[[2,2',2''-[29H,31H-phthalocyaninetriyltris(methylene)]tris[1H-isoindole-1,3(2H)-dionato]](2-)-N29,N30,N31,N32]copper

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance is not genotoxic in the Ames test, in the HPRT test and in the chromosome abberration test in vitro. All studies were performed with well characterized test material accoring to OECD guidelines and under GLP. There is no concern for genotoxic properties.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-2015

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)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

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:

Identifier: CAS 59160-79-1
physical state: solid, blue
Batch identification: 120001P040
Purity: >= 97%
Homogeneity:given
Stability: unlimited
Storage conditions: ambient (room temperature); dry storage; protect against humidity

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HGPRT)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthinesupplemented with 10% (v/v) fetal calf serum (FCS).
- Treatment medium (without S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum.
- Treatment medium (with S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine.
- Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with: hypoxanthine (13.6 x 10-3 mg/mL), aminopterin (0.18 x 10-3 mg/mL), thymidine (3.88 x 10-3 mg/mL), 10% (v/v) fetal calf serum (FCS).
- Selection medium ("TG" medium): Hypoxanthine-free Ham's F12 medium supplemented with: 6-thioguanine (10 μg/mL), 1% (v/v) stable glutamine (200 mM), 10% (v/v) fetal calf serum (FCS).
- All media were supplemented with: 1% (v/v) penicillin/streptomycin and 1% (v/v) amphotericine B
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes; During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

1st Experiment
without S9 mix: 0; 3.13, 6.25, 12.5, 25, 50, 100 μg/mL
with S9 mix: 0; 5, 10, 20, 40 μg/mL
2nd Experiment
without S9 mix: 0; 0; 3.13, 6.25, 12.5, 25, 50, 100 μg/mL
with S9 mix: 0; 0; 5, 10, 20, 40 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, dimethyl sulfoxide was selected as the vehicle which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

400 μg/mL EMS (with S9 mix), 1.25 μg/mL DMBA (without S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9)
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures were used for all experimental groups.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range of 0.00 – 16.43 mutants per 10E6 clonable cells
• The positive controls both with and without S9 mix have to induce distinctly increased mutant frequencies (historical positive control data).
• At least 4 dose levels should be tested ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Assessment criteria
A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10E6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a 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 vehicle control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: yes

Historical negative control data range: MFcorr.: 0.00 – 16.43 per 106 cells
Historical positive control data range: MFcorr.: 47.35 – 812.14 per 106 cells

Remarks on result:

other: all strains/cell types tested

Exp.	Exposure period	Test groups	S9 mix	Prec.*	Genotoxicity**	Cytotoxicity***
					MFcorr.	CE1	CE2
					[per 106cells]	[%]	[%]
1	4 hrs	Acetone 1%	-	-	1.84	100.0	100.0
		3.13 µg/mL	-	-	n.c.1	98.8	n.c.1
		6.25 µg/mL	-	-	n.c.1	91.8	n.c.1
		12.50 µg/mL	-	-	2.42	94.7	102.5
		25.00 µg/mL	-	+	1.64	91.3	103.7
		50.00 µg/mL	-	+	2.08	84.7	105.1
		100.00 µg/mL	-	+	1.84	82.8	99.9
		EMS 400 μg/mL	-	-	111.67	87.7	82.1
2	4 hrs	Acetone 1%	-	-	4.15	100.0	100.0
		5.00 µg/mL	-	-	4.07	96.3	93.3
		10.00 µg/mL	-	-	1.17	91.9	108.7
		20.00 µg/mL	-	+	1.56	94.4	104.3
		40.00 µg/mL	-	+	1.92	84.7	99.6
		EMS 400 μg/mL	-	-	91.04	92.7	94.1
1	4 hrs	Acetone 1%	+	-	0.85	100.0	100.0
		3.13 µg/mL	+	-	n.c.1	93.8	n.c.1
		6.25 µg/mL	+	-	n.c.1	103.4	n.c.1
		12.50 µg/mL	+	-	0.30	90.5	95.6
		25.00 µg/mL	+	+	1.21	84.1	89.4
		50.00 µg/mL	+	+	1.15	82.9	94.3
		100.00 µg/mL	+	+	0.64	82.2	84.2
		DMBA 1.25 μg/mL	+	-	215.85	103.0	70.3
2	4 hrs	Acetone 1%	+	-	9.22	100.0	100.0
		5.00 µg/mL	+	-	5.24	94.0	105.8
		10.00 µg/mL	+	-	0.86	86.7	98.4
		20.00 µg/mL	+	+	3.66	99.3	100.8
		40.00 µg/mL	+	+	0.31	87.1	97.1
		DMBA 1.25 μg/mL	+	-	113.73	99.1	90.3

* 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

n.c.1 Culture was not continued since a minimum of only four analysable concentrations are required

Conclusions:

