Tetramethyl 2,2'-[1,4-phenylenebis[imino(1-acetyl-2-oxoethane-1,2-diyl)azo]]bisterephthalate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro: Ames: negative; OECD 471, (Prival-Ames is ongoing)
Clastogenicity in human lymphocytes: non clastogenic (OECD 487, nanomaterial protocol)

Mouse lymphoma assay: negative; OECD 476

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:

2021-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

adapted for nanomaterials

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016, specific adaptions for nanomaterials as of - NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Issued by Landesamt für Umwelt, Rheinland-Pfalz, Germany,

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

yellow solid
batch 13007010
purity 95.7%
storage at room temperature

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

For lymphocytes:
- 31 year old male donor, non-smoking
- Buffy coat cells were isolated from whole blood
- Whether blood from different donors were pooled or not: not applicable; single donor
- Mitogen used for lymphocytes:Cytochalasin B

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Based on the solubility properties of the test substance 256 µg/mLwas used as top concentration. Higher concentrations led to inhomogeneous formulations.
1, 3, 10, 30, 60, 100, 256 mg/L

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 is 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: 100, 60 and 30 µg/mL Tungsten Carbide-Cobalt (Nanostructures and Amorphous Material Inc.; Houston, Texas)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : At least 2 cultures were prepared per test group (referred to as A and B), and at least 1000 cells per culture were evaluated for the occurrence of micronucleated cells.

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 20h
- Harvest time after the end of treatment (sampling/recovery times): see treamtent with cyokinesis blocking substance

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:

- Identity of cytokinesis blocking substance: cytoB, 20h exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells are transferred into tubes, centrifugated at 900 g for 5 min and washed with HBSS. After washing, the cells are 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 are fixed by adding of fixative (19 parts methanol and 1 part acetic acid). The cells are centrifuged (900 g, 5 min, 4°C) and fixed suspended in fresh fixative and incubated for 20 min at 4°C. The fixation step is repeated twice. After the last fixation step, the cells can be centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fresh fixative and spread on slides. The slides are dipped in deionized water, the cells are pipetted on the slide and fixed by passing through a flame. The cells are stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2, 20 min).


- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): criteria of Countryman and Heddle.
- The diameter of the micronucleus is less than 1/3 of the main nucleus
- The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
- Only binucleated cells will be scored.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: 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

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The slides were scored microscopically for micronuclei.

OTHER:
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 (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 in 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.

Rationale for test conditions:

Pre-experiments were performed to characterize the concentration depandent aggllomeration of particles.

Evaluation 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 is obtained.
• A dose-related increase in the number of cells containing micronuclei is observed.
• The number of micronucleated cells exceeds 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 is observed under any experimental condition.
• The number of micronucleated cells 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).

Species / strain:

lymphocytes: human

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no cytotoxicity, but tested up the dispersibility limit

Remarks:

poorly soluble nanoparticle

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: At the beginning of the treatment period, the pH was measured at least for the top concentration and for the vehicle control, each. No influence was observed.
- Data on osmolality: not determined
- Possibility of evaporation from medium: none
- Water solubility: Insoluble; see adaption for pooly soluble nanoparticles

RANGE-FINDING/SCREENING STUDIES :
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. The percentiles of the size distribution (D10, D50, D90) did show a trend with dose. The dissolved content at the end of the incubation time of 20h was around 0.05%.

STUDY RESULTS
- Concurrent vehicle negative and positive control data are shown in table 1
- Cytotoxicity and number of micronucleated cells are shown in table 1
- Historical control data are provided in the attachment.

