2,9-bis(3,5-dimethylphenyl)anthra[2,1,9-def:6,5,10-d'e'f']diisoquinoline-1,3,8,10(2H,9H)-tetrone

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 compliant, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA, with and without metabolic activation, 1998, K1

 

HPRT: negative, according to OECD TG 476, GLP compliant, Chinese hamster ovary (CHO), with and without metabolic activation, negative, 2020, K1

 

Micronucleus test: negative, according to OECD TG 478, GLP compliant, primary human lymphocytes (buffy coat cells), without metabolic activation, negative, 2021, 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:

12 Feb 2021 - 23 Jul 2021

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:

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

14 Feb 2017

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Version 1.2, dated 06 May 2018

Principles of method if other than guideline:

The test substance is an insoluable organic 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 nanomaterial and is largely insoluable. Therefore, the selection of the concentration to be tested and scored is based either on the induced cytotoxicity or the homogeneity of the dispersion in the vehicle.
2) Metabolic activation: nanoparticles do not generally require metabolic activation (Elspuru, 2018). 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 (Elspuru, 2018).
4) The compatibility of the used test procedure for the assessment of the putative mutagenic potential of a nanomaterial is confirmed by the additional testing of the nanomaterial positive control Tungsten-Carbide-Cobalt (WC-Co). This compound has been shown to be a suitable nanomaterial positive control (Moche; 2014)

Elespuru R (2018) Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods. Toxicological Sciences 164(2), 391-416
Moche H, Chevalier D, Barois N, Lorge E, Claude N, Neslany F (2014) Tungsten Carbide-Cobalt as a nanoparticulate reference positive control in in vitro genotoxicity assays. Toxicological Sciences 137(1), 125-134

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Supplier: BASF
Batch identification: 100071P040
CAS No.: 4948-15-6
Purity: ≥ 99 %
Date of production: 21 Dec 2010
Physical state, appearance: Solid, red
Mass-specific surface area (BET): 97.1 m²/g

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Storage conditions: ambient (RT)
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
Storage stability: The stability of the test substance under storage conditions was guaranteed until 30 Mar 2023 as indicated by the sponsor, and the sponsor holds this responsibility.

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

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

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED

- Sex, age and number of blood donors:
1st experiment: one male, 34 year old;
2nd experiment: one female, 24 year old
- Whether whole blood or separated lymphocytes were used: Buffy coat cells were isolated from whole blood and cultures thereof were treated with the test substance.
- Mitogen used for lymphocytes: Phytohemagglutinin (PHA) / Phytohemagglutinin M form (PHA-M)
- The lymphocytes of each donor have previously shown to respond well to stimulation of proliferation with PHA and to the used positive control substances.

MEDIA USED
All media were supplemented with:
• 1% [v/v] penicillin/streptomycin (final concentration 100 μg/mL)
• 20% [v/v] fetal calf serum (FCS)
For the stimulation the medium was supplemented with:
• 0.5% [v/v] Phytohemagglutinin (PHA, stock solution 0.6 mg/mL, final concentration
3 μg/mL) for the 1st Experiment and 1.5% Phytohemagglutinin M form (PHA-M) for the 2nd
Experiment
For the Cytochalasin B treatment the medium was supplemented with:
• 30 μL Cytochalasin B (Cyt B, stock solution: 2 mg/mL in DMSO, final concentration: 6
μg/mL)
Culture medium
RPMI 1640 medium containing stable glutamine supplemented with 20% [v/v] FCS.

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

Cytokinesis block (if used):

Cytochalasin B (Cyt B)

Metabolic activation:

without

Test concentrations with justification for top dose:

The selection of the top concentration to be used were based on the homogeneity of the test substance in the vehicle.
The stock dispersions 20 and 5 mg/mL could not be prepared, due to inhomogenous formulation ot the test substance. Therefore, the highest stock dispersion, which could be used was 2.56 mg/mL. Thus, 256 μg/mL was the highest tested concentration.
Selected doses: 1, 3, 10, 30, 60, 100, 256 µg/mL.

