Polychloro copper phthalocyanine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non mutagenic in bacteria (OECD 471, GLP) and mammalian cells in vitro (OECD 476, GLP), non clastogenic in mammalian cells in vitro (OECD 487 and 473).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (Draft Proposal), 13 Dec 2007

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Batch No: BW 137 (Partien 616-619)
- Analytical purity: > 98 % (analytical report dated Feb 15, 2000)
- Appearance, consistency: green powder
- Storage: room temperature

Target gene:

The micronucleus assay is a method independent of the karyotype of the cells used for an indirect detection of damage of chromosomes or the mitotic apparatus. Micronuclei are formed either from acentric chromosome fragments as a result of a chromosome-breaking (clastogenic) effect or by entire chromosomes as a consequence of impairments of chromosome distribution in the course of mitosis (aneugenic effect).

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 ceII line has a high proliferation rate (doubling time of about 12 - 16 hours), a high plating efficiency (~ 90 %) as well as a stable karyotype (modal number of 22 chromosomes).
Stocks of the V79 cell line (1-ml portions) were maintained at -196 °C in liquid nitrogen using 7 % DMS0 in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for mycoplasma contamination, karyotype stability and plating efficiency (incl. vital staining).

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of male SD rats (treated i.p. with 500 mg/kg bw Aroclor 1254 five days before sacrifice), mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH7.4).

Test concentrations with justification for top dose:

- Mixed population method:
24 hours exposure, 24 hours harvest time, without S9-mix: 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25.0 and 50.0 µl/ml;
4 hours exposure, 24 hours harvest time, with S-9 mix: 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25.0 and 50.0 µl/ml
- Mitotic shake off method:
24 hours exposure, 27 hours preparation time, without S-9 mix: 0, 1.56, 3.156, 6.25, 12.5, 25.0, 50.0 and 75.0 µg/ml;
4 hours exposure, 27 hours preparation time, with S-9 mix: 0, 1.560, 3.125, 6.25, 12.5, 25.0, 50.0 and 75.0 µg/ml;

Vehicle / solvent:

In comparison to other commonly used vehicles (e.g. water, acetone), DMSO is the most suitable. Therefore DMSO was selected as the vehicle which had been demonstrated to be suitable in the V79 in vitro micronucleus assay and for which historical control data is available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethyl-Methane-Sulfonate (EMS) without S9-mix and Cyclophosphamide (CPP) with S-9 mix

Details on test system and experimental conditions:

Pretests for dose selection:
- Mixed Population Method: (24 hours sampling time); tests with cultures exposed to a wide dose range, i.e. 0.5 - 2500 µg/ml culture medium both without S-9 mix (24 hours continuous treatment) and after adding a metabolizing system (pulse treatment of 4 hours); 50 µg/ml was (with and without S-9 mix) the top dose selected. Due to strong test substance precipitation which interferes with evaluation of cells higher doses could not be evaluated.
- Mitotic Shake Off Method: 75 µg/ml (with and without S-9 mix) was selected as top dose, because a strong test substance precipitation was found at higher concentration levels, which interferes with evaluation of cells.

Cell cycle time: ca. 13-14 hours under the selected culture conditions

Preparation of test cultures:
- Mixed Population Method:
The experimental procedure was carried out based an the method of Kalweit. et al. (Mut. Res. 439: 183-190, 1999). Logarithmically growing cultures (after the 6th passage) which were more than 50 % confluent were trypsinized (0.25 % trypsin solution and Ca-Mg-free Hanks' balanced salt solution, HBSS). Prior to trypsin treatment, the cells were rinsed once with 5 ml Ca-Mg-free HBSS. This process was stopped by adding MEM supplemented with 10 % FCS. A single ceII suspension was prepared, and about 5 ml MEM supplemented with 10 % FCS and containing about 30000 - 80000 cells were seeded in each chamber of Quadriperm dishes. Two chambers of a Quadriperm dish were used for one test culture. The Quadriperm dishes were incubated with 5 % CO2 at 37 °C and > 90 % humidity.
Treatment of test cultures:
Without S-9 mix: About 6 hours after seeding and incubating the cells, the medium was replaced by 4 ml fresh medium with FCS. The test article, suspended in 50 µI DMS0 was added to the culture medium. Concurrent negative and positive controls were tested. The cultures were incubated for 24 hours with 5% CO2 at 37°C and > 90 % humidity.
With S-9 mix: About 6 hours after seeding and incubating the cells, the medium was replaced by 3 ml of fresh medium (without FCS). The test article, suspended in 50 µI, was added to the culture medium along with 1 ml S-9 mix. Concurrent negative and positive controls were tested. After incubation (5 % CO2, 37 °C and > 90 % humidity) for 4 hours, the serum-free medium was replaced by 5 ml MEM supplemented with 10 % FCS after being rinsed twice with 5 ml HBSS. Subsequently, the Quadriperm dishes were incubated again with 5 % CO2 at 37 °C and > 90 % humidity for another 20 hours until the cells were harvested.