Table 1: Summary of results

Test groups [μg/mL]	Micronucleated cells* [%]	Cytotoxicity Proliferation index cytostasis [%]
vehicle (0.05% BSA-water (w/v))	0.9	0.0
1	n.d.	5.3
3	n.d.	3.6
10	0.5	2.4
30	0.7	9.5
60	n.d.	3.4
100	0.5	6.9
256	0.9	9.4
WC-Co 30	1.1	49.7
WC-Co 60	1.2	59.5
WC-Co 100	n.d.	74.0
Positive control (MMC 0.04 μg/mL)	10.3^S	18.1
Positive control (Col 0.05 μg/mL)	3.0^S	19.8

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

S Frequency statistically significantly higher than corresponding control values

The increase in the frequencies of micronuclei induced by the positive control substances MMC and Colchicine demonstrated the sensitivity of the test system. The values were

compatible to the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study. The nanomaterial positive control also induced an

increase in the micronucleus frequency, which was in the range of the historical control data, nevertheless the values were not statistically significant. This was due to the

high vehicle control value, which was the same as the upper 95% control limit value. This does not affect the validity of the study.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-2012

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

- Expiry date: 23 Mar 2021
- Storage conditions: Room temperature
- Batch identification: DEB2 146870

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium including stable glutamine supplemented with
1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL)
1% (v/v) sodium pyruvate (10 mM) (= RPMI-0)
For 4-hour exposure interval:
RPMI-0 supplemented with 5% (v/v) fetal calf serum (FCS) (= RPMI-5)
For 24-hour exposure interval and subculturing cells:
RPMI-0 is supplemented with 10% (v/v) fetal calf serum (= RPMI-10)
For cloning efficiency and selection medium:
RPMI-0 supplemented with 20% (v/v) fetal calf serum (= RPMI-20)

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from rats pretreated with phenobarbital i.p. and β-naphthoflavone orally

Test concentrations with justification for top dose:

1st Experiment
without S9 mix (4-hour exposure period)
0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0; 20.0 μg/mL

with S9 mix (4-hour exposure period)
0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0; 20.0 μg/mL

2nd Experiment
without S9 mix (24-hour exposure period)
0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0 μg/mL

with S9 mix (4-hour exposure period)
0; 0.25; 0.5; 1.0; 2.0; 4.0; 8.0 μg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 4 - 5 days
- Exposure duration: 4h or 24h
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): trifluorthymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, relative growth


OTHER: During the week prior to treatment, spontaneous TK deficient mutants (TK-/-) were eliminated from the stock cultures by incubating 1.5 x 10E6 cells per 75 cm² flask (5 x 104 cells/mL) for 1 day in “THMG" medium (pretreatment medium A), and for the following 3 days in “THG" medium (pretreatment medium B).

Evaluation criteria:

The MLTK assay is considered valid if the following criteria are met considering the international guidelines and the current recommendations of the IWGT:
- The absolute cloning efficiency obtained at the time of mutant selection (CE2) of the negative/vehicle controls should fall in the range of 65 - 120%.
- The suspension growth (SG) of the negative/vehicle controls referring to the expression period following treatment should fall in the range of 8 - 32 for 4-hour exposure and 32 - 180 for 24-hour exposure.
- The mutant frequency of the negative/vehicle controls should fall within the range of 50 - 170 x 10E-6 colonies.
- The positive controls should yield an absolute increase in total MF that is an increase above the spontaneous background MF (an induced MF [IMF]) of at least 300 x 10E-6 colonies. The small colony MF should account for at least 40% of that IMF, means a small colony IMF of at least 120 x 10E-6 colonies. Alternatively, the positive controls should induce at least 150 small colonies. The upper limit of cytotoxicity observed in the positive controls should have a relative total growth (RTG) that is greater than 10%.
- The highest applied concentration of the test substance should be 5 mg/mL, 5 μL/mL or 10 mM, unless limited by cytotoxicity or solubility of the test substance. If toxicity occurs, the highest concentration should lower the cloning efficiency 1 (CE1) or the relative total growth (RTG) to 10 to 20% of survival. If precipitation occurs, the highest evaluated concentration should be the lowest concentration where precipitation is observed by the unaided eye.