Vehicle / solvent:

In accordance to the “SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 6 May 2018, 0.05% w/v bovine serum albumin water (BSA-water) was used as vehicle.
The final concentration of the vehicle 0.05% w/v BSA-water in culture medium was 10% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

mitomycin C

other: Tungsten Carbide-Cobalt

Details on test system and experimental conditions:

TIME SCHEDULE:
Day 1: Activation of the cells with Phytohemagglutinin
Day 3: Test substance incubation (approx. 48 hours after activation)
Day 4: Removal of test substance by intense washing; treatment with Cyt B
Day 5: Preparation of the slides

Since a pulse treatment (as described in OECD 487) does not allow enough time for the nanoparticles to enter the cell, only continuous treatment protocol was used.

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

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

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 20 h
- Harvest time after the end of treatment: 20 h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- cytokinesis blocking: 6 µg/mL Cytochalasin B (Cyt B), 20 h
- Methods of slide preparation and staining technique: The cells were centrifuged (900 g, 5 min, 4°C) and suspended in fresh fixative and incubated for 20 min at 4°C. The fixation step was repeated twice. After the last fixation step, the cells were centrifugated directly (900 g, 5 min, 4°C), suspended in 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, 10 min) and mounted.
- Number of cells spread and analysed per concentration: At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.
- Criteria for scoring micronucleated cells: The analysis of micronuclei was carried out according to the following criteria of Countryman and Heddle (1976).
- The diameter of the micronucleus was less than 1/3 of the main nucleus
- The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
- Only binucleated cells were scored.
Slides were coded randomly before microscopic analysis with an appropriate computer program. Cultures with few isolated cells were analyzed for micronuclei.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it was determined in 500 cells per culture (1000 cells per test group). This value indicates the average number of cell cycles per cell during the period of exposure to the actin polymerization inhibitor Cyt B.

CBPI = ((No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)) / (Total number of cells)

The CBPI was used to calculate the % cytostasis (relative inhibition of cell growth compared to the respective vehicle control group) - a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.

% Cytostasis = 100 - 100 {(CBPIT - 1) / (CBPIC - 1)}

T = test substance treated culture
C = vehicle control culture

pH value:
At the beginning of the treatment period, the pH was measured at least for the top concentration and for the vehicle control, each.

Evaluation criteria:

Acceptance criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells in the control groups (vehicle/positive) and in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.

Assessment criteria:
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).

Statistics:

An appropriate statistical analysis was performed. The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i. e. one-sided Fisher's exact test).
If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (s) were printed in the tables.
In addition, a statistical trend test (SAS procedure REG) was performed to assess a possible dose-related increase of micronucleated cells. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report.
The dependent variable was the number of micronucleated cells and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05.
However, both, biological and statistical significance were considered together.

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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

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

CYTOTOXICITY
In this study, no reduced proliferative activity was observed after 20 hours continuous test substance treatment in the test groups scored for cytogenetic damage.
Reduced proliferation was obtained in the 2nd Experiment using the nanomaterial positive control at 100.0 μg/mL (CBPI cytostasis: 60.0%). Therefore, this control group was not scored for micronucleated cells. At 30 and 60 μg/mL reduced proliferation (cytostasis: 51.2 and 58.1%, respectively) was obtained.

STUDY RESULTS
In the 1st and 2nd Experiment the values (0.4 – 0.9% micronucleated cells) were within the 95% upper control limit of the historical negative data range (0.2 – 0.9% micronucleated cells). A statistical significance compared to the concurrent vehicle control value (0.5 and 0.7% micronucleated cells) was not observed (one-sided Fisher's exact test).
In the 1st Experiment the micronucleus rates were dose-relatedly increased at 10.0, 30.0, 100.0 and 256 μg/mL (0.6%, 0.6%, 0.5% and 0.9% micronucleated cells, respectively). However, all values were clearly within the range of the 95% control limit of our historical negative control data range. To clarify the results a repeat experiment was carried out, which showed no positive response as assessed by trend analysis. Therefore, these findings are regarded as biologically irrelevant.
The positive control substances MMC and Colchicine induced statistically significantly increased micronucleus frequencies in all independently performed experiments. In this study, the frequencies of micronucleated cells (MMC: 9.7% and 5.3%; Colchicine: 2.4% and 2.9%) were compatible to the historical positive control data range.
The nanomaterial positive control substance Tungsten Carbide-Cobalt (WC-Co) induced statistically significantly increased micronucleus frequencies in all independently performed experiments. In this study, the frequencies of micronucleated cells (1st Experiment at 100
μg/mL: 1.3% and 2nd Experiment at 60 μg/mL: 1.5%) and exceeded the 95% upper control limit
of the historical negative data range (0.2 – 0.9% micronucleated cells)

