Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No signs of genotoxic effects in-vitro were observed, neither in bacterial systems nor in mammalian cells.


- Ames test: according to OECD TG 471, GLP, K1, negative


- HPRT test: read across from CAS 574-93-6 and CAS 1328-53-6, according to OECD TG 476, GLP, K1 negative


- CA study: read across from 69987-63-3, according to OECD TG 473, GLP, K1 negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 Jan 2021 - July 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, Germany
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 14 Feb 2023
- Purity: 98.5%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under storage conditions: stable
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Due to the use of culture medium (HAM´s F12) as vehicle the verification of the stability of the test substance in the vehicle was not required.

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 0.5vol% 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 (2.56 mg/mL and 2.0 mg/mL).
The stock dispersion of 2.0 mg/mL was handled separately. For further dilutions only the stock dispersion of 2.56 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, blue
- molecular weight: 515 g/mol
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:
1
st 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
2
nd Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 1000.00 µg/mL
with S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 1000.00 µg/mL

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 both main Experiments
were defined as followed. 0.05% w/v bovine serum albumin water (BSA-water) was used as
vehicle.
2000.0 µg/mL was an inhomogenous suspension and thus is regarded as invalid.
1000.0 µg/mL was used as top concentration in the second repeat experiment which was a homogeneous suspension when applied to the test culture.
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: 3

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.

In the pre-test the pH value was not relevantly altered by the test substance. The osmolality was not relevantly influenced by the addition of the test substance preparation to the culture
medium at the concentrations measured. The highest applied concentration of 0.1 mg/mL (Test group: 100 μg/mL) was a homogeneous
dispersion. Test substance dispersions were obtained from 0.025 mg/mL (Test group:25 μg/mL) onward.
After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced RS of about or below 20%.
The test substance is a nanoparticle. Therefore, some considerations need to be taken for the performance of this assay in order to adhere to the current version of the OECD guideline.
The substance is tested at precipitating levels, the highest concentration is based on homogeneity of the formulation.
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Osmolality and pH values were not relevantly influenced by test substance treatment.
In the 1st Experiment in the absence of S9 mix, test substance precipitation was observed in culture medium at the end of treatment at 25.00 μg/mL and above. In the presence of S9 mix
test substance precipitation was observed from 1.00 μg/mL onward. In the 2nd Experiment in the presence and absence of S9 mix test substance precipitation occurred from 10.00 μg/mL
onward.

After 4 hours treatment neither in the absence nor presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test
group tested for gene mutations.

The results of the characterization confirmed that the study was performed with the compound as nanoparticles. Since the test substance is an insoluble nanoparticle, the visual assessment of concentration of test substance precipitation is misleading. As described in the characterization report the test substance did not dissolve within the tested period. Thus, the documentation of test substance precipitation merely shows that the particulate matter could be observed visually and does not mean that the test substance at lower concentrations were dissolved.

  Table 3:   Summary of results – experimental parts without S9 mix

Exp. period         Test groups
[h]               [µg/mL]		 S9
mix		 Prec.* MFcorr.
[per 106 cells]		 RS [%] CE2 [%]
1 4 Vehicle control (BSA-water 0.05% (v/v)) - n.d. 0.00 100.0 100.0
0.125 - - 2.69s 124.6 108.1
0.25 - - 3.05s 129.3 104.9
0.50 - - 1.29 110.6 89.8
1.00 - - 1.82 118.8 95.9
10.00 - - 0.00 110.6 86.6
25.00 - + 1.26 125.5 92.2
50.00 - + 3.36s 127.3 86.6
100.00 - + 1.25 130.0 93.0
Positive control (EMS 400 μg/mL) - n.d 110.04s 87.8 72.4
2 4 Vehicle control (BSA-water 0.05% (v/v)) - n.d. 2.27 100.0 100.0
0.125 - - 0.00 128.8 82.2
0.250 - - 1.40 113.4 81.0
0.500 - - 1.36 112.1 83.6
1.000 - - 0.34 122.3 83.0
10.000 - + 2.01 121.1 98.6
25.000 - + 0.00 100.1 88.4
50.000 - + 3.09 101.7 82.4
100.000 - + 1.62 103.5 87.3
1.000.000 - + 1.99 81.8 85.3
    Positive control (EMS 400 μg/mL) - n.d. 118.18s 77.1 77.9

*       Macroscopically visible 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

s Mutant frequency statistically significantly higher than corresponding control values (p ≤ 0.05)

