevant toxic effects were observed up to the maximal analysable concentration of 312.5 µg/ml. Higher concentrations led to very heavy precipitation of the test item in the suspension cell cultures precluding cell counting.

No relevant toxic effects occurred in both main experiments with the exception of culture II at the maximal concentration in the first experiment with metabolic activation.

Precipitation of the test item visible to the unaided eye was observed at 20.0 µg/ml and above in both main experiments. No relevant and reproducible increase in mutant colony numbers was observed in both experiments in the absence and presence of metabolic activation. In the first experiment, the threshold of twice the colony count of the corresponding solvent control was reached in the second culture with metabolic activation at the maximal concentration under heavy

precipitation. Since no comparable effects occurred in the first parallel culture under identical conditions, this increase was judged as an artefact based upon precipitation of the test item.

The ratio of small versus large colonies was not shifted up to the maximal analysable concentration.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large colonies.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep. 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 3089-16-5
- Purity: approx. 98 %
- Expiration date: August 19, 2005
- Test material form: solid: particulate/powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in a well closed box at room temperature
- Stability of the test substance in the solvent/vehicle: for hours

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment, the test item was dissolved in DMSO (purity > 99 %, MERCK, D-64293 Darmstadt).

FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in DMSO

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (supplemented with cofactors) derived from phenobarbital (i.p.) and β-naphthoflavone (orally) induced rat liver

Test concentrations with justification for top dose:

33; 100; 333; 1000; 2500; and 5000 µg/plate

For each strain and dose level, including the controls three plates were used.

In a pre-experiment the concentration range of the test item was 3 - 5000 pg/plate. No relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2.5 μg/plate (10 µg/plate in TA102), in DMSO; with S9); 4-nitro-o-phenylenediamine (10 μg/plate in TA98 and 50 µg/plate in TA1537, in DMSO; without S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at testing: not specified

DURATION
- Preincubation period: 60 min
- Exposure duration: at least 48 hours

DETERMINATION OF CYTOTOXICITY
- Toxicity detected by a
• reduction in the number of revertants
• clearing or diminution of the background lawn

Evaluation criteria:

Acceptance criteria:
The experiment was considered valid if the following criteria were met:
• regular background growth in the negative and solvent control
• the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
• the positive control substances should produce a significant increase in mutant colony frequencies

Assessment criteria:
The test substance was considered positive in this assay if one of the following criteria was met:
• the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100, and TA 102 or thrice in strains TA 1535 and TA 1537
• a dose-dependent and reproducible increase in the number of revertants was induced

A test substance was generally considered non-mutagenic in this test if:
• neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is induced

Statistics:

A statistical analysis of the data was not performed.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated weakly at 2500 µg/plate and above in the overlay agar in experiment I with S9 mix. The undissolved particles of the test item had no influence on the data recording.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

Appropriate reference mutagens showed a distinct increase in induced revertant colonies.

HISTORICAL CONTROL DATA
- Positive historical control data: see table 3
- Negative (solvent/vehicle) historical control data: see table 3

Tab. 1: Summary of results (without S9 mix)

Concentration µg/plate	Revertants/plate, mean from three plates
	TA1535  I                 II		TA1537   I              II		TA98   I       II		TA100    I                II		TA102  I                    II
Negative control Solvent control Positive control* 33 100 333 1000 2500 5000	16 15 1070 17 16 16 18 18 16	10 10 841 8 8 10 6 5 10	7 9 48 7 7 6 11 11 14	5 4 58 5 5 5 7 5 5	21 20 244 17 22 22 16 19 19	16 17 177 12 15 15 15 18 15	147 130 766 136 130 143 155 134 108	105 91 820 105 88 101 92 102 91	251 240 1383 309 264 286 303 305 289	207 149 872 142 158 171 161 139 143

* Sodium azide (10 µg/plate) strains TA 1535 and TA 100;

4-nitro-o-phenylene-diamine strains TA 1537 (50 µg/plate) and TA 98 (10 µg/plate);

Methyl methane sulfonate (5 µl/plate) strain TA 102

Tab. 2: Summary of results (with S9 mix)

Concentration µg/plate	Revertants/plate, mean from three plates
	TA1535   I           II		TA1537   I              II		TA98     I       II		TA 100    I                II		TA102      I              II
Negative control Solvent control Positive control* 33 100 333 1000 2500 5000	15 15 187 15 16 16 18 18 9	11 9 85 6 6 8 7 11 8	10 10 125 9 13 14 18 14 8	8 5 64 9 4 6 7 5 5	22 25 637 18 24 21 26 22 16	19 20 598 15 14 20 21 19 24	142 146 939 155 155 140 153 169 144	118 102 573 116 121 97 109 122 116	362 400 1152 424 358 372 422 372 286	221 239 835 268 257 260 252 255 232