- Mitotic Shake 0ff Method:
The experimental procedure was carried out based on the method of Seelbach et al. (Toxicol. in Vitro 7: 185-193, 1993). Logarithmically growing cultures (after the 3rd passage) which were more than 50 % confluent were trypsinized (0.25% trypsin solution and Ca-Mg-free Hanks' balanced salt solution, HBSS). Prior to trypsin treatment, the celis were rinsed once with 5 ml Ca-Mg-free HBSS. This process was stopped by adding MEM supplemented with 10 % FCS. A single cell suspension was prepared, and about 25 ml MEM supplemented with 10 % FCS and containing about 2 x 10exp+6 cells were seeded in each culture flask. One flask was used per test culture. The flasks were incubated with 5 % CO2 at 37 °C and > 90 % humidity.
Treatment of test cultures:
Without S-9 mix: About 16 - 24 hours after seeding and incubating the cells, the medium was replaced by 20 ml fresh medium with FCS. The test article, suspended in 250 µI DMS0, was added to the culture medium. Concurrent negative and positive controls were tested. The cultures were incubated for 24 hours with 5 % CO2 at 37 °C and > 90 % humidity.
With S-9 mix: About 16 - 24 hours after seeding and incubating the cells, the medium was replaced by 15 ml of fresh medium without FCS. The test artcle, suspended in 250 µl DMS0, was added to the culture medium along with 5 ml S-9 mix. Concurrent negative and positive controls were tested. After incubation (5% CO2, 37 °C and > 90 % humidity) for 4 hours, the serum-free medium was replaced by 25 ml MEM supplemented with 10 % FCS after being rinsed twice with 10 ml HBSS. Subsequently, the flasks were incubated again with 5% CO2 at 37 °C and > 90 % humidity for another 20 hours until mitotic shake off.
Mitotic Shake 0ff:
24 hours following the treatment of the cells, the mitotic cells were shaken off the monolayer by hitting the flasks and were transferred into sterile centrifuge tubes. The cells were centrifuged at 100 x g for 5 minutes and afterwards the supernatant was removed and the ceII pellet was resuspended in 2 ml MEM with 10 % FCS. Approx. 0.5 ml of the cdl suspension-was transferred onto microscope slides in Quadriperm dishes (2 slides per test culture) and 2.5 ml of medium with 10 % FCS was added. The dishes were then incubated with 5 % CO2 at 37 °C and > 90 % humidity for 3 hours until harvesting.
CeII harvest and preparation of slides
The cells were prepared based on the methods described byKalweit et al. (Mut. Res. 439: 183-190, 1999). After incubation the culture medium was completely removed. For hypotonic treatment, 5 ml of a 1.5 % Sodium citrate solution at 37 °C was added for about 5 minutes. Following aspiration of the hypotonic solution, 5 ml of fixative (ethanol : glacial acetic acid Formaldehyde (37 %)/3: 1: 0.0125) which was at 4 °C was added for ca. 5 minutes and then removed and 5 ml of fresh fixative was added for at least another 5 minutes at room temperature for complete fixation. The sildes were taken out of the Quadriperm chambers, briefly allowed to drip and then rapidly passed through a Bunsen burner flame. The preparations were dried in the air and subsequently stained in Wrights solution (modified Mav-Gründwald solution) for ca. 3 minutes. After being rirised once in Titrisol pH 7.2, the slides were stained in a 2.6 % Giemsa solution (5.2 ml Giemsa, 195 ml Titrisol pH 7.2) for ca. 20 minutes. After being rirsed twice in Titrisol pH 7.2 and clarified in xylene, the preparations were mounted using Gorbit-Balsam.

