Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, (1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)methyl derivs.

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. A related substance was not genotoxic 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 bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Substance type: blue solid
- Analytical purity: commercial grade, >80%
- Lot/batch No.: A 80905
- Expiration date of the lot/batch: November 07, 2004
- Stability under test conditions: stable
- Storage condition of test material: room temperature

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:

liver of phenobarbitone/B-naphtoflavone treated rats

Test concentrations with justification for top dose:

0.005, 0.01, 0.05, 0.1 and 0.2 mg per plate (exp I)
0.0003, 0.001, 0.003, 0.01 and 0.03 mg per plate (exp II)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminofluorene, 2-Aminoanthracene, Methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

According to the OECD Guideline TG 471, the recommended maximum test item concentration for soluble, non-cytotoxic test items is 5 mg or 5 pl/plate. In case of a cytotoxic effect or any insolubility of the test item (formation of precipitates) the maximum test item concentration is
given by the minimum concentration which leads to the corresponding effect. In pretests the test item showed signs of precipitation at concentrations above 0.05 mg/plate. No cytotoxic effects on the test systems were observed. Due to this facts the maximum test item concentration set in the test reported here was 0.2 mg/plate with and without metabolic activation.

Evaluation criteria:

A mutagenic potential of a test item was assumed if one quotient of the mean value of the respective revertant colonies over the mean value of the respective negative control is 2.0 or higher. A dose effect relationship could underline this conclusion. A possible mutagenic potential of a test item was assumed if one quotient of the mean value of the respective revertant colonies over the mean value of the respective negative control ranges between 1.7 to 1.9 in combination with a dose effect relationship. No mutagenic potential of a test item was assumed if all quotients of the mean value of the respective revertant colonies over the mean value of the respective negative control range between 1.0 (or lower) to 1.6. A nonexistent dose effect relationship could underline this conclusion.

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:

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:

The visual control of the plates showed signs of precipitation at all five test item concentrations applied (main study), and at 0.03 mg/plate and 0.01 mg/plate (repeat study) of test item at all strains with and without metabolic activation.
At all tested concentrations no cytotoxic effects on the test systems were observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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:

mammalian cell gene mutation assay

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

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:

not mutagenic

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 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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

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

Remarks:

Migrated from field 'Test system'.

				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 (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The target substance has been tested for mutagenicity in the Ames test (OECD 471, GLP) (GPT 2004). It was found to be non mutagenic and not cytotoxic. Experimental data on mutagenicity and clastogenicity in mammalian cells in vitro is available for the UVCB analogue with the slightly average substitution grade (2.48 versus 1.4). Both substances share the same components; the analogue UVCB substance contains more of the higher substituted components. All components are poorly soluble and have a molecular weight greater than 500 g/mol. A mode of action for genotoxicity is reactivity with DNA or proteins In this case, the structural features are identical and any potential for reactivity present in the target substance would be identified from the higher substituted analogue. Therefore, it is acceptable to use the data of the analogue for read-across.

A general read-across justification with structures and data matraces has been added to the toxikokinetic section and the Chemical Safety Report.

The GLP-study was performed according to the OECD Guideline TG 471 for the bacterial reverse mutation test with Salmonella typh.TA98, TA100, TA1535, TA1537, and E. coli WP2 uvrA (GPT 2004).Exposure of the test system was performed using the pre-incubation method. The test item was applied at concentrations of 0.2 mg, 0.1 mg, 0.05 mg, 0.01 mg, and 0.005 mg/plate (main study) and 0.03 mg, 0.01 mg, 0.003 mg, 0.001 mg and 0.0003 mg/plate (repeat study) with and without metabolic activation (S9 rat liver). Concentrations were chosen so low because the OECD guideline recommends testing non-precipitating concentrations. The visual control of the plates showed signs of precipitation at all five test item concentrations applied (main study), and at 0.03 mg/plate and 0.01 mg/plate (repeat study) of test item at all strains with and without metabolic activation. At all tested concentrations no cytotoxic effects on the test systems were observed.

For none of the strains - with and without metabolic activation - a clear increase in the number of revertant colonies was observed. Furthermore, no dose-effect relationship could be determined. The respective negative controls produced a low number of revertants. The strain-specific positive controls were shown to produce the expected increase in revertant colonies, with and without metabohc activation, respectively. The substance was found to be non mutagenic.

Another Ames test with a crude version of the substance (GPT 2004b) also showed absence of mutagenicity.

The following data is available for the substance with the average higher substitution grade:

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, 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 fifteenth time in Directive EC2020/1182.