For further details see table 2.

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

Table 1: Linear trend-test 

Linear trend-test	Slope	One-sided p-value*
1st Experiment	0.00128	0.01325
2nd Experiment	0.00056848	0.15946

* The linear trend-test testing for an increased number of micronucleated cells is significant (significance level of 5%), if the one-sided p-value is lower than 0.05.
Values indicating a statistically significant trend are printed in bold characters.

 

Table 2: Summary of results

Exp.	Exposure/ Recovery/ Preparation intervall	Test groups	Micronucleated cells *	Cytotoxicity Proliferation index cytostasis
	[h]	[µg/mL]	[%]	RS [%]
1	20/0/20	VC^1	0.5	0.0
		1.0	n.d.	-1.9
		3.0	n.d.	1.5
		10.0	0.6	1.7
		30.0	0.6	4.3
		60.0	n.d.	4.1
		100.0	0.5	3.7
		256.0	0.9	3.4
		WC-Co 30	n.d.	32.9
		WC-Co 60	n.d.	40.4
		WC-Co 100	1.3^S	44.2
		PC^2	9.7^S	14.2
		PC^3	2.4^S	32.5
2	20/0/20	VC^1	0.7	0.0
		1.0	n.d.	-4.6
		3.0	n.d.	-2.5
		10.0	0.4	-6.7
		30.0	0.6	-11.4
		60.0	n.d.	-5.4
		100.0	0.7	-9.1
		256.0	0.7	0.7
		WC-Co 30	0.9	51.2
		WC-Co 60	1.5^S	58.1
		WC-Co 100	n.s.	60.0
		PC^2	5.3^S	0.4
		PC^3	2.9^S	24.8

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

^s Frequency statistically significantly higher than corresponding control values

n.d. Not determined; n.s. Not scorable due to strong cytotoxicity

VC vehicle control; PC positive control

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

 

Table 3: HISTORICAL NEGATIVE CONTROL DATA

Period: April 2018 - November 2020

	Micronucleated cells [%]
Exposure period	20 hrs
Mean	0.5
Minimum	0.2
Maximum	1.2
Standard Deviation	0.17
95% Lower Control Limit	0.2
95% Upper Control Limit	0.9
No. of Experiments	54

  

Table 4: HISTORICAL POSITIVE CONTROL DATA

Period: April 2018 – November 2020

	Micronucleated cells [%]
	Mitomycin C (0.04 µg/mL)	Colchicin (0.05 µg/mL)
Exposure period	20 hrs	20 hrs
Mean	4.1	4.0
Minimum	2.1	2.4
Maximum	7.1	7.2
Standard Deviation	0.93	1.05
No. of Experiments	48	45

 

Conclusions:

Under the experimental conditions chosen here, the conclusion is drawn that the test material has no potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence of metabolic activation.

Executive summary:

The test substance was tested for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Two independent
experiments were carried out, incubating the cells for 20 h (20 h harvest time) with the test substance at concentrations in the range of 1.0 to 256 µg/mL. A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. In this study, 0.05% w/v BSA-water was selected as vehicle. The characterization of the nanomaterial in cell culture medium showed, that the particles were successfully dispersed into a stable suspension with partial agglomeration, that did not change significantly during the treatment period. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicine (Col) and the nanomaterial positive control Tungsten Carbide-Cobalt (WC-Co), led to the expected increase in the number of cells containing micronuclei.

