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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pigment Red 221 is an insoluble nanomaterial. It was found to be non mutagenic in the Ames test in 1994 (OECD 471, GLP), noting that the Ames test is not recommended for nanomaterials in the ECHA guidance ( Appendix R7-1 for nanoforms applicable to Chapter R7a and R7c Endpoint specific guidance, Draft public version 3.0 of June 2020). It also caused no genotoxicity in the micronuclues test in vivo (OECD 474, GLP) after gavage dosing. The latter study was not considered suitable for hazard assessment as systemic update had not been not investigated and may actually be impossible to demonstrate for an insoluble pigment.

Instead, the assessment shall be made using mammalian cells; however the OECD testing guidelines have not yet been adapted to include nanomaterials. The above mentioned guidance on information requirements recommends that for in-vitro testing, additional examinations are included to locate and characterize the nanoparticles. Particularly the dose setting is a challenge as the standard requirements of the guideline regarding the presence / absence of precipitates cannot be applied to insoluble nanomaterials. As a consequence, testing for gene mutations in mammalian cells in vitro has been postponed until testing conditions will have been established.

As the state of art is further advanced with the nanoprotocol for the in-vitro micronucleus test (OECD 487 and NANOGENOTOX-Project), Pigment Red 221 was tested in this assay in 2022. The study included the investigation of the dispersion stability in test medium at the beginning of the incubation and at the end of the 20h exposure. Pre-tests were carried out to identify the highest concentration with a stable dispersion and not leading to agglomeration.

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). Tested test material concentrations were: 1, 3, 10, 30, 60, 100 and 256 µg/mL.

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

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).

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).

At the above described conditions, Pigment Red 221 was tested in form of a stable dispersion, with particles ranging from 20 to 500 nm in size. No cytotoxicity and no formation of micronuclei were observed. The dissolved material was below the limit of quantification. Pigment Red 221 was found to be non genotoxic in the in-vitro micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021- 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:

- 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

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

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

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

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

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

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

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

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

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

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

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

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

Cytokinesis block (if used):

Cytochalasin B (Cyt B)

Metabolic activation:

without

Metabolic activation system:

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

Test concentrations with justification for top dose:

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

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

mitomycin C

other: Tungsten Carbide-Cobalt

Details on test system and experimental conditions:

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

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

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

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

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

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

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- cytokinesis blocking: 6 µg/mL Cytochalasin B (Cyt B), 20 h
- Methods of slide preparation and staining technique: The cells were centrifuged (900 g, 5 min, 4°C) and suspended in fresh fixative and incubated for 20 min at 4°C. The fixation step was repeated twice. After the last fixation step, the cells were centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fresh fixative and spread on slides. The slides were dipped in deionized water, the cells were pipetted on the slide and fixed by passing through a flame. The cells were stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2, 10 min) and mounted.
- Number of cells spread and analysed per concentration: At least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.
- Criteria for scoring micronucleated cells: The analysis of micronuclei was carried out according to the following criteria of Countryman and Heddle (1976).
- The diameter of the micronucleus was less than 1/3 of the main nucleus
- The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
- Only binucleated cells were scored.
Slides were coded randomly before microscopic analysis with an appropriate computer program. Cultures with few isolated cells were analyzed for micronuclei.

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

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

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

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

T = test substance treated culture
C = vehicle control culture

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

Rationale for test conditions:

The test substance is a nanomaterial and is largely insoluable.

Evaluation criteria:

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

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

Statistics:

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

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity, tested up to homogenous nanoparticle disperspersion

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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

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

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

STUDY RESULTS
The values (0.4 – 0.7% micronucleated cells) were within the 95% upper control limit of the historical negative data range (0.2 – 0.9% micronucleated cells; see table). A statistical significance compared to the concurrent vehicle control value (0.6% micronucleated cells) was not observed.
The experimental part described above showed no positive dose response as assessed by a trend analysis.
The positive control substances MMC (0.04 μg/mL) and Colchicine (0.05 μg/mL) induced statistically significantly increased micronucleus frequencies in all independently performed experiments. In this study, the frequencies of micronucleated cells (5.3% and 2.9% micronucleated cells (MMC and Col, respectively) were compatible to the historical positive control data range. The nanomaterial positive control substance Tungsten Carbide-Cobalt (WC-Co) induced statistically significantly increased micronucleus frequencies. In this study, the frequencies of micronucleated cells (60 μg/mL: 1.5%) and exceeded the 95% upper control limit of the historical negative data range (0.2 – 0.9% micronucleated cells)

For further details see table 2.