* 2-aminoanthracene (2.5 µg/plate) strains TA 1535, TA 1537, TA 98, and TA 100;

2-aminoanthracene (10 µg/plate) strain TA 102

Tab. 3: Historical Control Data

Negative Control (without metabolic activation)
1535	1537	98	100	102
9-29	5-28	15-57	77 -189	121 - 293

 

Solvent Control (without metabolic activation)
1535	1537	98	100	102
9-25	4-28	14-58	77-225	121 - 295

 

Positive Control (without metabolic activation)
1535	1537	98	100	102
68- 814	42 -191	86-450	460-910	751- 1395

Conclusions:

In the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was

observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Mutagenicity

 

In a reliable GLP-conform study according to OECD TG 471 (Klimisch score 1), the target substance was tested for its potential to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102 in two different experiments (plate incorporation test and pre-incubation test). The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

 

Mammalian Mutagenicity

 

In a reliable GLP-conform study according to OECD TG 476 (Klimisch score 1), the target substance was tested for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was solely performed in the absence of metabolic activation with a treatment period of 24 hours. The test item was evaluated at the following concentrations: 5.0; 10.0; 20.0; 100.0; and 320.0 µg/ml. According to the pre-experiment on toxicity and the solubility of the test item the concentration range was selected. In the pre-experiment precipitation visible to the unaided eye occurred already at 39.1 µg/ml and above with and without metabolic activation during pulse and continuous treatment. No relevant toxic effects were observed up to the maximal analyzable concentration of 312.5 µg/ml. Higher concentrations led to very heavy precipitation of the test item in the suspension cell cultures precluding cell counting. No relevant toxic effects occurred in both main experiments with the exception of culture II at the maximal concentration in the first experiment with metabolic activation. Precipitation of the test item visible to the unaided eye was observed at 20.0 µg/ml and above in both main experiments. No relevant and reproducible increase in mutant colony numbers was observed in both experiments in the absence and presence of metabolic activation. In the first experiment, the threshold of twice the colony count of the corresponding solvent control was reached in the second culture with metabolic activation at the maximal concentration under heavy precipitation. Since no comparable effects occurred in the first parallel culture under identical conditions, this increase was judged as an artefact based upon precipitation of the test item. The ratio of small versus large colonies was not shifted up to the maximal analyzable concentration. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large colonies.

 

Chromosomal Damage

 

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) - 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.

 

Further toxicological data of category members:

 

Additionally, several reliable data for genetic toxicity in vitro as well as in vivo are available for other quinacridone pigments (Pigment Violet 19 and Pigments Red 122, 202, 209 and 282).

 

Bacterial Mutagenicity:

In a reliable GLP-conform study according to OECD TG 471 (Klimisch score 1), Pigment Violet 19 was tested for its mutagenicity in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and Escherichia coli strain WP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate using the plate incorporation assay. Additionally, a preincubation assay with or without metabolic activation was performed using the concentrations 33, 100, 333, 1000, 2500, and 5000 µg/plate. The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

 

In a reliable GLP-conform study according to OECD TG 471 (Klimisch score 1), Pigment Red 122 was tested for its mutagenicity in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. Slight toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in the absence of metabolic activation in strain TA 1535 at 5000 µg/plate and in strain TA 1537 at 2500 and 5000 µg/plate in experiment I, and in strain TA 1537 from 1000 - 5000 µg/plate in experiment II. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In a reliable GLP-conform study similar to OECD TG 471 (Klimisch score 2), Pigment Red 202 was tested for its mutagenicity in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 with (induced rat liver S9 mix) and without metabolic activation at concentrations of 100, 333, 667, 1000, 3330 and 5000 µg/plate using the plate incorporation assay. The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects. In another reliable GLP-conform study similar to OECD TG 471 (Klimisch score 2) the Pigment Red 202 was again tested for its mutagenicity in Escherichia coliWP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate using the plate incorporation assay. Here as well, the test item did not reveal any mutagenic activity under the conditions tested.

 

In a reliable GLP-conform study according to OECD TG 471 (Klimisch score 1), Pigment Red 209 was tested for its mutagenicity in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and Escherichia coli strain WP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 50, 160, 500, 1600, and 5000 µg/plate using the plate incorporation assay as well as an independently performed preincubation assay. The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

 

In a reliable GLP-conform study according to OECD TG 471 (Klimisch score 1), Pigment Red 282 was tested for its mutagenicity in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and Escherichia coli strain WP2uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate using the plate incorporation assay. Additionally, a preincubation assay with or without metabolic activation was performed using the concentrations 33, 100, 333, 1000, 2500, and 5000 µg/plate. The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects, thus confirming the validity of this assay.