Table 4: Summary of results – experimental parts with S9 mix

Exp. Exposure
period [h]		 Test groups [µg/mL] S9
mix		 Prec.* Genotoxicity**
MFcorr.
[per 106 cells]		 Cytotoxicity***
RS
[%]		 CE2
[%]
1 4 Vehicle control (BSA-water 0.05% (v/v)) + n.d. 3.41 100.0 100.0
  0.125 + - 0.00 129.5 94.4
  0.25 + - 1.56 122.1 99.1
  0.50 + - 0.00 128.0 91.0
  1.00 + + 0.69 118.8 89.8
  10.00 + + 0.82 125.9 75.9
  25.00 + + 1.24 100.2 74.6
  50.00 + + 2.60 97.9 71.5
  100.00 + + 0.87 95.3 71.5
  Positive control (DMBA 1.25 μg/mL) + n.d. 78.87s 81.5 65.9
2  4 Vehicle control (BSA-water 0.05% (v/v)) + n.d. 5.96 100.0 100.0
0.125 + - 1.45 155.9 96.8
0.25 + - 2.45 105.4 100.4
0.50 + - 6.41 113.5 109.5
1.00 + - 1.61 102.3 87.4
10.00 + + 4.15 106.6 93.0
25.00 + + 1.79 100.4 98.2
50.00 + + 3.08 94.0 91.2
100.00 + + 1.15 115.9 91.2
1000.00 + + 5.11 101.9 96.1
  Positive control (DMBA 1.25 μg/mL) + n.d. 114.75s 80.1 76.1

 

 * Macroscopically visible 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

s Mutant frequency statistically significantly higher than corresponding control values

Particle Characterization in cell culture medium

The size distribution is polydisperse, ranging from below 50 nm to 1000 nm. Due to the selective tracking of pigment particles by the UVVis optics the colloidal components of the cell culture

medium (proteins etc) do not interfere with the analysis.

Across all doses tested (10 - 100 mg/L), the median diameter D50 remained constant. The D90 percentile, representating the largest agglomerates, shows a minimal trend towards larger agglomerates at higher doses (Table 5).

The re-characterisation after 20h finds no change. The size distributions overlap, and consequently also the percentile diameters shows that the particles have not significantly changed their state of agglomeration.

The dissolved content at the end of the incubation time of 20h is below the limit of quantification (below 0.05% of 0.01 mg/mL).

 

Table 5: Percentile diameters of the measured particle size distributions in cell culture medium

  t = 0h t = 20h
Dose c=0.01 mg/ml c=0.03 mg/ml c=0.10 mg/ml c=0.10 mg/ml
D10 [nm] 31 39 41 41
D50 [nm] 65 69 70 64
D90 [nm] 110 118 134 90
(d90-d10)/d50 1.21 1.15 1.33 0.76
Conclusions:
Pigment Blue 16 was not mutagenic in the HPRT test.
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:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Batch no. 21993
- Storage conditions: Room temperature
- Name: Fastogen Blue 10GN
- Fine violet powder
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
obtained from the JCRB Cell Bank.
Metabolic activation:
with and without
Metabolic activation system:
liver S9 from Aroclor1254-induced rats
Test concentrations with justification for top dose:
8, 40, 200, 1000 and 5000 μg/ml (preliminary test)
1250 - 5000 μg/ml (main test)
Vehicle / solvent:
Cell culture medium

The test material was found to be poorly soluble in dimethyl sulphoxide and acetone, but to form a doseable suspension in culture medium at concentrations of 50 mg/ml and below. Accordingly, suspensions ofthe test material for use in testing were prepared in culture medium immediately before addition to test cultures. The suspensions of maximum concentration was prepared initially and lower concentrations were prepared by dilution in culture medium. All concentrations cited in this report are stated in terms of the material as supplied
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (suspension)

DURATION
- Preincubation period: 24h
- Exposure duration: without S-9 mix cells were exposed continuously for 6, 24 or 48 hours, with S-9 mix exposure was limited to 6 hours; cells exposed for 6 hours were cultured in fresh medium for a further 18 hours before harvesting.
- Expression time (cells in growth medium): 18h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48h


SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1.5

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS: A sample ofthe laboratory stock culture was tested in December 1996 and was found to be mycoplasma free.
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Rationale for test conditions:
The test material was found to be poorly soluble in dimethyl sulphoxide and acetone, but to form a doseable suspension in culture medium at concentrations of
50 mg/ml and below. Accordingly, suspensions ofthe test material for use in testing were prepared in culture medium immediately before addition to test
cultures. The suspensions of maximum concentration was prepared initially and lower concentrations were prepared by dilution in culture medium. All
concentrations cited in this report are stated in terms of the material as supplied; no correction has been made for purity or activity below 100%.
Evaluation criteria:
The test material is considered to be clastogenic in this test if the following conditions are
met:
- statistically significant increases in the frequency of metaphases with aberrant
chromosomes (excluding gap-type aberrations) are observed at one or more test
concentrations
- the increases exceed the historical negative control range at this laboratory
- the increases are reproducible between replicate cultures and between tests
- the increases are not associated with !arge changes in pH or osmolarity of the treatment
medium or extreme toxicity (which can cause non-specific effects)
- evidence of a dose-response relationship, or increases at both sampling times will be
considered to support the conclusion.
The biological significance of gap-type aberrations is questionable and increases observed
only when gaps are included in the analysis will not be considered to be conclusive
evidence of clastogenic activity.
Statistics:
The Fisher Exact Probability test is a useful technique for analysing data when comparing
two independent samples. lt is used when the observed events all fall into one or other of
two mutually exclusive classes. The test determines whether the two groups differ in the
proportions with which they fall into the two classifications.
The frequency of aberrant metaphases for each treatment group was compared with the
corresponding solvent control group value using a one-tailed test.
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Water solubility: substance is insoluble
- Precipitation: substance is applied as a suspension
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: yes, substance is not cytotoxic

Preliminarv toxicity test - Mitotic indices: Presence of S-9 mix - 6 hour exposure. 24 hour sampling time
Treatment (µg/ml) Culture number Number of cells Number of metaphases Mitotic index* Mean mitotic index %
Reduction
Culture medium 1 1005 76 7.6 5.8
(-) 2 1002 39 3.9
Test material 3 1028 40 3.9 4.1 29
(8) 4 1047 45 4.3
Test material 5 1018 52 5.1 5.4 7
(40) 6 1033 59 5.7
Test material 7 1018 48 4.7 3.6 38
(200) 8 1032 25 2.4
Test material 9 1011 37 3.7 4.9 16
(1000) 10 1028 63 6.1
Test material 11 1040 85 8.2 6.8 Increase
(5000) 12 1027 55 5.4
Mitotic index = number of metaphases x 100

Preliminary toxicity test - Mitotic indices: Absence of S-9 mix - 6 hour exposure. 24 hour sampling time
Treatment (µg/ml) Culture number Number of cells Number of metaphases Mitotic index* Mean mitotic index %
Reduction
Culture medium 13 1014 305 30.1 25.5
(-) 14 1011 210 20.8
Test material 15 1005 220 21.9 19.3 24
(8) 16 1021 170 16.7
Test material 17 1001 480 48.0 35.9 increase
(40) 18 1002 238 23.8
Test material 0.19 1016 376 37.0 23.8 7
(200) 20 1005 107 10.6
Test material 21 1030 302 29.3 26.7 increase
(1000) 22 1019 245 24.0
Test material 23 1034 130 12.6 16.4 36
(5000) 24 1058 214 20.2

Preliminary toxicity test - Mitotic indices: Absence of S-9 mix - 24 hour exposure. 24 hour sampling time
Treatment (µg/ml) Culture number Number of cells Number of metaphases Mitotic index* Mean mitotic index %
Reduction
#
Culture medium 25 1009 432 42.8 42.5 -
(-) 26 1028 434 42.2
Test material 27 1016 321 31.6 34.7 18
(8) 28 1020 385 37.7
Test material 29 1.010 344 34.1 35.8 16
(40) 30 1013 380 37.5
Test material 31 1018 443 43.5 39.1 8
(200) 32 1013 351 34.6
Test material 33 1014 416 41.0 35.2 17
(1000) 34 1021 299 29.3
Test material 35 1009 222 22.0 23.9 44
(5000) 36 1007 260 25.8

 Preliminary toxicity test - Mitotic indices: Absence of S-9 mix - 48 hour exposure. 48 hour sampling time
Treatment (µg/ml) Culture number Number of cells Number of metaphases Mitotic index* Mean mitotic index %
Reduction
#
Culture medium 37 1026 64 6.2 5.5                -
(-) 38 1045 49 4.7
Test material 39 1019 48 4.7 4.1 25
(8) 40 1037 35 3.4
Test material 41 1029 81 7.9 7.8 increase
(40) 42 1021 78 7.6
Test material 43 1018 68 6.7 5.9 increase
(200) 44 1008 50 5.0
Test material 45 1018 43 4.2 4.6 16
(1000) 46 1019 50 4.9
Test material 47 1007 66 6.6 7 increase
(5000) 48 1085 80 7.4
Conclusions:
non clastogenic in vitro
Executive summary:

The effects on chromosomal structure of exposure to the substance were investigated in cultured CHL (Chinese hamster lung) cells. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activation system (S-9 mix):

without S-9 mix cells were exposed continuously for 6, 24 or 48 hours, with S-9 mix exposure was limited to 6 hours; cells exposed for 6 hours were cultured in fresh medium for a further 18 hours before harvesting.

