2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine

Endpoint summary

Description of key information

In a reliable in vitro skin corrosion study human skin tissue (eipdermis keratinocytes) was exposed to undiluted substance for 3 and 60 minutes. There was 94% and 100 % tissue viability following the 3- and 60-minutes exposure, respectively. Resultantly, the substance considered as non-corrosive to human skin.

In a reliable in vitro skin irritation study conducted on the substance the mean value of relative tissue viability was 74% following a 15-minutes exposure to undiluted substance. This value is above the threshold for skin irritation (50%). Therefore, the substance is considered to be as non-irritant to human skin.

In a reliable in vitro eye irritation study employing bovine cornea were exposed to the undiluted substance for 4 hours. Opacity and permeability values were measured. There was a mean IVIS score of -0.4 following the 4-hour exposure point. Resultantly, the test material is considered to be non-irritant to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th February 2019 - 8th February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

Adopted 29th July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch (Lot) Number: 2715/18/01
Expiry date: 01 July 2020
Physical Description: Dark green solid
Purity/Composition: 99%
Storage Conditions: At room temperature protected from light

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Cultured to form a multilayered, highly differentiated model of the human epidermis

Source strain:

other: Not applicable

Details on animal used as source of test system:

EpiDerm Skin Model (EPI-200, Lot no.: 29944 Kit I and J).

Justification for test system used:

Recommended system according to the OECD 431 and EC TG.

Vehicle:

unchanged (no vehicle)

Details on test system:

The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

Substance: 25.4 to 41.6 mg per tissue
Positive control: 50 μL per tissue
Negative control: 50 μL per tissue

Duration of treatment / exposure:

3 minutes and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours at 37°C in air containing 5% CO2.

Number of replicates:

Duplicate

Irritation / corrosion parameter:

% tissue viability

Remarks:

3-minute time point

Run / experiment:

mean of two samples

Value:

ca. 94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 minute time point

Run / experiment:

Mean of two samples

Value:

ca. 100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No indication of corrosion

Other effects / acceptance of results:

The non-specific reduction of MTT by the substance was -7.4% and 2.2% of the negative control tissues after 3 minutes and 1 hour, respectively.
In addition to the normal 3-minute and 1-hour procedure, two tissues were treated with substance instead of MTT solution these tissues were incubated with DMEM. The color interference by the substance was 5.4% and 7.3% of the negative control tissues after 3 minutes and 1 hour respectively.
For the true tissue viability to be calculated, in addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item were used but instead of MTT solution these tissues were incubated with DMEM. The nonspecific color in freeze-killed tissues by the substance was 2.8% and 4.4% of the negative control tissues after 3 minutes and 1 hour respectively.

Mean Absorption in the in vitro Skin Corrosion Test

	3 -minute application viability (%)				1 -hour application viability (%)
	A (OD570)	B (OD570)	Mean (OD570)	SD (+/-)	A (OD570)	B (OD570)	Mean (OD570)	SD (+/-)
Negative control	1.375	1.511	1.443	0.097	1.316	1.538	1.427	0.157
Substance*	1.279	1.423	1.351	0.101	1.281	1.562	1.422	0.199
Positive control	0.511	0.324	0.087	0.132	0.250	0.117	0.183	0.094

SD = Standard deviation

Duplicate exposures are indicated by A and B.

* For the 3 minute exposure the substance values are corrected for the color interference (5.4%).

For the 1 hour exposure the substance values are corrected for the non-specific MTT reaction and color interference (2.2%

and 7.3 %, respectively). In addition these values are corrected for the nonspecific color in freeze-killed tissues (4.4%).

The values are corrected for background absorption (0.0475). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in the in vitro Skin Corrosion Test

 

	3 -minute application viability (% of control)	1 -hour application viability (% of control)
Negative control	100	100
Substance	94	100
Positive control	29	13

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this study.

Executive summary:

In a reliable in vitro skin corrosion study, conducted according to the OECD Guideline 431, 'In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method', the undiluted substance ( 25.4 to 41.6 mg) was applied onto reconstructed human skin tissue (epidermal model, EpiDerm tissue (0.6 cm²)) in duplicate for a period of 3 or 60 minutes.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the substance compared to the negative control tissues was 94 % and 100 %, respectively. Because the mean relative tissue viability for the substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the substance is considered to be non corrosive.

 

In conclusion, the substance is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this study.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th March 2019 - 1st April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

OECD Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Adopted 28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

No. 440/2008. Official Journal of the European Union No. L142; Amended by EC No. 640/2012 OJ No. L193, 20 July 2012

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch (Lot) Number: 2715/18/01
Expiry date: 01 July 2020 (expiry date)
Physical Description: Dark green solid
Purity/Composition: 99%
Storage Conditions: At room temperature protected from light

Test system:

human skin model

Source species:

other: Not applicable

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small Model TM

Source strain:

other: Not applicable

Justification for test system used:

One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN Small ModelTM (EPISKIN-SMTM), 0.38 cm2.
This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes are cultured for 13-days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Source.

SkinEthic Laboratories, Lyon, France.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg of substance (tissue moistened with 5 µL Milli-Q water prior to the application).

Duration of treatment / exposure:

15 ± 0.5 minutes at room temperature.

Duration of post-treatment incubation (if applicable):

42 ± 1 hours at 37°C.

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Remarks:

maen of three

Run / experiment:

1

Value:

74

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Because a color change was observed in aqueous conditions and by adding MTT-medium it was concluded that the substance did interact with the MTT endpoint.
In addition to the normal procedure, three freeze-killed tissues treated with substance and three freeze-killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the substance was 12% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the substance treated viable tissues.
In addition to the normal procedure, three tissues were treated with substance. Instead of MTT solution the tissues were incubated with assay medium. The non-specific color by the substance was 10% of the negative control tissues. The OD of the tissue incubated with assay medium was subtracted from the ODs of the substance treated tissues incubated with MTT medium.
For the true tissue viability to be calculated, three freeze-killed tissues treated with substance were measured but instead of MTT solution these tissues were incubated with assay medium.
The nonspecific color in freeze-killed tissues by the substance was 9.7% of the negative control tissues.

Mean absorption in the In Vitro Skin Irritation Test with substance

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)	SD (+/-)
Negative Control	1.0820	1.010	1.063	1.052	0.037
Substance	0.862	0.908	0.579	0.783	0.178
Positive Control	0.055	0.045	0.118	0.072	0.040

OD = optical density

SD = Standard deviation

* The substance values are corrected for the non-specific MTT reaction and color interference.

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.046). Isopropanol was used to measure the background absorption.

Mean Tissue Viability in the In Vitro Skin Irritation Test with the substance

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative Control	100	3.5
Substance	74	17
Positive Control	6.9	3.8

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is non-irritant in the in vitro skin irritation test under the experimental conditions described in this study.

Executive summary:

In an in vitro skin irritation study conducted according to OECD TG 439 ‘In vitro Skin Irritation: Reconstructed Human Epidermis Test Method’ human skin tissue (eipdermis keratinocytes) was exposed to the undiluted (10 mg) substance for 15-minutes at room temperature. Following the exposure, the substance was rinsed and incubated for further 42 ± 1 hours at 37°C in fresh medium. There was 74 % tissue viability following the 15-minute exposure point. This value is above the threshold for skin irritation (50%). Resultantly, the substance is considered to be a non-irritant to human skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th February 2019 - 19th February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted October 09, 2017

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Batch (Lot) Number: 2715/18/01
Expiry date: 01 July 2020 (expiry date)
Physical Description: Dark green solid
Purity/Composition: 99%
Storage Conditions: At room temperature protected from light

Additional information.

Test Facility test item number: 210026/A
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum foil

Species:

cattle

Strain:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

356 to 369 mg

Duration of treatment / exposure:

for 240 +/- 10 minutes at 32 +/- 1 °C.

Duration of post- treatment incubation (in vitro):

Opacity determined directly after the treatment; Permiability determined after post-treatment incubation of 90 +/- 5 minutes at 32 +/- 1 °C.

Number of animals or in vitro replicates:

Triplicate.

Details on study design:

Test System.

Test System: Bovine eyes were used as soon as possible after slaughter.

Source:
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.

Transport:
Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Preparation of Corneas.

The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1 °C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1 °C.

Cornea Selection and Opacity Reading.

After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Substance Preparation.
No correction will be made for the purity/composition of the substance.
Since no workable suspension of the substance in physiological saline could be obtained, the substance was used as delivered by the sponsor and added pure on top of the corneas.
To protect the substance from light, amber-colored glassware or tubes wrapped in tin-foil were used for substance preparations.

Treatment of Corneas and Opacity Measurements.

The medium from the anterior compartment was removed and 750 ul of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (356 to 369 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1°C. After the incubation the solutions and the substance were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the substance on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

-2.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

0.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Opacity Score.

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	4.1	5.4	1.2			0.7
	3.4	3.5	0.1
	4.3	5.3	0.9
Positive control	4.6	160.2	155.6	155		137
	3.2	113.1	109.9	109
	3.1	149.6	146.5	146
Substance	3.4	4.3	1.0	0.2		-0.6
	3.4	2.3	-1.1	-1.8
	4.5	5.0	0.5	-0.3

¹  Final Opacity = Opacity after treatment – Opacity before treatment.

²  Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

In vitro Irritancy Score.

Treatment	Final Opacity^2	Final OD490^2	In vitro Irritancy Score^1
Negative control	1.2	0.001	1.2
	0.1	0.023	0.4
	0.9	0.010	1.1
Positive control	155	1.225	173
	109	2.577	148
	146	1.733	172
Substance	-0.2	-0.008	0.1
	-1.8	-0.019	-2.1
	-0.3	0.072	0.8

¹  In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

²  Positive control and substance are corrected for the negative control.

Permeability Score (uncorrected).

Treatment	Dilution factor	OD490 1	OD490 2		OD490 3		Average OD		Final OD		Mean final negative control
Negative control	1	0.001		0.001		0.001		0.001		0.001		0.011
	1	0.026		0.028		0.016		0.023		0.023
	1	0.007		0.007		0.016		0.010		0.010
Positive control	1	1.229		1.240		1.241		1.237		1.237
	6	0.442		0.440		0.441		0.441		2.646
	6	0.300		0.300		0.301		0.300		1.802
Substance	1	0.003		0.003		0.004		0.003		0.003
	1	-0.008		-0.008		-0.007		-0.008		-0.008
	1	0.086		0.082		0.082		0.083		0.083

Interpretation of results:

GHS criteria not met

Conclusions:

The results of an eye corrosion/irritation study employing isolated bovine cornea exposed to undiluted substance for 240-minutes indicate that it is a non-irritant to the eye.

Executive summary:

In a reliable in vitro eye irritation study, conducted according to OECD Guideline 437, ‘Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage’, the ability of the substance to induce opacity and permeability in an isolated bovine cornea were determined.

The substance was applied undiluted (356 - 369 mg) onto corneas (n=3) for a period of 240 minutes, followed by rinsing of the substance. The opacity of the corneas was then determined. The permeability measurements were determined after 90 minutes of incubation with fluorescein. The results of an eye corrosion/irritation study show that the mean in vitro irritance score was -0.4, indicating that that the substance is non-irritant to the eye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the findings of a reliable in vitro skin irritation, skin corrosion and eye irritation studies conducted on the substance, classification of the substance is not justified.