Interpretation of results: negative

In the HPRT in vitro gene mutation test with CHO cells no mutagenicity was observed after treatment with the test substance when tested up to clearly precipitating test substance concentrations. All values were nearby the concurrent vehicle control values and clearly within our laboratory’s historical negative control data range.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identifier: CAS 59160-79-1
physical state: solid, blue
Batch identification: 120001P040
Purity: 97,2g/100g
Homogeneity:given
Stability: unlimited
Storage conditions: ambient (room temperature); dry storage; protect against humidity

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

1st Experiment

4-hour exposure, 18-hour sampling time, with and without S9 mix
0; 3.13; 6.25; 12.5; 25; 50 μg/mL


2nd Experiment
18-hour exposure, 18-hour sampling time, without S9 mix
0; 1.56; 3.13; 6.25; 12.5; 25; 50 μg/mL

18-hour exposure, 28-hour sampling time, without S9 mix
0; 1.56; 3.13; 6.25; 12.5; 25; 50 μg/mL

4-hour exposure, 28-hour sampling time, with S9 mix
0; 1.56; 3.13; 6.25; 12.5; 25; 50 μg/mL

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 - 30 hours
- Exposure duration: 4h or 18h
- Expression time (cells in growth medium): 10, 14 or 24 hours

- Fixation time (start of exposure up to fixation or harvest of cells): 28h


SPINDLE INHIBITOR (cytogenetic assays): colcemide
STAIN (for cytogenetic assays): 7.5% (v/v) Giemsa/Titrisol solution pH 7.2

NUMBER OF REPLICATIONS:2

NUMBER OF CELLS EVALUATED: 100 metaphases per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

The V79 in vitro cytogenetic assay is considered valid if the following criteria are met:
• The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable metaphases.
• The numbers of cells with structural/numerical aberrations in the negative control has to be within the range of the historical negative control data.
• The positive control substances both with and without S9 mix have to induce a distinct increase of structural chromosome aberrations.

The test substance is considered as “positive” if the following criteria are met:
• A statistically significant, dose-related and reproducible increase in the number of cells
with structural chromosome aberrations (excl. gaps).
• The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle
control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met:
• The number of cells with structural aberrations (excl. gaps) in the dose groups is not
statistically significant increased above the concurrent negative/vehicle control value and
is within the historical negative control data range.

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni- Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) are printed in the tables.

Species / strain:

Chinese hamster lung fibroblasts (V79)

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: none
- Effects of osmolality: none
- Evaporation from medium: not relevant
- Water solubility: poorly soluble
- Precipitation:yes

Remarks on result:

other: all strains/cell types tested

				Aberrant cells [%]				Cytotoxicity*
	Test groups	S9 mix	Precipitation	incl. gaps#	excl. gaps#	with exchanges	Polyploid cells [%]	Cell number [%]	Mitotic index [%]
4/18 hrs	Vehicle control (Acetone)	-	n.d.	8.0	4.0	0.5	0.0	100.0	100.0
	3.13 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	101.8	n.d.
	6.25 µg/mL	-	-	5.5	2.5	0.5	0.0	117.9	101.2
	12.50 µg/mL	-	-	4.0	3.0	1.0	0.0	113.9	100.0
	25.00 µg/mL	-	-	3.0	2.5	1.0	1.0	110.0	99.2
	50.00 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	112.9	n.s.
	100.00 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	98.9	n.s.
	EMS 500 μg/mL	-	n.d.	26.0s	24.0s	13.0s	0.0	n.t.	89.4
18/18 hrs	Vehicle control (Acetone)	-	n.d.	2.0	1.5	0.5	0.0	100.0	100.0
	1.56 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	129.3	n.d.
	3.13 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	115.9	n.d.
	6.25 µg/mL	-	-	5.5	1.5	0.0	0.0	113.4	96.8
	12.50 µg/mL	-	-	5.0	3.0	2.5	0.0	108.4	89.8
	25.00 µg/mL	-	+	4.5	1.5	0.0	0.0	126.0	81.1
	50.00 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	93.9	n.s.
	EMS 500 μg/mL	-	n.d.	25.0s	25.0s	19.0s	0.0	n.t.	61.4
18/28 hrs	Vehicle control (Acetone)	-	n.d.	4.5	1.5	0.5	0.0	100.0	100.0
	1.56 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	96.7	n.d.
	3.13 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	92.0	n.d.
	6.25 µg/mL	-	-	n.d.	n.d.	n.d.	n.d.	105.4	n.d.
	12.50 µg/mL	-	+	3.5	1.0	0.5	0.0	111.8	121.8
	25.00 µg/mL	-	+	6.5	3.0	2.0	0.0	113.6	120.3
	50.00 µg/mL	-	+	n.s.	n.s.	n.s.	n.s.	94.6	n.s.
	EMS 500 μg/mL	-	n.d.	34.0s	31.0s	23.0s	0.0	n.t.	74.2
4/18 hrs	Vehicle control (Acetone)	+	n.d.	8.5	5.0	1.0	0.0	100.0	100.0
	1.56 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	96.0	n.d.
	3.13 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	95.6	n.d.
	6.25 µg/mL	+	-	4.5	3.0	1.0	0.0	104.4	143.9
	12.50 µg/mL	+	-	4.5	1.5	1.0	0.0	87.9	127.5
	25.00 µg/mL	+	-	5.0	2.5	0.5	1.0	81.9	116.4
	50.00 µg/mL	+	+	n.s.	n.s.	n.s.	n.s.	62.8	n.s.
	CPP 0.5 μg/mL	+	n.d.	27.0s	27.0s	19.0s	0.0	n.t.	90.1
4/28 hrs	Vehicle control (Acetone)	+	n.d.	4.0	1.0	0.5	0.0	100.0	100.0
	1.56 µg/mL	+	-	n.d.	n.d.	n.d.	n.d.	91.2	n.d.
	3.13 µg/mL	+	-	2.0	1.0	0.5	0.0	99.1	113.5
	6.25 µg/mL	+	-	4.0	3.0	1.0	0.0	116.8	122.4
	12.50 µg/mL	+	-	6.0	3.5	1.0	0.0	119.8	118.5
	25.00 µg/mL	+	+	n.s.	n.s.	n.s.	n.s.	93.6	n.s.
	50.00 µg/mL	+	+	n.s.	n.s.	n.s.	n.s.	113.2	n.s.
	CPP 0.5 μg/mL	+	n.d.	26.0s	23.0s	12.0s	0.0	n.t.	123.1