 

Mammalian Mutagenicity

 

In a reliable GLP-conform study according to OECD TG 476 (Klimisch score 1), Pigment Violet 19 was tested for its genotoxic potential using the HPRT and XPRT genes in CHO AA8 cells (with and without metabolic activation) using test concentrations of 15.625, 32.25, 62.5 and 125 µg/mL based on initial cytotoxicity test. The test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system. The test item was considered to be non-mutagenetic under the conditions of the test. The appropriate reference mutagenes  showed distinct positive mutagenic effects, thus confirming the validity of this assay.

 

In a reliable GLP-conform study according to OECD TG 476 (Klimisch score 1), Pigment Red 122 was tested for its potential to induce gene mutations at the HGPRT locus in V 79 cells of the Chinese hamster (with and without metabolic activation) using test concentrations of  2, 5, 10 and 20 µg/mL based on initial cytotoxicity test. The test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system. The test item was considered to be non-mutagenetic under the conditions of the test. The appropriate reference mutagenes  showed distinct positive mutagenic effects, thus confirming the validity of this assay.

 

In a reliable GLP-conform study similar to OECD TG 476 (Klimisch score 2), Pigment Red 202 was tested for its ability to induce forward mutations at the thymidine kinase (TK) locus in the L5178Y mouse lymphoma cell line. The mutation assays were performed at concentrations up to 5000 µg/mL with and without metabolic activation (S9 liver homogenate from Aroclor-induced rats), based on the results of a cytotoxicity pretest. Without metabolic activation, treatments at 3500 µg/mL and 5000 µg/mL were moderately cytotoxic and the remaining four treatments (78.1 µg/mL to 1250µg/mL) were non-cytotoxic. None of the six analyzed treatments induced a mutant frequency that exceeded the minimum criterion for a positive response. In the presence of metabolic activation, treatments from 78.1 µg/mL to 5000 µg/mL were also evaluated. Concentrations of 3500 µg/mL and 5000 µg/mL were weakly cytotoxic and the remaining four treatments were non-cytotoxic. No evidence for mutagenicity was observed at any concentration.

 

Chromosomal Damage

 

In a reliable GLP-conform study according to OECD TG 473 (Klimisch score 1), Pigment Violet 19 was tested for its ability to induce chromosomal aberrations in human lymphocytes following in vitro exposure in the presence and absence of a metabolizing system with test concentrations of 0.015625, 0.03125 and 0.0625 mg/mL based on initial cytotoxicity results. The test substance did not cause a relevant increase in the number of structurally aberrant metaphases. No increase in the frequency of cells containing numerical aberrations was demonstrated either. The test substance is considered not to be a chromosome-damaging (clastogenic) agent. The appropriate reference mutagenes  showed distinct positive mutagenic effects, thus confirming the validity of this assay.

 

In a reliable GLP-conform study according to OECD TG 473 (Klimisch score 1), Pigment Red 282 was tested for its potential to induce chromosome aberrations in V79 cells of the Chinese hamster in vitro exposure in the presence and absence of a metabolizing system with test concentrations between 0.31 and 10 µg/mL based on initial cytotoxicity results. The test substance did not cause a relevant increase in the number of structurally aberrant metaphases. No increase in the frequency of cells containing numerical aberrations was demonstrated either. The test substance is considered not to be a chromosome-damaging (clastogenic) agent. The appropriate reference mutagenes  showed distinct positive mutagenic effects, thus confirming the validity of this assay.

 

In a reliable GLP-conform study according to OECD TG 474 (Klimisch score 1), Pigment Red 122 was tested in the micronucleus test. The test compound was suspended in sesame oil and dosed once oral at 2500 mg/kg bw to male and female mice. The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes was statistically not different from the control values. The positive control substance induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system.

 

In a reliable GLP-conform study similar to OECD TG 474 (Klimisch score 2), Pigment Red 202 was tested in the micronucleus test. The test item was suspended in corn oil and applied to CD-1 mice by oral gavage at 1250, 2500, and 5000 mg/kg, based upon results of a range finding pre-test. The test item did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay. The positive control group showed a significant positive response.

 

Therefore, no classification for genotoxicity is necessary for the members of the Quinacridone Pigments category.

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 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP) and the mouse lymphoma assay (OECD 476, GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the 17th time in Regulation (EC) No. 2021/849.