Mutagenicity criteria:
- MF exceeds a threshold of 126 mutant colonies per 10exp6 cells above the concurrent negative/vehicle control value.
- Evidence of reproducibility of any increase in mutant frequencies
- A statistically significant dose-related increase in mutant frequencies

Statistics:

An appropriate statistical trend test (SAS procedure PROC REG; 9) was performed to assess a possible dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative/vehicle control groups. A trend was judged as statistically significant whenever the p-value (probability value) was below 0.10 and the slope was greater than 0.
However, both, biological and statistical significance has been considered together.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH value was not influenced by test substance treatment.
- Effects of osmolality: Osmolarity was not influenced by test substance treatment.
- Precipitation: was found from 4 µg/plate onwards

ADDITIONAL INFORMATION ON CYTOTOXICITY: In both experiments, no cytotoxic effects indicated by reduced cloning efficiencies or reduced relative total growth of below 20% of control were observed neither in the presence nor in the absence of S9 mix.

According to the results of the present in vitro study, the test substance did not lead to a relevant increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. The mutant frequencies at any concentration were close to the range of the concurrent negative control values and within the range of our recent negative control data set.

Unfortunately, not all acceptance criteria mentioned were fully fulfilled. In the 1st Experiment in the absence of S9 mix the absolute cloning efficiency of the vehicle control determined at the time of mutant selection (CE2) was slightly higher than the proposed upper border of 120% as recommended by IWGT. However, due to missing cytotoxicity and the fact that dose selection was based on the solubility properties of the test substance this finding has no impact on the outcome of this experiment. In addition, either in the 1st Experiment or in the 2nd Experiment in the absence of metabolic activation the corrected mutation frequencies of the vehicle controls (35.7 x 10E-6 colonies or 46.6 x 10E-6 colonies, respectively) were slightly lower than proposed by IWGT (50 – 170 x 10E-6 colonies). However, the mutation frequencies of the vehicle control groups were clearly within our laboratory’s historical negative control data range and, thus, these observations have to be regarded as irrelevant.

Finally, it has to be considered that the minor deviations from the criteria of acceptance described above have no detrimental impact on the validity of this study. The increase in the frequencies of mutant colonies induced by the positive control substances MMS and CPP clearly demonstrated the sensitivity of the test method and of the metabolic activity of the S9 mix employed. The values were within or above the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

Conclusions:

negative

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

with Prival modification

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reliable data from several studies on genetic toxicity are summarized in the table below:

	PY 93	PY 94	PY 95	PY 128	PY 155
	5580-57-4	5580-58-5	5280-80-8	79953-85-8	68516-73-4
Bacterial mutagenicity	Non mutagenic K1 Non Prival	Non mutagenic K1 Prival	Non mutagenic K2 Non Prival	Non mutagenic K1 Non Prival	Non mutagenic, four strains K2 Non Prival Non mutagenic, all strains, lower concentrations Prival Ames ongoing
Clastogenicity in vitro	Non clastogenic (read across)	Non clastogenic (read across)	Non clastogenic K1	Not clastogenic in human lymphocytes K1 Nanoprotocol	No indications of clastogenicity seen in the MLA Not clastogenic in human lymphocytes K1 Nanoprotocol
Mutagenicity in mammalian cells in vitro	Non mutagenic (read across)	Non mutagenic (read across)	Non mutagenic K1	Non mutagenic (read across)	Non mutagenic K1

 

Bacterial gene mutation:

Mutagenicity in bacteria

Mutagenicity in bacterial reverse mutation assays (Ames test, standard protocol) was investigated in 1997 and in 2022. Negative results were obtained in all tests with and without metabolic activation. All relevant tester stains were tested and test item concentrations were adequate. An Ames test using the Prival modifications for Azo compounds is ongoing.
 