Treatments were established by the addition oftest suspensions (in culture medium) to 24-hour cultures. Cell division was arrested by the addition ofthe spindle poison, Colcemid, two hours before the cells were harvested; slides were then prepared for microscopic analysis.

All slides were scored for chromosomal aberrations. The highest concentration scored for chromosomal aberrations (5000 μg/ml) is the highest required by EC, OECD, US and Japanese regulatory test guidelines.

One hundred metaphases were analysed from all selected cultures. Treatment with the test substance did not produce biologically or statistically significant increases in the frequency of metaphases with aberrant chromosomes at any concentration tested,

compared to vehicle control values (p>0.05 both including and excluding gap-type aberrations), at any sampling time, either in the presence or absence of S-9 mix.

The known clastogens, Mitomycin C and cyclophosphamide, induced significant increases in the frequency of metaphases with aberrant chromosomes, compared to the vehicle control values, at both sampling times in both cytogenetic tests (p<0.001 in all cases), thus demonstrating the sensitivity ofthe test procedure, and the metabolic activity of the S-9 mix employed.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No signs of genotoxic effects in-vivo were seen, neither in the Nucleus Anomaly Test or the Mouse Spot test.


- Nucleus Anomaly Test: read across from CAS 147-14-8, similar to OECD TG 484, GLP, K2 negative


- Mouse Spot test: read across from CAS 147-14-8, similar to OECD TG 474, GLP, K2 negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 484 (Genetic Toxicology: Mouse Spot Test)
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
26 September 2014
Deviations:
yes
Remarks:
No plasma analytics. Tested doses exceeded limit dose, 1000 cells per animal scored (but acceptable, since two groups above limit dose scored)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
- Name of test material (as cited in study report): TK 13 143 (crude copper phthalocyanine)
- Analytical purity: commercial grade
- Lot/batch No.: F 53 / H 91375
Species:
hamster, Chinese
Strain:
other: random outbred strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Ciba-Geigy Tierfarm, Sisseln, Switzerland
- Age at study initiation: females 6 to 10 weeks, males 4 to 9 weeks
- Weight at study initiation: females 21 to 32 g, males 22 to 33 g in tolerability test; females 20 to 27 g, males 20 to 26 g in mutagenicity test
- Diet: NAFAG No. 924
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 23 °C
- Humidity: 40 - 46 %
- Housing in air conditioned rooms
- Photoperiod: 12hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
0.5 % Carboxymethylcellulose (CMC), Hercules Comp., USA
Details on exposure:
Tolerability test:
A preliminary test was conducted to determine the highest dosage of the test material to be applied in the mutgenicity test (Doses / Concentrations:
200, 1000 and 5000 mg/kg bw). Three groups of 4 chinese hamsters were treated with 3 different single doses. The observation period corresponded to the interval between administration and sacrifice of the animals in the mutagenicity test, plus one day. The highest dose survived by all animals was used in the second part of the tolerability test.
In the second part, the animals were treated according to the scheme used in the mutagenicity test with consecutive doses. The observation period corresponded to the interval between administration and sacrifice of the animals in the mutagenicity test, plus one day. Depending on the outcome the highest dose causing no deaths was used as the highest in the mutagenicity test.

Mutagenicity test:
The test material was administered orally to groups of 6 female and 6 male animals each. Treatment consisted of daily one application on 2 consecutive days. 24 h after the second application the animals were sacrificed.
Duration of treatment / exposure:
48 h
Frequency of treatment:
two treatments on 2 consecutive days
Post exposure period:
24 h after the second application
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
Dose / conc.:
2 500 mg/kg bw/day (actual dose received)
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Tolerability test: 2 animals per sex per dose
Mutagenicity test: 6 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (ENDOXAN): 128 mg/kg bw in 20 ml/kg bw 0.5 % CMC
Details of tissue and slide preparation:
Bone marrow was harvested from the shafts of both femurs and homogenized. Small drops were transferred on the end of a slide and spread out. 3 h later, the slides were stained in undiluted May-Grünwald solution/water for 2 min and in Giemsa´s 40 % for 20 min. After being rinsed in methanol 55 % for 5-8 sec and washed with water, the slides were cleaned in xylene and mounted in Eukitt.
The slides of three female and three male animals each of the negative and positive control group and of the groups treated with various doses of the test material were examined. 1000 bone marrow cells each were scored per animal and the following anomalies were registered: single jolly bodies, fragments of nuclei in erythrocytes, micronuclei in leucopoietic cells and polyploid cells.
Statistics:
The significance of difference was assessed by x2-test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In all dose groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control (0.1 %).
By contrast, the positive control yielded in a marked increase of the percentage of cells with anomalies (9.48 %).

