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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

261-638-5

EC Name:

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

Cas Number:

59160-79-1

Molecular formula:

C59 H31 Cu N11 O6

IUPAC Name:

[[2,2',2''-[(29H,31H-phthalocyanine-C,C,C-triyl-kN29,kN30,kN31,kN32)tris(methylene)]tris[1H-isoindole-1,3(2H)-dionato]](2-)]-copper

Test material form:

solid: nanoform

Details on test material:

BET: 17.5 m2/g
TEM (min. Feret): 46.2 nm (D50)

Method

Target gene:

not applicable

Species / strain

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

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Controls 2

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

				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

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative