Low chlorinated copper, [29H,31Hphthalocyaninato(2-)-N29,N30,N31,N32]-, wherein the number of chlorines is more than or equal to 0 and less than or equal to 7

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- Particle size distribution: D50 = 28.9 nm (TEM)
- Shape of particles: elongated, rod
- Surface area of particles: BET = 100.3
- Crystal structure: crystalline
- Coating: none
- Density: Rel density = 1.442; Bulk density 125 kg/m3

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

Method

Target gene:

HPRT

Species / strain

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).

Controls

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

  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

Applicant's summary and conclusion

Conclusions:

Pigment Blue 16 was not mutagenic in the HPRT test.