Evaluation:
As a rule, 2000 cells from each dose group, each dose group consisting of 2 test cultures, were evaluated and the number of micronucleus-containing cells was recorded.
The analysis of micronuclei was carried out, observing the following criteria:
- the micronucleus consists of an area less than 1/3 of the area of the main nucleus
- the micronucleus and main nucleus retain the same staining
- the micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell
- for evaluation, only cells clearly surrounded by a nuclear membrane were scored.
Slides were coded before microscopic analysis. Cultures with few isolated cells were not analysed for micronuclei.
Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative values can occur.
- Mitotic index (Ml):
A mitotic Index based on 1000 cells/culture was determined for all test groups both in the Mixed Population Method and Mitotic Shake 0ff Method.
- Cell count
For the determination of cytotoxicity, additional cell cultures (using 25 cm2 plastic flasks) were treated in the same way as in the Mixed Population Method. Growth inhibition was estimated by counting the number of cells in the dose groups in comparison with the concurrent vehicle control at the end of the culture period using a counting chamber.
- Cell morphology
About 3 - 4 hours after test substance treatment with S-9 mix and about 22 - 24 hours after treatment without S-9 mix, the cell morphology, which is an indication of attachment of the cells to the slides or flasks, was checked for each culture in all test groups using both modifications of the assays (with the exception of the positive controls).
- Fragmentation
During evaluation of 1000 cells/culture the occurance of fragmentation (fragmented nuclei or multinucleated cells and cells with > 6 micronuclei) was recorded.
Evaluation of the preparations:
Before analysis, the finished preparations were checked for the following
- possible reduction in the quality of the cells
- number of arialyzable cells
- amount of fragmented cells
The analyzable dose groups were determined based on the findings of this control check.
- Proliferal:ion Index (PI)
In addition to the evaluation of micronuclei and mitotic index in the Mixed Population Method, the proliferation index was determined as a measure of cytotoxicity. The PI, based on 1000 cells per culture (2000 cells per dose group), was determined for all test groups with or without S-9 mix. The number of clones (packs) consisting of 1 cell, 2, 4 or 8 cells was recorded and the Pl was calculated using the following formula:
PI = ((ncl-1) x 1 + (ncl-2) x 2 + (ncl-4) x 3 + (ncl-8) x 4) / total no. of clones
cl-1 = cell clone with 1 cell
cl-2 = cell clone with 2 cells
cl-3 = cell clone with 3 or 4 cells
cl-4 = cell clone with 5, 6, 7 or 8 cells

Controls:
- Vehicle control: The vehicle controls with and without S-9 mix only contained the vehicle for the test substance at the same concentration and volume used in the test culture.
- Positive controls:
The following positive control substances were used to demonstrate the sensitivity of the test method and the activity of the S-9 mix:
* Ethyl-Methane-Sulfonate (EMS): For the detection of clastogens without metabolic activation, 350 pg EMS (SIGMA, M-0880)/ml culture medium added in a volume of 1 ml (Mixed Population method) or 5 ml (Mitotic Shake 0ff method).
* Cyclophosphamide (CPP): For the detection of clastogens with metabolic activation, 2.5 pg CPP Endoxan®, ASTA MEDICA, Reg. Nr. E 432-1 )/ml culture medium added in a volume of 1 ml (Mixed Population method) or 5 ml (Mitotic Shake 0ff method).
The stability of EMS and CPP is well-defined under the selected culture conditions, since both positive control articles are well-established reference clastogens.

Evaluation criteria:

Acceptance criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed, at least to a large extent, the identification and evaluation of a sufficient number of analyzable cells.
- The proportion of cells with micronuclei in negative control (vehicle control) cultures was within the normal range ofthe historical control data.
- The positive control chemicals (with and without S-9 mix) induced a significant increase in the number of cells with micronuclei.

Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of cells containing micronuclei.
- The proportion of micronucleus-containing cells exceeded both the concurrent negative control range and the negative historical control range.
A test substance is generally considered nongenotoxic in this test system if:
- There was no significant increase in the number of micronucleus-containing cells at any dose above concurrent negative control frequencies and within the historical control data.

Statistics:

Due to the clear negative finding, a statistical analysis was not carried out.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MIXED POPULATION METHOD
- Micronucleus Frequency:
An increase in the number of cells containing micronuclei was not observed either without S-9 mix or after the addition of a metabolizing system.
- Mitotic Index (MI):
The mitotic Index was based on 1000 cells per culture for the different test groups with and without metabolic activation. According to the results of the determination of the mitotic index, no suppression of the mitotic activity was observed under all experimental conditions.
- Proliferation Index (PI):
The proliferation Index is based on 1000 cells per culture (2000 cells per test group) for the different test groups without and with metabolic activation and allows the measurement of colony sizes. According to the results of the determination of the PI, no cytotoxic response was observed under any of the experimental conditions.
- CelI Count:
According to the results of the ceIl count, no growth inhibition was observed under all experimental conditions.
- CeII Morphology:
CeIl attachment was not influenced at any dose evaluated for micronuclei.
- Treatment Conditions:
Osmolality and pH values were not influenced by test substance treatment.
- Solubility:
Concentrations >10.0 µg/ml (without S-9 mix) or > 12.5 mg/ml (with S-9 mix) led to strong precipitation which interferes with evaluation of cells.

MITOTIC SHAKE 0FF METHOD
- Micronucleus Frequency:
An increase in the number of micronucleated cells was not found either without S-9 mix or after the addition of a metabolizing system.
- Mitotic Index:
The mitotic index was based on 1000 cells per culture for the different test groups with and without metabolic activation. According to the results of the determination af the mitotic index, no suppression of the mitotic activity was observed under any of the experimental conditions.
- CeIl Morphology:
Cell attachment was not influenced at any dose evaluated for micronuclei.
- Treatment Conditions:
Osmolality and pH values were not influenced by test substance treatment.
- Solubility:
Strong test substance precipitation in the vehicle was observed in all dose groups from about 6.25 µg/ml onward.

The increase in the frequencies of micronuclei induced by the positive control agents, EMS and CPP, cIearly demonstrated the sensitivity of the test method and of the metabolic activity of the S-9 mix employed.

Micronucleus frequency (absolute) for Mixed Population Method based on  
1000 cells per culture (2000 cells per test group; 24 hours mitotic shake off) 
observed are summarised below:
- without activation:
-----------------------------------------------------------
Test            /Dose    /mean from   /mean   /mean mitotic
substance       /(µg/ml) /2 cultures  /%      /index (abs.)
-----------------------------------------------------------
DMSO            /        /13.0        /1.3    /28

Positive        /350     /35.5        /3.55   /16

Test substance  /0.78    /8.5         /0.85   /28
                /1.56    /5.0         /0.5    /15
                /3.125   /8.0         /0.8    /19
                /6.25    /5.0         /0.5    /20
                /10.0    /8.0         /0.8    /16
-----------------------------------------------------------

- with activation:
----------------------------------------------------------
Test            /Dose    /mean from   /mean  /mean mitotic
substance       /(µg/ml) /2 cultures  /%     /index (abs.)
----------------------------------------------------------
DMSO            /        /17.0        /1.7    /21

Positive        /350     /124         /12.4   /30

Test substance  /0.78    /7.0         /0.7    /18
                /1.56    /6.0         /0.6    /16
                /3.125   /8.0         /0.8    /23
                /6.25    /5.5         /0.55   /18
                /10.0    /7.5         /0.75   /21
----------------------------------------------------------

Micronucleus frequency (absolute) for Mitotic Shake off Method based on 1000 
cells per culture (2000 cells per test group; 24 hours mitotic shake off) 
observed are summarised below:
- without activation:
----------------------------------------------------------
Test            /Dose    /mean from   /mean  /mean mitotic
substance       /(µg/ml) /2 cultures  /%     /index (abs.)
----------------------------------------------------------
DMSO            /        /4.5         /0.45  /28

Positive        /350     /46.5        /4.65  /16

Test substance  /6.25    /2.0         /0.20  /20
                /12.5    /9.5         /0.95  /24
                /25.0    /5.5         /0.55  /24
                /50.0    /7.0         /0.70  /14
                /75.0    /7.5         /0.75  /18
----------------------------------------------------------

- with activation:
----------------------------------------------------------
Test            /Dose    /mean from   /mean  /mean mitotic
substance       /(µg/ml) /2 cultures  /%     /index (abs.)
----------------------------------------------------------
DMSO            /        /3.5         /0.35  /8

Positive        /2.50    /57          /5.70  /20

Test substance  /1.56    /4.5         /0.45  /12
                /3.125   /6.5         /0.65  /12
                /6.25    /2.0         /0.20  /9
                /12.50   /4.5         /0.45  /19
                /25.0    /2.5         /0.25  /10
----------------------------------------------------------

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Analytical purity: 99.1 %

Target gene:

The purpose of the in vitro chromosomal aberration test is to identify agents that cause structural chromosomal aberrations in cultured mammalian cells. Structural aberrations may be of chromosome or chromatid type.

Species / strain / cell type:

other: CHL/IU cells

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

- without S9-mix (6 hour short-term treatment): 0, 125, 250, 500 µg/ml
- with S9-mix (6 hour short-term treatment): 0, 125, 250, 500 µg/ml
- without S9-mix (24 hour continuous treatment): 0, 125, 250, 500 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9-mix: Mitomycin C; +S9-mix: Benzo[a]pyrene

Details on test system and experimental conditions:

200 cells were analyzed in each group for chromatid braks, chromatid exchange, chromosome break, chromosome exchange (dicentric and ring).
Judgement was done on the basis of criteria of Ishidate et al. 1987.

Species / strain:

mammalian cell line, other: Chinese hamster CHL/IU cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance did not induce chomosomal aberrations or polyploidy with or without metabolic activation.
At the end of the treatment period at 6 h and at 24 h, the test substance foamed membrane-like sheet onto the medium and precipitation of the test substance in the medium was also observed.
At the end of the treatment period at 24 h the colour of the medium was changed to green.