Table 1: Percent of cells with anomalies of nuclei

 

Animal No.

Sex (m/f)

Single Jolly Bodies

Fragments of nuclei in erythrocytes

Micronuclei in erythrocytes

Micronuclei in leucopoietic cells

Polyploid cells

Total

negative control

1

f

 

 

 

 

 

0.0

2

f

0.2

 

 

 

 

0.2

3

f

0.2

 

 

 

 

0.2

4

m

0.1

 

 

 

 

0.1

5

m

0.1

 

 

 

 

0.1

6

m

 

 

 

 

 

0.0

cyclophosphamide

1

f

11.6

2.3

2.0

 

 

15.9

2

f

4.5

1.0

1.6

0.1

 

7.2

3

f

9.1

2.2

1.3

0.1

 

12.7

4

m

6.7

0.7

1.0

0.3

0.3

9.0

5

m

4.0

1.2

0.8

 

 

6.0

6

m

4.4

0.6

0.9

0.2

 

6.1

1250 mg/kg bw

1

f

 

 

 

 

 

0.0

2

f

 

 

 

 

 

0.0

3

f

0.1

 

 

 

 

0.1

4

m

 

 

 

 

 

0.0

5

m

0.1

 

 

 

 

0.1

6

m

0.1

 

 

 

 

0.1

2500 mg/kg bw

1

f

 

 

 

 

 

0.0

2

f

0.2

 

 

 

 

0.2

3

f

0.1

 

 

 

 

0.1

4

m

0.3

 

 

 

 

0.3

5

m

0.1

 

 

 

 

0.1

6

m

 

 

 

 

 

0.0

5000 mg/kg bw

1

f

 

 

 

 

 

0.0

2

f

0.2

 

 

 

 

0.2

3

f

 

 

 

 

 

0.0

4

m

0.1

 

 

 

 

0.1

5

m

 

 

 

 

 

0.0

6

m

0.1

 

 

 

 

0.1
Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 484 (Genetic Toxicology: Mouse Spot Test)
Version / remarks:
adopted 23 Oct 1986
Deviations:
yes
Remarks:
Historical control data not included in the report. MDS not scored, Limit dose exceeded by factor of 5
GLP compliance:
yes
Type of assay:
mouse spot test
Specific details on test material used for the study:
- Name of test material (as cited in study report): TK 13 143 (crude copper phthalocyanine)
- Analytical purity: commercial grade
- Lot/batch No.: F 53 / H 91375
Species:
mouse
Strain:
other: C57/Bl/6, males: T-stock
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Bomholtgard Ltd. Denmark
- Age at study initiation: 3 - 4 months
- Weight at study initiation: females 20 - 23 g in toleerability test and 19 - 30 g in mutagenicity test; male body weight was not determined. (Males were not treated; males were included to mate with females and then only pregnant females were treated.)
- Assigned to test groups randomly: yes
- Diet: NAFAG No. 890 pellets standard diet, ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 23 °C
- Humidity: 48 - 56 %
- Air conditioned room
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
intraperitoneal
Vehicle:
Sesame oil was used as vehicle for the test material, Hank´s BSS was used as vehicle control for the positive control.
Details on exposure:
Tolerability test:
A preliminary test was conducted to determine the highest dosage of the test material to be applied in the mutgenicity test. 3 groups of 4 female mice were treated with 3 different single doses.The observation period lased 2 weeks. Depending on the outcome, the highest dose causing no deaths was used as the highest in the mutagenicity test or, if neccessary, the test was repeated with lower doses. Doses tested for tolerability were 200, 1000 and 5000 mg/kg bw.

Mutagenicity test:
One untreated male was placed in a cage with 2 untreated females. The females were inspected daily for successful mating. The day on which a vaginal plug was observed was designated as "day 1/2 of gestation". The females presumed to be pregnant were removed and the procedure was repeated for 4 consecutive days (4 mating nights). Subsequently the presumably pregnant females were uniformely distributed among the respective groups by random.
The test material preparation was administered intraperitoneally to groups of 71 successfully mated females. All presumably pregnant females were treated on the 10th day after conception. Treatment consisted of a single i.p. injection of the respective dose. The animals of the control group received vehicle only.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
once
Post exposure period:
until the birth of the offspring
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
Dose / conc.:
2 500 mg/kg bw/day (actual dose received)
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Tolerability test: 4 females per group
Mutagenicity test: 48 males and 96 females per group
Control animals:
yes, concurrent vehicle
Positive control(s):
N-Nitroso-N-ethylurea (ENU), 50 mg/kg bw in Hank´s BSS was administered intraperitoneally in parallel.
Tissues and cell types examined:
Animals treated as embryos were allowed to come to birth. The number of live and dead offspring was listed. The pups were inspected for external visible morphological changes. The examination upon spots began at the age of 12 - 14 days and was carried out twice per week during 3 weeks. 2 classes fo spots were distinguished and registered: pigmented and white spots, randomly distributed on the coat (recessive spots RS) and white mid-ventral spots (WMVS) within 5 mm of the mid-ventral line presumably arising from cell killing and thus not a result of mutagenic effects. Yellow, agouti-like spots in the vicinity of the mammae, genitalia, throat, axillary and inguinal areas and on the mid-forehead, which are presumed to result from misdifferentiation (MDS) are omitted from scoring. The pelts from the animals with spots were preserved.
Statistics:
The statisitcal analysis was conducted in 2 parts, Firstly the numbers of recessive spots in the control group and in the treatment groups were compared by a chi-squared test. Secondly, a test was carried out to determine whether the frequency of recessive spots increases with increasing doses.
If the effect of the substance increased with the dose, the trend test was preferable. The tests were applied on the condition that the proportions of recessive spots were constant over litters.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Tolerability test:
The dose of 5000 mg/kg bw was found to be the highest applicable in the mutagenicity test and was administered, together with 2 further doses, diminishing by a factor of 0.5.

Mutagenicity test:
From 71 presumably pregnant females per dose group, the following numbers actually were pregnant and gave birth to litters: Control: 56; 1250 mg/kg bw: 45, 2; 2500 mg/kg bw: 48; 5000 mg/kg bw: 44.
The average littersizes registered were: Control: 6.45; 1250 mg/kg bw: 5.93; 2500 mg/kg bw: 5.29; 5000 mg/kg bw: 6.07. 343 animals from the control group, 216, 171 and 169 animals from the groups, treated with 1250, 2500 and 5000 mg/kg bw were examined for colour spots.
The following percentages of animals with recessive (RS) and mid-ventral (WMVS) spots were recorded from gross observations:
RS: Control 0.29 %, 1250 mg/kg bw: 0.93 %, 2500 mg/kg bw: 0 %, 5000 mg/kg bw: 0 %
WMVS: Control 1.17 %, 1250 mg/kg bw: 3.24 %, 2500 mg/kg bw: 2.34 %, 5000 mg/kg bw: 1.78 %
In the positive control, the mean percentage of RS spots was 4.75 and of WMVS spots 2.71.
Statistical analysis for RS revealed the following results: Overall test X2 (3) = 3.82,p = 0.2819; Trend test (one-sided): Z = -0.7637, p = 0.7775

Table 1: THE EFFECT ON OFFSPRING FROM C57 Bl/6 x T CROSS TREATED IN UTERO

dose vehicle route of application treated females with vaginal plug % females with litters average litter size number of offspring examined offspring with recessive spots (total) % number of offspring with intraventral spots (total) %
negative control Sesame oil i .p . 71 62.0 6.1 169 0.0 0.0 3 1.8
1250 Sesame oil i .p . 71 63.4 5.9 216 2.0 0.9 7 3.2
2500 Sesame oil i .p . 71 67.6 5.3 171 0.0 0.0 4 2.3
5000 Sesame oil i .p . 71 62.0 6.1 169 0.0 0.0 3 1.8
50 mg/kg positive control Hank's i .p . 71 66.2 6.5 295 14.0 4.8 8 2.7