The test substance was formulated in the given vehicle according to the 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, test material is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 May 2020 - 13 Jul 2020

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 using the Hprt and xprt genes)

Version / remarks:

29 Jul 2016

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Batch identification: 100071P040
CAS No.: 4948-15-6
Content: Sum.: 99.7 g/100 g
Date of production: 21 Dec 2010
Date of expiry: 30 Mar 2023
Molecular weight: 598.66 g/mol
Physical state, appearance: Solid, orange-red
Mass-specific surface area (BET): 97.1 m²/g
Storage conditions: Room temperature
Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.
Storage stability: The stability of the test substance under storage conditions was guaranteed until 30 Mar 2023 as indicated by the sponsor, and the sponsor holds this responsibility.

Target gene:

hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO (Chinese hamster ovary)
- Suitability of cells: not specified

For cell lines:
- Absence of Mycoplasma contamination: each batch checked
- Number of passages if applicable: at least 2 passages before experiment; a further passage to prepare test cultures
- Methods for maintenance in cell culture: Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and subcultured twice weekly (routine passage in 75 cm² plastic flasks).
- Cell cycle length, doubling time or proliferation index : high proliferation rate (doubling time of about 12 - 16 hours)
- Modal number of chromosomes: karyotype with a modal number of 20 chromosomes
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- All media were supplemented with penicillin/streptomycin (100 IU / 100 μg/mL), amphotericine B (2.50 μg/mL)
- Culture medium/Treatment medium(without S9 mix): Ham's F12 medium with stable glutamine, hypoxanthine, 10% (v/v) fetal calf serum (FCS)
- Treatment medium (with S9 mix): Ham's F12 medium with stable glutamine, hypoxanthine
- Pretreatment medium ("HAT" medium): Ham's F12 medium with hypoxanthine (136 µg/mL), aminopterin (1.8 µg/mL), thymidine (38.8 µg/mL), 10% (v/v) FCS
- Selection medium ("TG" medium): Ham's F12 medium with stable glutamine, 6-thioguanine (10 μg/mL), 10% (v/v) FCS

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)

The S9 fraction was prepared according to Ames et al. (1975):
- At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days.
- During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am – 6 pm and darkness from 6 pm – 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum.
- 24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C.
- The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- The S9 mix was prepared freshly prior to each experiment (Ames, 1975). For this purpose, a sufficient amount of S9 fraction was thawed at room temperature; 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors) in the pre-experiment and main experiments. This preparation, the S9 mix (10% S9 fraction), was kept on ice until used. The concentrations of the cofactors in the S9 mix were: 8 mM MgCl2, 33 mM KCL, 5 mM glucose-6-phosphate, 4 mM NADP, 15 mM phosphate buffer (pH 7.4).
- The phosphate buffer (DeMarini, 1989) is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.

Ames, B.N. et al. (1975): Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mut. Res. 31, 347-364.
DeMarini, D.M. et al. (1989): Cytotoxicity and effect on mutagenicity of buffers in a microsuspension assay. Ter. Carc. Mut. 9, 287-295.

Test concentrations with justification for top dose:

Based on the data and the observations from the pre-test and taking into account the current
guidelines, the following doses were selected in this study:
Dose selection (with and without S9 mix), 4 h exposure: 0.05, 0.08, 0.15, 0.26, 0.48, 0.86, 1.54, 2.78, 5.00 µg/mL

The highest tested concentration (5.00 μg/mL) was based on test substance precipitation in culture medium.