Mammalian gene mutation:
Mutagenicity in mammalian cells has been investigated in two reliable studies according to OECD guideline 476. Pigment Yellow 155 was tested for its ability to induce gene mutations at the thymidine kinase (TK) locus in L5178Y TK+/- mouse lymphoma cells in vitro with the microwell method (BASF 2012). The study was performed under GLP following OECD testing guideline 476 using well characterized test material and it is therefore valid without restrictions. Two independent experiments were carried out with and without the addition of induced rat liver S9 mix (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested and evaluated in this study: 1st Experiment without S9 mix (4-hour exposure period) 0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0; 20.0 μg/mL with S9 mix (4-hour exposure period) 0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0; 20.0 μg/mL 2nd Experiment without S9 mix (24-hour exposure period) 0; 0.31; 0.63; 1.25; 2.5; 5.0; 10.0 μg/mL with S9 mix (4-hour exposure period) 0; 0.25; 0.5; 1.0; 2.0; 4.0; 8.0 μg/mL.After a treatment period of 4 hours both with and without metabolic activation and of 24 hours without metabolic activation, an expression phase of about 48 hours and a selection period of about 10 days, the colonies of each test group were counted and the number of large and small colonies was determined. The negative controls gave mutant frequencies within the range expected for the L5178Y TK+/- mouse lymphoma cell line. Both positive controls, MMS and CPP, led to the expected increase in the frequencies of forward mutations. No cytotoxicity indicated by either reduced relative cloning efficiency 1 or reduced relative total growth of below 20% of control was observed in both main experiments. The test substance was poorly soluble. Thus, dose selection was performed with regard to the solubility properties of the test substance in culture medium. At least the highest applied concentrations tested for gene mutations were clearly above the border of test substance solubility in culture medium. On the basis from the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies both either without S9 mix or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions described, the test substance did not induce forward mutations in vitro in the mouse lymphoma assay with L5178Y TK+/- cells in the absence and the presence of metabolic activation.

Cytogenicity study in mammalian cells:

Pigment Yellow 155 was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). One experiment was carried out. The test material fulfilled the criteria of a nanomaterial according to the EU REACH definition. Thus, in accordance to the OECD 487 guideline the following modifications were made:

1) Solubility properties: the test substance is a poorly soluble nanomaterial. 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 (Elespuru R. (2018) Genotoxicity Assessment of Nanomaterials: Recommendations on

Best Practices, Assays, and Methods. Toxicological Sciences 164(2), 391-416). 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 was omitted. Cells were treated for a period of 20h corresponding to approx. 1 cell cycle (Elespuru R (2018) Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods. Toxicological Sciences 164(2), 391-416).

4) The compatibility of the used test procedure for the assessment of the putative mutagenic potential of a nanomaterial was 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 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).

Previous assessments have shown that isolated buffy coat cells are more suitable for nanomaterial testing as compared to whole blood (Burgum M J, Ulrich C, Partosa N, Evans S J, Gomes C, Beritt-Seifert S, Landsiedel R, Bogni A, Honarvar N, Doak S. Adapatation of the in vitro micronucleus assay for nanomaterial testing (manuscript in preparation)). Therefore, in this study buffy coat cells isolated from whole blood were used.

Concentrations of 0; 1; 3; 10; 30; 60; 100; 256 µg/mL were selected based on a pre-test on homogeneity of the dispersions. The highest used concentration was 256 µg/mL. Higher concentrations could not be homogenously formulated. Test groups 0; 10; 30; 100 and 256 µg/mL were evaluated for the occurrence of micronuclei and concentrations of 10, 30 and 100 µg/mL were investigated for particle size distribution (non GLP).

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. 0.05% w/v BSA-water was used as vehicle. 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. The characterization of the nanomaterial in cell culture medium showed that the particles were successfully dispersed into a stable suspension with partial agglomeration which did not change significantly during the treatment period. The vehicle controls gave frequencies of micronucleated cells within the 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 as indicated by reduced proliferation index (CBPI) was observed up to the highest applied test substance concentration. The test substance did not cause any biologically relevant increase in the number of cells containing micronuclei.

Analysis of the pigment particles in the cell culture medium showed a polydisperse size distribution, ranging from less than 20nm to 2000 nm. D50, D10 and D90 percentiles showed a trend to higher values with dose. Comparison of the size distribution before and after 20h done at the concentration of 100 showed no change in particle size distribution.

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 (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP).