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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Polychloro copper phthalocyanine
EC Number:
215-524-7
EC Name:
Polychloro copper phthalocyanine
Cas Number:
1328-53-6
Molecular formula:
C32HxClyCuN8
IUPAC Name:
polychloro copper phthalocyanine
Test material form:
solid
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

Method

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
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.
Controls
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.

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
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.

Any other information on results incl. tables

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
----------------------------------------------------------

Applicant's summary and conclusion