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

Table 1: Linear trend-test 

Slope	One-sided p-value*
0.00037368	0.25058

* The linear trend-test testing for an increased number of micronucleated cells is significant (significance level of 5%), if the one-sided p-value is lower than 0.05.

 

Table 2: Summary of results

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

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

^s Frequency statistically significantly higher than corresponding control values

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

VC vehicle control; PC positive control

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

 

Table 3: HISTORICAL NEGATIVE CONTROL DATA

Period: April 2018 - November 2020

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

  

Table 4: HISTORICAL POSITIVE CONTROL DATA

Period: April 2018 – November 2020

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

 

Fractionating techniques with selective detection (AUC, UIVVis) were used to characterise the test item preparations in the cell culture medium. Compared to the size of the constituent particles determined independently by TEM, the particles were successfully dispersed into a stable suspension with partial agglomeration that did not change significantly during the genotoxicity testing.

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

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

Conclusions:

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

Executive summary:

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

The test substance was formulated in the given vehicle according to the NANOGENOTOX Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018.

In this study, no cytotoxicity indicated by reduced proliferation index (CBPI) was observed up to the highest applied test substance concentration. On the basis of the results of the present study, the test substance did not cause any biologically relevant in the number of cells containing micronuclei.
Thus, under the experimental conditions described, test material is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Pigment Red 221 (CAS 71566-54-6, 926 g/mol)

Pigment Red 221 was tested for mutagenic effects in vitro in five histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli (Ciba-Geigy Ltd 1994). The following strains were used: S. typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537 and E. coli WP2 uvrA. The study was performed under GLP and according to OECD

testing guideline 471. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was suspended in ethanol and tested at five concentrations in the range of 312.5 to 5000.0 mg/plate in the presence and absence of a metabolic activation system. In order to confirm the results, the experiments were repeated with and without metabolic activation at the concentrations of 61.7 to 5000.0 mg /plate. In the first and second mutagenicity test, it was tested as a suspension at all concentrations. In order to clarify if solutions of the test material yield different results, a further repetition was performed at concentrations in the range of 1.6 to 5000.0mg/plate. In this repetition the lowest substance concentration was completely dissolved in ethanol. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control. In all three experiments, performed with and without metabolic activation, none of the tested concentrations led to an increase in the incidence of either histidine- or tryptophan prototrophic mutants by comparison with the negative control.

Pigment Red 221 was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity) (BASF 2022). The test substance is an insoluable 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 (19). 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. Higher concentrations than 256 µg/mL could not be homogenously formulated. Test groups printed in bold type were evaluated for the occurrence of micronuclei: 20 hours exposure 0; 1; 3; 10; 30; 60; 100; 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 order to confirm the results of the cultures treated with Tungsten Carbide-Cobalt (WC-Co) additional 1000 cells per culture were scored for cultures treated with 60 and 100 µg/mL (WC-Co) as well as the vehicle control 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 dissolved content at the end of the incubation time of 20h was about 0.05%. 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 CarbideCobalt (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 relevant 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, Pigment Red 221 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

 

Pigment Red 221 was investigated for clastogenic (and/or aneugenic) effects on mouse bone marrow cells in vivo in a GLP compliant study following OECD testing guideline 474 (Ciba-Geigy Ltd 1995). The test substance was administered once by gavage to groups of 5 male and 5 female Tif MAGf (SPF) mice at doses of 5000, 2500 and 1250 mg/kg. Additional groups of animals were treated with the vehicle alone or with the positive control cyclophosphamide (64 mg/kg). From the high dose group and from the negative control group animals were sacrificed 24 and 48 hours thereafter. From the intermediate and the low dose group and from the positive control group animals were sacrificed 24 hours after application. Subsequently femoral bone marrow cells were prepared and polychromatic erythrocytes were scored for micronuclei. The high dose applied (limit dose) was toxic as manifested in the tolerability test by various symptoms. In all dosage groups assessed at the different periods post treatment, no biologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective negative control group.

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. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.