Chromosomal analysis of the treated cells :
- after 6 hours treatment (without S-9 mix)
-------------------------------------------------------------------------- ------------
                                 /Number of cells
Treatment  /Dose    /Cell growth ----------------------------------------  /Polyploid
           /(µg/ml) /index       /analysed /with aberrations /with gaps    /(%)
-------------------------------------------------------------------------- ------------
Test       /0      /100          /200      /3                /0            /0.0
Substance  /125    /101          /200      /0                /0            /1.0
           /250    /123          /200      /2                /1            /0.0
           /500    /110          /200      /2                /0            /1.0

MMC        /0.1    /87           /200      /90               /0            /0.0
-------------------------------------------------------------------------- ------------

- after 6 hours treatment (with S-9 mix)
-------------------------------------------------------------------------- ------------
                                 /Number of cells
Treatment  /Dose    /Cell growth ----------------------------------------  /Polyploid
           /(µg/ml) /index       /analysed /with aberrations /with gaps    /(%)
-------------------------------------------------------------------------- ------------
Test       /0      /100          /200      /1                /0            /0.0
Substance  /125    /97           /200      /1                /1            /0.5
           /250    /62           /200      /1                /0            /0.0
           /500    /61           /200      /0                /0            /0.5

BP         /20     /75           /200      /199              /1            /0.0
-------------------------------------------------------------------------- ------------

- after 24 hours treatment (without S-9 mix)
-------------------------------------------------------------------------- ------------
                                 /Number of cells
Treatment  /Dose    /Cell growth ----------------------------------------  /Polyploid
           /(µg/ml) /index       /analysed /with aberrations /with gaps    /(%)
-------------------------------------------------------------------------- ------------
Test       /0      /100          /200      /2                /0            /0.0
Substance  /125    /94           /200      /1                /0            /0.0
           /250    /94           /200      /1                /0            /0.5
           /500    /90           /200      /0                /0            /0.5

MMC        /0.03   /68           /200      /72               /5            /0.0
-------------------------------------------------------------------------- ------------

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Analytical purity: 99.1 %

Target gene:

Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

without S9 mix: 9.77, 19.5, 39.1, 78.1, 156, 313 and 625 µg/plate (S. typhimurium TA100, TA1535, TA98 and TA1537)
without S9 mix: 313, 625, 1250, 2500 and 5000 µg/plate (E. coli WP2 uvrA)
with S9 mix: 156, 313, 625, 1250, 2500 and 5000 µg/plate (S. typhimurium TA100, TA1535, TA98 and TA1537)
with S9 mix: 313, 625, 1250, 2500 and 5000 µg/plate (E. coli WP2 uvrA)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: see details on test system

Details on test system and experimental conditions:

A Preincubation test was conducted.
Plates per test: 3
Number of replicates: 2

POSITIVE CONTROLS:
without S9-mix: AF-2 (TA 100, WP2, TA98), sodium azide (TA 1535) and 9-aminoacridine (all strains)
with S9-mix: 2-aminoanthracene (all strains)

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see details in "additional information on results"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY:
No increase in the number of his+ revertants was observed, with or without the addition of S9 mix in all S. typhimurium strains tested (TA102, TA100, TA98, TA97) as well as in E. coli WP2 uvr A.
The results of the negative and positive controls were as expected and confirmed the validity and sensitivity of the test system used.

CYTOTOXICITY:
Toxicity was observed at 313 µg/plate or more (TA100 and TA1535) and at 625 µg/plate (TA98 and TA1537) without metabolic activation, and at 5000 µg/plate (TA100, TA1535, TA98 and TA1537) with metabolic activation.

The maximum revertants/plate occurring at the non-cytotoxic (lowest) 
concentrations of the test substance were the following:
- First experiment
--------------------------------------------------------------------------------------
Strain  /Dose   /Maximum revertants /Positive controls   /Cytotoxic concentration(µg)
        /(µg)   /-S-9     /+S-9     /-S-9      /+S-9     /-S-9     /+S-9
--------------------------------------------------------------------------------------
TA-100  /0      /97+-4    /97+-4    /478+-27   /1154+-62 />=313    /5000
        /156    /105+-16  /         /          /         /         /
        /313    /         /100+-3   /          /         /         /

TA-1535 /0      /9+-1     /11+-4    /440+-10   /222+-37  />=313    /5000
        /9.77   /11+-4    /         /          /         /         /
        /2500   /         /11+-4    /          /         /         /

WP2 uvrA/0      /23+-6    /31+-8    /682+-30   /1435+-66 /-        /-
        /313    /25+-5    /         /          /         /         /
        /1250   /         /33+-7    /          /         /         /

TA-98   /0      /15+-2    /28+-2    /467+-44   /526+-30  /625      /5000
        /19.5   /22+-2    /         /          /         /         /
        /625    /         /32+-5    /          /         /         /

TA-1537 /0      /9+-1     /21+-2    /387+-88   /177+-25  /625      /5000
        /19.5   /11+-2    /         /          /         /         /
        /2500   /         /21+-3    /          /         /         /
--------------------------------------------------------------------------------------

- Second experiment
--------------------------------------------------------------------------------------
Strain  /Dose   /Maximum revertants /Positive controls   /Cytotoxic concentration (µg)
        /(µg)   /-S-9     /+S-9     /-S-9      /+S-9     /-S-9     /+S-9
--------------------------------------------------------------------------------------
TA-100  /0      /100+-10  /97+-7    /690+-41   /1480+-20 />=313    /5000
        /39.1   /115+-16  /         /          /         /         /
        /2500   /         /103+-6   /          /         /         /

TA-1535 /0      /7+-2     /9+-2     /411+-63   /262+-16  />=313    /5000
        /39.15  /9+-4     /         /          /         /         /
        /625    /         /11+-3    /          /         /         /

WP2 uvrA/0      /28+-1    /33+-4    /861+-67   /1313+-149/-        /-
        /625    /28+-3    /         /          /         /         /
        /625    /         /31+-4    /          /         /         /

TA-98   /0      /17+-4    /36+-3    /579+-24   /579+-18  /625      /5000
        /156    /         /28+-2    /          /         /         /
        /313    /24+-5    /         /          /         /         /

TA-1537 /0      /11+-2    /11+-1    /545+-145  /224+-15  /625      /5000
        /156    /16+-3    /         /          /         /         /
        /1250   /         /13+-2    /          /         /         /
--------------------------------------------------------------------------------------

Conclusions:

The test substance is not mutagenic in the Ames test.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25.1.2021 - June 2021

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 using the Hprt and xprt genes)

Version / remarks:

29 Jul 2016

Deviations:

yes

Remarks:

Additional investigations for nanomaterials included

GLP compliance:

yes (incl. QA statement)

Remarks:

certified by Landesamt für Umwelt Rheinland-Pfalz

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:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 11 Feb 2022
- Purity: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under storage conditions: stable


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance preparation was performed 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 12 June, 2018.
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. Two stock dispersions were prepared (xx mg/mL and xxmg/mL).
The stock dispersion of xx mg/mL was handled separately. For further dilutions only the stock dispersion of xx mg/mL were used.
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 test substance formulations was diluted according to the planned doses.
All test substance formulations were prepared immediately before administration.
To keep the test substance homogeneously in the vehicle, the test substance preparation was carefully pipetted before the removal.


FORM AS APPLIED IN THE TEST: Homogeneous dispersion

OTHER SPECIFICS
- physical state, appearance: Solid, green
- molecular weight: 1057 - 1127 g/mol (UVCB)

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type of cells: CHO (Chinese hamster ovary) cell line
- Source: cell stock of testing facility
- Suitability of cells: yes, as recommended in OECD TG 476
- Normal cell cycle time (negative control): not specified

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: 3
- Methods for maintenance in cell culture: Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mgfree). Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25% [w/v]) to remove the cells from the bottom of the plastic flasks. This reaction was stopped by adding 6 mL culture medium incl. 10% (v/v) FCS. Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension. Cells were counted in a counting chamber or using a cell counter. Cell suspensions were diluted with complete culture medium to the desired cell count.
- Doubling time: of about 12 - 16 hours
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL) and 1% (v/v) amphotericine B (stock solution: 250 μg/mL); 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity

Metabolic activation:

with and without

Metabolic activation system:

- type and composition of metabolic activation system: exogenous metabolic activation by cofactor-supplemented postmitochondrial fraction (S9 mix)
- source of S9: liver S9 mix from phenobarbital- and β-naphthoflavone induced rats
- method of preparation of S9 mix: the S9 mix was prepared freshly prior to each experiment. 1 part S9 fraction was mixed with 9 parts S9 supplement (consisting of 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 15 mM phosphate buffer (pH 7.4))
- concentration or volume of S9 mix and S9 in the final culture medium: 8 mL S9 mix and 32 mL test substance preparation (final volume 40 mL)

Test concentrations with justification for top dose:

1st Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; (2000.00) µg/mL
with S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; (2000.00) µg/mL

2nd Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; (2000.0)0 µg/mL
with S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; (2000.00) µg/mL

3rd Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 256.00 µg/mL
with S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 256.00 µg/mL

4th Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 256.00 µg/mL

Doses in brackets lead to inhomogeneous suspensions and were not investigated. In lower doses, the suspensions were homogenous.

Vehicle / solvent:

- Vehicle used: 0.05% w/v bovine serum albumin water (BSA-water)
- Justification for choice of solvent/vehicle: 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 12 June 2018, 0.05% w/v bovine serum albumin water (BSA-water) is used as vehicle.
The final concentration of the vehicle 0.05% w/v BSA-water in culture medium is be 10% (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

- DMBA with metabolic activation (1.25 μg/mL) - EMS without metabolic activation (400 μg/mL)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 4

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 20x10^6 cells in 40 mL medium/flask


TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20-24 hours
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7-9 days
- Selection time: 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7-9 days (cloning efficiency 1); 16 days (cloning efficiency 2)
- Method used: colonies were fixed with methanol and stained with Giemsa
- Selective agent: 6-thioguanine; 10 μg/mL; 6-7 days exposure
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2x10^6 cells from every treatment group were seeded in 20 mL selection medium (175 cm^2 flasks) and the remaining colonies were counted at the end of the selection period
- Criteria for small (slow growing) and large (fast growing) colonies: not applicable

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- Mutant frequency

- OTHER: Check or determination of further parameters:
- pH, osmolality, solubility, cell morphology
-Analytical Ultracentrifugation (AUC) method to determine the agglomeration in genotoxicity test medium.
- Incubation and filtration with analysis by Inductively-Coupled-Plasma Mass Spectrometry (ICPMS) or by UVVis-Spectrometry, to determine the static solubility in genotoxicity test medium.