Vehicle / solvent:

Due to the insolubility of the test substance in culture medium, acetone was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available. The final concentration of the vehicle acetone in culture medium was 1% (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Time schedule:
- Day 1: Seeding of the cells pretreated with "HAT" medium: in 300 cm² flasks (20x10^6 cells in 40 mL)
- Day 2: Test substance incubation (20 – 24 hours after seeding); exposure period (4 hours); removal of test substance by intense washing; 1st passage of the treated cells in 175 cm² flasks (2x10^6 cells in 20 mL medium) and seeding of the cloning efficiency 1 (survival) in 60 mm petri dishes (200 cells in 5 mL medium).
- Day 5: 2nd passage of the treated cells (seeding of 2x10^6 cells in 20mL medium)
- Day 7 - 9: Drying, fixation, staining and counting of the cloning efficiency 1; 3rd passage of the treated cells; addition of selection medium ("TG" medium); and seeding of the cloning efficiency 2 (viability)
- From day 16: Drying, fixation, staining and counting of the selected colonies and cloning efficiency 2

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20x10^6 cells in 40 mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Attachment period of the cells: 20 - 24 hours
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time: 7-9 days (from day 2)
- Selection time: 6-7 days (from day 7 to 9)
- Selective agent: 6-thioguanine (10 μg/mL)
- Fixation time: from day 7-9 and 16
- Each test group were fixed with methanol, stained with Giemsa and counted.

METHODS FOR MEASUREMENT OF CYTOTOXICITY

- Cloning efficiency 1: For the determination of the influence of the test substance after the exposure period, 200 cells per concentration were reserved from the treated cells and were seeded in petri dishes (60 mm diameter) and coated with 5 mL Ham's F12 medium incl. 10% (v/v) FCS in parallel to the 1st passage directly after test substance incubation.
- Cloning efficiency 2: For the determination of the mutation rate after the expression period, two aliquots of 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two petri dishes (60 mm diameter) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS.
- In all cases, after seeding the flasks or petri dishes were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted.

The cloning efficiency (CE, %) was calculated for each test group as follows:

total number of colonies in the test group
CEabsolute = ————————————————————— x 100
total number of seeded cells in the test group

CEabsolute of the test group
CErelative = —————————————— x 100
CEabsolute of the vehicle control


The number of colonies in every petri dish was counted and recorded. Using the formula above the values of absolute cloning efficiencies (CEabsolute, CE1 absolute and/or CE2 absolute) were calculated. Based on these values the relative cloning efficiencies (CErelative, CE1 relative and/or CE2 relative) of the test groups were calculated and reported as a percentage of the respective CEabsolute value of the corresponding vehicle control (vehicle control = 100%).
In addition, with regard to cell loss while exposure period, relative survival (RS) is calculated based on CE of cells plated immediatedly after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in vehicle controls.

number of cells at the end of treatment
Adjusted CE = —————————————————— x CE1
number of seeded cells

adjusted CE of the test group
RS = ———————————————————— x 100
adjusted CE of the vehicle control


METHODS FOR MEASUREMENTS OF GENOTOXICIY

The number of colonies in each flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 10^6 cells seeded. The uncorrected mutant frequency (MFuncorr.) per 10^6 cells was calculated for each test group as follows:

total number of mutant colonies
MFuncorr. = —————————————–— x 10^6
number of seeded cells

The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MFcorr.):

MFuncorr.
MFcorr. = —–——–— x 100
CE2 absolute

OTHER:
- Check or determination of further parameters: pH, osmolality, solubility, cell morpology

Evaluation criteria:

Acceptance criteria:

- The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the vehicle controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• Concurrent positive controls both with and without S9 mix should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase in mutant frequencies compared with the concurrent vehicle control.


Assessment criteria:

- A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

- Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

- A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation
frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent
vehicle control value and within the range of our laboratory’s historical negative control data
(95% control limit).

Statistics:

A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose.
The calculation was performed using EXCEL function RGP.
The used model is one of the proposed models of the International Workshop on Genotoxicity
Test procedures Workgroup Report (Moore, 2003).
A pair-wise comparison of each test group with the control group was carried out using Fisher's exact test with Bonferroni-Holm correction (Holm, 1979 and Siegel, 1956). The calculation was performed using EXCEL function HYPGEOM.VERT.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables.
However, both, biological and statistical significance are considered together.