Table 2: Results of positive control experiment

dose (mg/kg bw) vehicle or N-Nitroso-N-ethylurea vehicle route of application treated females with vaginal plug % females with litters average litter size number of offspring examined offspring with recessive spots (total) % number of offspring with intraventral spots (total) %
negative control Hank's BSS i .p . 66 68.2 5.6 277 0.0 0.0 3 1.1
25 Hank's BSS i .p . 66 74.2 7.4 350 14.0 4.0 10 2.9
50 Hank's BSS i .p . 66 66.7 7.2 298 20.0 6.7 5 1.7
75 Hank's BSS i .p . 66 71.2 7.2 270 27.0 10.0 18 6.7
Conclusions:
Mated female mice received a single intraperitoneal injection of 1250, 2500 or 5000 mg/kg bw on day 10 of pregnancy. An similar number of litters and litter size was observed for all treatment groups indicating absence of embryotoxicity. The number of offspring with recessive spots (indicators of mutagenicity) was increased in the positive control group, but not in the treatment groups. The number of intraventral spots (indicators of toxicity) showed a higher a variability without dose-dependency.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Tests for unscheduled DNA synthesis (Ciba 1985a and b) with Pigment Blue 15 (CAS-No. 147-14-8)


Unscheduled DNA synthesis was examined in primary rat hepatocytes and in a human fibroblast cell line CRL 1121. Each experiment was reported separately. Primary rat hepatocytes are metabolically competent whereas permanent cell lines such as the human fibroblasts have generally very little metabolic capacity. Both experiments did not show an induction of DNA repair activity.


 


Study in human fibroblast cell line


Based on dose-range-finder experiments, 60.0, 12.0, 2.40 and 0.48 µg/mL were used as test concentrations. The highest dose resulted in visible precipitation. DMSO was used as vehicle.  The incubation period with the test material was five hours. DNA repair activity was detected by incorporation of 3H-thymidine, which was visualized by autoradiography.


In the experiments performed, comparison of the mean number of silver grains per nucleus in the negative controls and in the cultures treated with the various concentrations of Pigment Blue 15 revealed no marked deviations. By contrast, "positive control" experiments with 4NQ0 (5 µM) yielded a mean value of 34.3 silver grains per nucleus. This value differs greatly from the two negative controls, by factors of 28.6 and 29.3.


Since no independent repeat experiment was performed, this study is assigned a validity score of 2. The study report contains a Quality Unit statement.


 


Study in primary rat hepatocytes


In a preliminary toxicity test, eleven concentrations increasing from 0.62 µg/mL to 100 µg/mL were tested to determine the highest applicable concentration in the DNA-repair assay. At the three highest concentrations (60, 80 and 100 µg/mL) a precipitation was visible in the culture medium. The two highest concentrations proved toxic. The highest usable concentration was found to be 60 µg/mL. DMSO was used as vehicle. Freshly isolated hepatocytes from a male, adult rat were used.


In the experiments performed, comparison of the mean number of silver grains per nucleus in the negative controls and in the cultures treated with the various concentrations of Pigment Blue 15 revealed no marked deviations. By contrast, "positive control" experiments with DMN (100 mM) yielded a mean value of 20.1 silver grains per nucleus. This value differs greatly from the two negative controls, by factors of 13.7 and 15.6. The study report contains a Quality Unit statement. It is valid without restriction.


 


HPRT Test (BASF 2021) with Pigment Blue 16 (CAS-No. 574-93-6)


The test 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. Two valid experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and b-naphthoflavone induced rats (exogenous metabolic activation). 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 both main Experiments were defined as followed. 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 Experiment. 2000.0 µg/mL was an inhomogenous suspension and thus is regarded as invalid (data not shown, however archived in the raw data of this study). In the 2nd Experiment 1000.0 µg/mL was used as top concentration which was a homogeneous suspension when applied to the test culture. 1 st 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. 1 st Experiment with S9 mix 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 2000.00 µg/mL. 2 nd Experiment without S9: mix 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 1000.00 µg/mL. 2 nd Experiment with S9 mix 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 1000.00 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, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. In both 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 two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.


 


HPRT Test (BASF 2021) with Pigment Green 7 (CAS-No. 1328-53-6)


The test 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. Four independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and b-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 (data not shown, however archived in the raw data of this study). 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. 1 st 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. 1 st Experiment with S9 mix: 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 2000.00 µg/mL. 2 nd 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. 2 st Experiment with S9 mix: 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 2000.00 µg/mL. 3 rd 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. 3 rd Experiment with S9 mix: 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 256.00 µg/mL. 4 th 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. 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. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.


 


HPRT Test (Ciba 1986) with Pigment Blue 15 (CAS-No. 147-14-8)


Crude copper phthalocyanine was tested in the HPRT-Test 11 years prior to the establishment of the OECD testing guideline. As a major difference to the test guideline, the doses were chosen based on cytotoxicity and not on substance precipitation, which is a known cause for false positive findings. The highest concentration resulted in cell survival of 10 %. At the time when the assay was performed, that had not yet been established. As a consequence, the insoluble pigment was tested at inappropriate doses (62.5 µg/mL – 2500 µg/mL in the presence of S9 mix and 25 – 1000 µg /mL in the absence of S9 mix), and a systematic observation of precipitates was not included. In the cytotoxicity experiment, it was noted that with the removal of the test substance with cell culture medium change, not all precipitates were removed. In the UDS in vitro study, 60 µg/mL was reported at the lowest precipitating concentration.


Another issue of this study is that cytotoxicity was determined in a separate experiment from the survival of thinly seeded cells. The determination of cloning efficiency was not performed, especially not in parallel to the mutant selection experiment. Instead, cloning efficiency was assumed to be 100% for all incubations.


Under the above described conditions, no increase in mutant frequency was observed in the presence of S9 mix. In the absence of S9-mix, one experiment showed a slight dose-dependent increase not reaching a threefold increase in mutant frequency. The repeat experiment, there was no dose-dependency, but individual concentrations showed a response of greater than 3.


Due to the inappropriate doses and lack of cloning efficiency determination, this experiment is considered to be unreliable. It was included in the dossier, because the same test materials were used for the mouse spot test and the nucleus anomaly assay. The study report contains a Quality Unit statement.


 


Chromosome Aberration Test with CAS-No. 68987-63-3


The effects on chromosomal structure of exposure to the substance were investigated in cultured CHL (Chinese hamster lung) cells. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activation system (S-9 mix): without S-9 mix cells were exposed continuously for 6, 24 or 48 hours, with S-9 mix exposure was limited to 6 hours; cells exposed for 6 hours were cultured in fresh medium for a further 18 hours before harvesting. Treatments were established by the addition of test suspensions (in culture medium) to 24-hour cultures. Cell division was arrested by the addition of the spindle poison, Colcemid, two hours before the cells were harvested; slides were then prepared for microscopic analysis. All slides were scored for chromosomal aberrations. The highest concentration scored for chromosomal aberrations (5000 μg/mL) is the highest required by EC, OECD, US and Japanese regulatory test guidelines.


One hundred metaphases were analysed from all selected cultures. Treatment with the test substance did not produce biologically or statistically significant increases in the frequency of metaphases with aberrant chromosomes at any concentration tested, compared to vehicle control values (p > 0.05 both including and excluding gap-type aberrations), at any sampling time, either in the presence or absence of S-9 mix. The known clastogens, Mitomycin C and cyclophosphamide, induced significant increases in the frequency of metaphases with aberrant chromosomes, compared to the vehicle control values, at both sampling times in both cytogenetic tests (p < 0.001 in all cases), thus demonstrating the sensitivity of the test procedure, and the metabolic activity of the S-9 mix employed.


 


Mouse spot test (Ciba 1986) with Pigment Blue 15 (CAS-No. 147-14-8)


The mouse spot test was initiated in the year that the OECD testing guideline 484 was adopted and when draft OECD guidelines were available. The study report contains a Quality Unit statement. Mated mice of the T strain received a single intraperitoneal injection of 5000, 2500 or 1250 mg/kg bw in sesame oil ten days after detection of the insemination plug. The tested amounts exceed the limit dose value of 1000 mg/kg bw given in the OECD testing guideline. The intraperitoneal injections of these high doses were however well tolerated. The other deviation to the other guideline is that MDS spots were not scored as they do not contribute to somatic mutagenicity. These yellow, agouti-like, spots associated with mammae, genitalia, throat, axillary and inguinal areas and mid-forehead, which are presumed to result from misdifferentiation (MDS). The positive control group received a single injection of 50 mg/kg bw N-Nitroso-N-ethylurea. Animals treated as embryos were allowed to come to birth. The number of live and dead offspring was listed. The pups were inspected for external visible morphological changes. The examination upon spots began at the age of 12 - 14 days and was carried out twice per week for 3 weeks. 2 classes of spots were distinguished and registered: pigmented and white spots, randomly distributed on the coat (recessive spots RS) and white mid-ventral spots (WMVS) within 5 mm of the mid-ventral line presumably arising from cell killing and thus not a result of mutagenic effects. Under the given experimental conditions, no evidence of mutagenic effects was obtained in the offspring of pregnant mice treated with the test material copper phthalocyanine. An increase in the recessive spots was observed in offspring of N-Nitroso-N-ethylurea treated mice. Treatment did not result in maternal toxicity and did not affect the number of females with litters or the mean number of pups per litter.

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 fourteenth time in Regulation (EC) No. 2020/217.