P Precipitation occured at the end of exposure period

* Relative values compared with the respective vehicle control

# Inclusive cells carrying exchanges

n.d. Not determined n.t. Not tested

n.s. Not scorable due to poor metaphase quality (precipitation)

S Aberration frequency statistically significant higher than corresponding control values

x Evaluation of a sample of 100 metaphase only due to strong clastogenicity

Conclusions:

Interpretation of results: negative

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 98.3 g / 100 g

Target gene:

His operon

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 ug - 5000 ug/plate (Standard Plate Test (SPT))
4 ug - 2500 ug/plate (Preincubation Test (PIT))

Vehicle / solvent:

Due to the limited solubility 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:

yes

Positive controls:

yes

Positive control substance:

other: with metabolic activation: 2-aminoanthracene; without metabolic activation: N-methyl-N' -nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine and N-ethyl-N' -nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

Standard Plate Test and Preincubation test

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions from about 500 - 2,500 ug/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions from about 500 - 2,500 ug/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions from about 500 - 2,500 ug/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions from about 500 - 2,500 ug/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions from about 500 - 2,500 ug/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

An increase in the nuinber of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results:negative

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The blue powder was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA in a reverse mutation assay (OECD 471 adopted 1997, GLP). Doses ranged from 0.02 - 5 mg and 0.004 - 2.5 mg per plate in the standard plate test and preincubation test, respectively. Aroclor-induced rat S9 mix served for metabolic activation.

Precipitation of the test substance was found from about 0.1 mg/plate onward. An increase in mutant frequency was not observed showing that the substance is not mutagenic in bacteria.

The positive and negative control experiments showed the expected results.

The substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (OECD 476, GLP). Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses the following concentrations were tested. Test groups printed in bold type were evaluated in this study:

1st Experiment

without S9 mix (4-hour exposure period)

0; 3.13; 6.25; 12.5; 25.00; 50.00; 100.00 μg/mL

with S9 mix (4-hour exposure period)

0; 3.13; 6.25; 12.5; 25.00; 50.00; 100.00 μg/mL

2nd Experiment

without S9 mix (4-hour exposure period)

0; 5.00; 10.00; 20.00; 40.00 μg/mL

with S9 mix (4-hour exposure period)

0; 5.00; 10.00; 20.00; 40.00 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa

and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line.

Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations.

The test substance was poorly soluble either in organic solvents or culture medium. In the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest applied test substance concentrations which were at the border of test substance solubility in culture medium. Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

The substance was assessed for its potential to induce structural chromosome aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro (OECD 473, GLP). Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test, the test substance did not exhibit any pronounced toxicity up to the highest applicable concentration of 1500 μg/mL (approx.1.38 mM), at which distinct test substance precipitation was observed. Test concentrations as indicated n the results table were chosen. A sample of 100 metaphases for each culture was analyzed for chromosomal aberrations, except for the positive control cultures where only 50 metaphases were scored due to clearly increased aberration rates.

The vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosome aberrations.

The test substance was poorly soluble either in organic solvents or culture medium. No clear cytotoxicity was observed up to the highest applied test substance concentrations which were at the border of test substance solubility in culture medium.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases at both sampling times either without S9 mix and/or after adding a metabolizing system.

No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either. Thus, under the experimental conditions described, the substance is considered not to have a chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Classification, Labeling, 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 genotoxicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.