Rationale for test conditions:

In the pre-test for toxicity based on 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 12 June 2018, 2.56 mg/mL 29H,31H-phthalocyanine was used as stock dispersion. The highest tested concentration was 100.0 μg/mL both with and without S9 mix at 4 hour exposure time.
The pre-test was performed following the method described for the main experiment. The Relative Survival (RS) was determined as a toxicity indicator for dose selection.
In the pre-test for dose selection the pH, osmolality and solubility were additionally determined for all or at least some selected doses.

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative control value and the range of our laboratory’s historical negative control data (95% control limit).

Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

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 corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies 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).

Statistics:

A linear dose-response is evaluated by testing for linear trend. The dependent variable is the corrected mutant frequency and the independent variable is the dose. The calculation is performed using EXCEL function RGP. The used model is one of the proposed models of the International Workshop on genotoxicity Test procedures Workgroup Report.

A pair-wise comparison of each test group with the control group is carried out using Fisher's exact test with Bonferroni-Holm correction. The calculation is performed using EXCEL function HYPGEOM.VERT.

If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: All doses contain insoluble particles, homogenicity of suspension as criterium

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The summary table is attached as a figure.

 

 

 

 

Conclusions:

Pigment Green 7 is not mutagenic in the HPRT test.

Executive summary:

The substance Polychloro copper phthalocyanine 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. Four independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and beta-naphthoflavone induced rats (exogenous metabolic activation). Since the test substance is a nanoparticle, dose selection for genotoxicity testing was based on 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 12 June 2018. Furthermore, to fulfill the requirements of the OECD Guidelines for the HPRT assay, the top concentrations in all main Experiments were defined as the highest homogenous suspension. 0.05% w/v bovine serum albumin water (BSA-water) was used as vehicle. Test groups printed in bold type were evaluated for gene mutations, except the highest tested concentration of the 1st and 2nd Experiment. 2000.0 µg/mL was an inhomogenous suspension and thus is regarded as invalid. In the 3rd and 4th Experiment 256.00 µg/mL was used as top concentration which was the highest homogeneous suspension which could be applied to the test culture.

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, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. In all experiments in the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations 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 four experiments performed independently of each other.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

GENOTOXICITY IN-VITRO:

 

Gene mutation in bacteria

Polychloro copper phthalocyanine was not mutagenic in a preincubation Ames test (tested up to 5000 μg/plate in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA with metabolic activation, up to 625 µg/plate in S. typhimurium TA100, TA1535, TA98 and TA1537 as well as up to 5000 µg/plate in E. coli WP2 uvrA without metabolic activation; metabolic activation: Rat liver, induced with phenobarbital and 5,6-benzoflavone. Cytotoxicity was observed at 313 µg/plate or more (TA100 and TA1535) and at 625 µg/plate (TA98 and TA1537) without metabolic activation, and at 5000 µg/plate (TA100, TA1535, TA98 and TA1537) with metabolic activation (acc. Guidelines for Screening Mutagenicity Testing of Chemicals Japan, JETOC 2001).

Polychloro copper phthalocyanine was also not mutagenic in a preincubation Ames test with and without metabolic activation (tested up to 5000 μg/plate in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA with and without metabolic activation; metabolic activation: Rat liver, induced with phenobarbital and 5,6-benzoflavone. Cytotoxicity was not observed.

In another Ames test a pre-incubation assay and a standard plate test were conducted, both with and without metabolic activation (tested up to 5000 μg/plate in Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537; metabolic activation: S9 fraction from the liver of male Sprague-Dawley rats (treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice) and mixed with a series of cofactors. Polychloro copper phthalocyanine was also not mutagenic in this assay (comp. OECD 471, BASF AG 1988). No cytotoxicity was observed.

In another Ames test, a weakly positive result was obtained at concentrations >= 333 µg/plate in S. typhimurium TA98 only with metabolic activation (comp. OECD 471, Zeiger 1988). The mutagenicity potential of the test substance was tested up to 10000 μg/plate in two Salmonella typhimurium strains TA100 and TA98; metabolic activation: S9-mix, prepared from the liver of either male SD rats or male Syrian hamsters, induced with Aroclor 1254.Without metabolic activation in the strain TA98 as well as in the strain TA100 (with or without metabolic activation), no increase of the number of his+ revertants was observed. Since the analyzed purity of the test substance used in this study was indicated to be 94 % and no further information on the remaining content was given, this result may be due to impurities of the test substance.

Another study provided also negative results for polychloro copper phthalocyanine in the Ames test (Milvy 1978, Val. 4).

A valid Ames Test reporting absence of mutagenicity was published in JETOC in 1997. It was performed with CAS no. 14832 -14 -5 which describes the fully chlorinated copper phthalocyanine. Pigment Green 7 is defined in the Colour Index as mixture of fully or almost fully chlorinated copperphthalocyanines (substitutation grades 14 -16).

 

Gene mutation in mammalian cells in vitro

The substance Polychloro copper phthalocyanine 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 (BASF 2021). Four independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and beta-naphthoflavone induced rats (exogenous metabolic activation). Since the test substance is a nanoparticle, dose selection for genotoxicity testing was based on 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 12 June 2018. Furthermore, to fulfill the requirements of the OECD Guidelines for the HPRT assay, the top concentrations in all main Experiments were defined as the highest homogenous suspension. 0.05% w/v bovine serum albumin water (BSA-water) was used as vehicle. Test groups printed in bold type were evaluated for gene mutations, except the highest tested concentration of the 1st and 2nd Experiment. 2000.0 µg/mL was an inhomogenous suspension and thus is regarded as invalid. In the 3rd and 4th Experiment 256.00 µg/mL was used as top concentration which was the highest homogeneous suspension which could be applied to the test culture.

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, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. In all experiments in the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations 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 four experiments performed independently of each other.

 

Cytogenicity in mammalian cells

In a Mammalian Cell Micronucleus Test (acc. OECD 487 draft proposal, BASF AG 2001) V79 cells were used, with and without metabolic activation. The test substance was found to be negative for causing cytogenicity at dose levels from 0.78 µg/ml up to 75 µg/ml. Cytotoxicity was not observed.

CHL/IU cells were used in two in-vitro chromosomal aberration tests (acc. Japanese Guidelines for Screening Mutagenicity Testing of Chemicals, JETOC 1997 and 2001), with and without metabolic activation. The test substance was found to be negative for causing cytogenicity at dose levels up to 5000 µg/ml. Cytotoxicity was not observed. The study published in JETOC in 1997 was performed with CAS no. 14832 -14 -5 which describes the fully chlorinated copper phthalocyanine. Pigment Green 7 is defined in the Colour Index as mixture of fully or almost fully chlorinated copperphthalocyanines (substitutation grades 14 -16).

 

GENOTOXICITY IN-VIVO with the non-chlorinated version Pigment Blue 15:1:

Valid experimental data were also available to assess the genetic toxicity in-vivo.

Crude Copper Phthalocyanine was administered by gavage to Chinese hamsters (Cricetulus griseus) of either sex. Treatment consisted of one daily dose of 1250, 2500 or 5000 mg/kg on each of two consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made. The experiment was performed to evaluate any mutagenic effect on somatic interphase cells in vivo (Ciba Geigy 1986, Val. 2). The bone marrow smears from animals treated with the various doses of the test material showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponded to the frequency observed in the control group. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg bw) yielded 9.48 % cells with anomalies of nuclei. This is significantly different from the controls (0.1 %) treated with the vehicle (0.5 % CMC) alone.

In a second in-vivo experiment, crude copper phthalocyanine was administered in a single intraperitoneal injection to pregnant female mice (C57 Bl/6) on the 10th day after conception. Doses of 1250, 2500 and 5000 mg/kg were given. The experiment was performed to ascertain whether the substance might have a mutagenic effect on somatic cells in vivo. The mouse spot test system permits the detection of induced point mutations and other genetic events in the melanoblasts of embryos exposed in utero. The induction of mutation is monitored post-natally by examination of the fur of young mice for recessive spots (RS) resulting from expression of recessive genes involved in coat-colour determination (Ciba Geigy 1986, Val. 2). The average litter size was not markedly affected by any of the doses administered. The survival rate of the young animals at the beginning of the observation period (approx. the 12 th day) was decreased with increasing doses. Altogether 899 animals were examined for spots. 0.29 % of the control animals showed RS. The frequencies of RS in the group treated with 1250, 2500 and 5000 mg/kg were respectively 0.93, 0 and 0 %. Thus, the relative incidence of RS among the animals treated with the various doses of the test material did not differ significantly from that of the control (sesame oil). By contrast, the positive control experiment with ethylnitrosourea (50 mg/kg) performed simultaneously yielded an statistically significant average RS frequency of 4.75 %.

 

 

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. No adverse findings on genotoxicity was observed in in-vitro or in-vivo studies. 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 Regulation (EC) No 2020/1182.