- Moore, M.M. et al. (2003) Mouse Lymphoma Thymidine Kinase Gene Mutation Assay: International Workshop on Genotoxicity Tests (IWGT) Workgroup Report – Plymouth, UK 2002. Mut. Res. 540, 127–140.
- Holm, S. (1979) A Simple Sequentially Rejective Multiple Test Procedure. Scand J Statist 6, 65-70.
- Siegel, S. (1956) Nonparametric statistics for the behavioral sciences. New York, NY, US: McGraw-Hill

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not affected
- Data on osmolality: not affected
- Possibility of evaporation from medium: no
- Water solubility: insoluble in water and cell culture medium
- Precipitation and time of the determination: test substance precipitation was observed at 1.25 μg/mL and above after 4 hours in the absence and presence of S9 mix
- Cell morphology and attachment of the cells: not adversely influenced (grade > 2) in any test group

RANGE-FINDING/SCREENING STUDIES:
An initial range-finding cytotoxicity test for the determination of the experimental doses was conducted.
The pre-test was performed following the method described for the main experiment. The relative survival (RS) was determined as a toxicity indicator for dose selection and various parameters were checked for all, or at least some, selected doses.
In the pre-test for toxicity based on the solubility properties of the test substance 10.00 μg/mL (approx. 0.02 mM) was used as top concentration both with and without S9 mix at 4 hour exposure time.

STUDY RESULTS
- The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study.
- The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed.

For all test methods and criteria for data analysis and interpretation:
- At least four concentrations were evaluated to describe a possible dose response relationship.
- Statistical analysis; statistical significance for p ≤ 0.05
- see table 1 in section "Any other information on results incl. tables"

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements and genotoxicity results: see table 2 in section "Any other information on results incl. tables"

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

Table 1: Linear trend-test

 

Linear trend-test	Slope*	One-sided p-value*
Corrected Mutation frequency without S9 mix	-0.71162	0.7360
Corrected Mutation frequency with S9 mix	-1.32571	0.8616

* The linear trend-test testing for an increased mutant frequency is significant (significance level of 5%), if the one-sided p-value is lower than 0.05 and the slope is greater than 0.

 

Table 2: Summary of results

 

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity** MFcorr.	Cytotoxicity***
	[h]	[µg/mL]			[per 10^6 cells]	RS [%]	CE2 [%]
1	4	VC^1	-	n.d.	1.05	100.0	100.0
		0.05	-	-	n.c.	125.6	n.c.
		0.08	-	-	n.c.	134.3	n.c.
		0.15	-	-	n.c.	135.3	n.c.
		0.26	-	-	3.39	128.5	103.5
		0.48	-	-	0.00	124.5	135.8
		0.86	-	-	0.68	132.5	103.5
		1.54	-	+	0.61	128.7	114.7
		2.78	-	+	n.c.^1	n.c.^1	n.c.^1
		5.00	-	+	n.c.^1	n.c.^1	n.c.^1
		PC^2	-	n.d	42.12^s	113.2	102.5
1	4	VC^1	+	n.d.	1.42	100.0	100.0
		0.05	+	-	n.c.	91.9	n.c.
		0.08	+	-	n.c.	89.1	n.c.
		0.15	+	-	n.c.	103.8	n.c.
		0.26	+	-	3.13	107.9	102.5
		0.48	+	-	1.69	68.1	105.3
		0.86	+	-	1.53	96.1	92.9
		1.54	+	+	0.72	82.6	98.9
		2.78	+	+	n.c.^1	n.c.^1	n.c.^1
		5.00	+	+	n.c.^1	n.c.^1	n.c.^1
		PC^3	+	n.d.	48.52^s	72.6	84.3

 

* Microscopically visible precipitation in culture medium at the end of exposure period

** Mutant frequency MFcorr.: mutant colonies per 10⁶ cells corrected with the CE2 value

*** Cloning efficiency related to the respective vehicle control

^s Mutant frequency statistically significantly higher than corresponding control values (p ≤ 0.05)

n.c. Culture was not continued since a minimum of only four analysable concentrations is required

n.c.¹ Culture was not continued since only one concentration beyond the solubility limit is required

n.d. Not determined

VC vehicle control

PC positive control

¹ Acetone 1% (v/v) ² EMS 400 μg/mL ³ DMBA 1.25 μg/mL

 

 

Table 3: HISTORICAL NEGATIVE CONTROL DATA

Summary (all vehicles)

Period: March 2016 - December 2019

	Without S9 mix all vehicles*	With S9 mix all vehicles*
Corrected Mutant Frequency**
Exposure period	4 hrs	4 hrs
Mean	2.76	2.93
Minimum	0.00	0.00
Maximum	8.00	9.93
Standard Deviation	1.73	2.09
95% Lower Control Limit	0.00	0.00
95% Upper Control Limit	6.21	7.08
No. of Experiments	130	132

* = culture medium, water 10% (v/v), DMSO 1% (v/v), acetone 1% (v/v)

** = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

 

 

Table 4: HISTORICAL POSITIVE CONTROL DATA

Summary (all vehicles)

Period: March 2016 - December 2019

	Without S9 mix 400 µg/mL ethyl methanesulfonate (EMS)	With S9 mix 1.25 µg/mL 7,12-Dimethylbenz[a]anthracene (DMBA)
	Corrected Mutant Frequency*	Corrected Mutant Frequency*
Exposure period	4 hrs	4 hrs
Mean	160.94	126.62
Minimum	42.47	21.52
Maximum	438.29	270.48
Standard Deviation	73.52	54.78
No. of Experiments	124	129

* = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in an in vitro mammalian cell gene mutation test (HPRT-locus) in absence and in the presence of metabolic activation.

Executive summary:

The test substance was evaluated for genotoxic potential in a HPRT locus assay using CHO cells according to OECD TG 476 (GLP compliant). In one experiment a dose range from 0.05 to 5 µg/ml was tested, both with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without or after the addition of the metabolizing system (S9 mix). No cytotoxicity could be observed, but a precipitation of the test materials was seen after 4 h exposure at dose 1.54 and above. Overall, the test item was considered to be non-mutagenetic under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Physical state: red powder
- Storage condition of test material: room temperature
- Lot/batch No.: Mg 20024

Target gene:

his and trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

20 - 5000 µg/plate (standard plate test) and 4 - 1000 µg/plate (preincubation test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility/insolubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix; all strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N' -nitro-N-nitrosoguanidine

Remarks:

without S9 mix; TA 1535 and TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

without S9 mix; TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix; TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix; E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
Plate incorporation method:
- Exposure duration: ca. 48-72 hours at 37°C
Preincubation method:
- Preincubation period: 20 minutes at 37°C
- Exposure duration: ca. 48-72 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth), reduction in the titer

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Toxicity: A slight decrease in the number of revertants was occasionally observed.
- Precipitation: Precipitation of the test substance was found from about 100 μg/plate onward.

Results of Experiment I (Standard Plate Test with and without rat liver S-9 mix):

	TA98		TA100		TA1535		TA1537		WP2 uvrA
concentration	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	24	37	113	114	19	20	9	10	33	40
20	23	33	101	113	20	20	9	8	27	37
100	23	28	73	107	20	21	9	9	27	35
500	22	26	104	110	19	21	8	9	23	30
2500	22	25	105	108	20	17	7	9	27	31
5000	22	24	98	106	20	19	6	7	31	27
MNNG			1145		1020
2-AA		1327		1058		219		103		291
AAC							777
NOPD	1164
ENNG									873

Results of Experiment II (Pre-incubation Test with and without rat liver S-9 mix):

	TA98		TA100		TA1535		TA1537		WP2 uvrA
concentration	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	28	36	103	112	18	18	10	11	28	35
4	20	29	101	110	14	15	11	11	28	29
20	19	28	105	121	14	15	10	11	28	32
100	18	22	103	101	11	15	9	10	31	31
500	16	24	95	101	11	12	8	10	28	31
1000	14	21	89	104	9	11	9	11	30	33
MNNG			931		1017
2-AA		959		1054		151		176		195
AAC							676
NOPD	675
ENNG									843

Controls:

MNNG: N-methyl-N'-nitro-N-nitrosoguanidine (5 µg/plate)

2 -AA: 2-aminoanthracene (2.5 µg/plate for TA 98, 100, 1535, 1537; 60 µg/plate for WP2 uvrA)

AAC: 9-aminoacridine (100 µg/plate)

NOPD: 4-nitro-o-phenylendiamnine (10 µg/plate)

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine (10 µg/plate)

Conclusions:

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Executive summary:

The test article was tested in the Ames reverse mutation assay (GLP-compliant study according to OECD guideline 471) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvr A at 20 to 5000 µg/plate in the standard plate test and at 4 to 1000 µg/plate in the preincubation test (vehicle: DMSO) with and without metabolic activation. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9mix or after the addition of a metabolising system.A slight decrease in the number of revertants was occasionally observed. Precipitation of the test substance was found from about 100 μg/plate onward. Under the conditions tested, the test substance is not mutagenic in the Ames test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Mutagenicity

The test article was tested in the Ames reverse mutation assay (GLP-compliant study according to OECD guideline 471) using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvr A at 20 to 5000 µg/plate in the standard plate test and at 4 to 1000 µg/plate in the preincubation test (vehicle: DMSO) with and without metabolic activation (BASF AG, 1998). An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9mix or after the addition of a metabolising system.A slight decrease in the number of revertants was occasionally observed. Precipitation of the test substance was found from about 100 μg/plate onward. Under the conditions tested, the test substance is not mutagenic in the Ames test.

In a supporting Ames reverse mutation assay (study equivalent to OECD guideline 471) the test substance was tested using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538 and Escherichia coli WP2 uvr A at 4 to 3500 µg/plate (vehicle: DMSO) with and without metabolic activation using the plate incorporation test (Hoechst AG, 1982). Precipitation of the test substance was found from about 500 μg/plate onward. Under the conditions tested, the test article was not mutagenic in the Ames test.

 

Mammalian Mutagenicity

The test substance was evaluated for genotoxic potential in a HPRT locus assay using CHO cells according to OECD TG 476 with GLP compliance (BASF, 2020). In one experiment a dose range from 0.05 to 5 µg/ml was tested, both with and without the addition of liver S9 mix from phenobarbital and β-naphthoflavone induced rats. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without or after the addition of the metabolizing system (S9 mix). No cytotoxicity could be observed, but a precipitation of the test materials was seen after 4 h exposure at dose 1.54 and above. Overall, the test item was considered to be non-mutagenetic under the conditions of the test.

 

Mammalian Cell Micronucleus Test

The test substance was tested for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Two independent experiments were carried out according to OECD TG 487 and in GLP compliance (BASF, 2021), incubating the cells for 20 h (20 h harvest time) with the test substance at concentrations in the range of 1.0 to 256 µg/mL. A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. In this study, 0.05% w/v BSA-water was selected as vehicle. The characterization of the nanomaterial in cell culture medium showed, that the particles were successfully dispersed into a stable suspension with partial agglomeration, that did not change significantly during the treatment period. The vehicle controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicine (Col) and the nanomaterial positive control Tungsten Carbide-Cobalt (WC-Co), led to the expected increase in the number of cells containing micronuclei.

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, test material is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes.

 

Further toxicological data of category members:

The test article belongs to the "perylene based organic pigments" category (see attached document for details on category members and for read across justification). Regarding the genetic toxicity, additional reliable data are available for other category members. All of the studies are taken into account for the evaluation and assessment of the toxicity of the test article.

At least one Ames tests per substance is available for all other category members. None of these tests gave any rise to concern for genotoxicity. Consequently, all substances of this category have been regarded as not genotoxic in the bacterial reverse mutation test.

The additional HPRT and CA assays, performed for other category members, each with and without metabolic activation, were also negative and lead no evidence for a mutagenic potential of the test substances.

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), the HPRT Test (OECD 476, GLP) and the in vitro Micronucleus test (OECD 487, GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.