Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[3-(cyclohexylamino)propyl]amino]sulfonyl derivs.

Endpoint summary

Description of key information

Justification of the classification of the substance for eye irritation/corrosion

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2017-04-18 to 2017-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test system:

human skin model

Source species:

human

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin SA, RHE/S/17
- Tissue batch number(s): 17-RHE-057 and 17-RHE-047
- Delivery date: 2017-05-16
- Date of initiation of testing: 2017-05-16

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: none


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800 absorbance microplate reader
- Wavelength: 570 nm


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The results were expressed as a viability percentage compared with the negative control : viability %= OD test item/OD negative control)x 100


CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: killed Human skin model surfaces
- Procedure used to prepare the killed tissues (if applicable): The 2 additional killed Human skin model surfaces (Episkin SA, RHE/S/17 Batch No. 17-RHE-047). The same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA, batch No. 17 MPE 041) during 2 hours and 05 minutes.
Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 μL of maintenance medium (Episkin SA, batch No. 17 MA 036).
- N. of replicates : 2
- Method of calculation used: As the test item is identified as producing both direct MTT reduction and colour interference:
True viability % = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT + OD of killed tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 100

Evaluation and interpretation of the results
The OD values obtained for each test item are used to calculate a percentage of viability relative to the negative control, which is arbitrarily set to 100%. The cut-off value of percentage cell viability distinguishing irritant from non-classified test items associated with the SkinEthic RHE model is given below:

The test item is considered as non-irritant to skin in accordance with UN GHS No Category:
- if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.

The test item is identified as requiring classification and labelling according to UN GHS (Category 2):
- if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.In accordance with the Regulation (CE) No.1272/2008 and with a classification non-corrosive on a
skin corrosion test, the item has to be classified in Category 2 “Irritant”. The corresponding hazard
statement is “H315: Causes skin irritation” with the signal word “Warning”.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg


NEGATIVE CONTROL
DPBS – VWR - Batch No. 6MB235

POSITIVE CONTROL
- Concentration: 5% SDS solution prepared by weighing 0.5 g of SDS (SIGMA Batch No. STBF1623V) in a 10 mL volumetric flask qsp 10 mL of distilled waterTEST MATERIAL

Duration of treatment / exposure:

42 minutes

Duration of post-treatment incubation (if applicable):

41 h 30 min

Number of replicates:

Reconstructed human epidermis: 3 replicates for test item, positive and negative controls
Killed human skin model surfaces (for non-specific MTT reduction): 2 replicates for test item, positive and negative controls
Killed human skin model surfaces (for non-specific colour controls): 2 replicates
Living human skin model surfaces (for non-specific colour controls): 2 replicates

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 86.8

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes
- Colour interference with MTT: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

GHS criteria not met

Conclusions:

In accordance with the Regulation EC No. 1272/2008, the test item has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.
No hazard statement or signal word is required.

Executive summary:

The aim was to evaluate the possible irritating effects of the test item after topical application on in vitro human reconstructed epidermis (SkinEthic RHE® model).

The test item was applied at the dose of 16 mL, during 42 minutes, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) previously moistened with 10 μL of distilled water. The application was followed by a rinse with 25 mL of DPBS and a 41 hours and 30 minutes post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Additionally, 2 killed Human skin model surfaces previously moistened with 10 μL of distilled water were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction. Moreover, 2 living and 2 killed Human skin model surfaces previously moistened with 10 μL of distilled water were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate non-specific living and killed color controls. The experimental protocol was established in accordance with O.E.C.D. Test Guideline No. 439 adopted 28 July 2015 and the Test method B.46 of Council regulation No. 761/2009 dated 23 July 2009 (EU Journal L220) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The mean corrected percent viability of the treated tissues was 86.8%, versus 1.7% in the positive control (5% Sodium Dodecyl Sulfate).

In accordance with the Regulation EC No. 1272/2008, the test item has to be considered as Non-irritant to skin. It corresponds to UN GHS No Category.

No hazard statement or signal word is required.

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:

From 2017-04-12 to 2017-04-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

yes

Remarks:

The eyes were incubated between 55 and 70 minutes instead of between 45 and 60 minutes, as initially scheduled. This deviation is considered as without impact on the conclusion of the study.

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

yes

Remarks:

The eyes were incubated between 55 and 70 minutes instead of between 45 and 60 minutes, as initially scheduled. This deviation is considered as without impact on the conclusion of the study.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

other: Eyes of chickens

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Etablissement Brun, 33820 Etauliers, France), chicken killed for human consumption have been used for this assay.
- Number of animals: 7 eyes
- Characteristics of donor animals (e.g. age, sex, weight): spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
* transport time : 1h35
* transport temperature : ambient temperature
* transport conditions : in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing:
1h35 for the enucleation,
55 and 70 minutes to equilibrate them to the test system prior to dosing, once all eyes had been examined and approved

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg, (powder, as supplied)

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Test item: 3 replicates
Concurrent negative control : 1 replicate
Concurrent positive control : 3 replicates

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a
bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.

The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip
(in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.6°C and 32.4°C.

After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp
microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.



EQUILIBRATION AND BASELINE RECORDINGS
Selected eyes were incubated between 55 and 70 minutes to equilibrate them to the test system prior to dosing.
Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0).
The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES : 3 replicates for treatment and positivie controls/1 replicates for negative control

NEGATIVE CONTROL USED
Physiological saline – Dutscher Batch No. 3012316

POSITIVE CONTROL USED
sodium hydroxide – Sigma, Batch No. MKBP7805V

APPLICATION DOSE AND EXPOSURE TIME
30 mg of test item and positive control (powder) for 10 seconds evenly covering the surface of the cornea.
30 µL of negative control for 10 seconds evenly covering the surface of the cornea.


OBSERVATION PERIOD : 240 minutes (observation post-treatment)

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: the eyes were rinsed four times with 10 mL of physiological saline

SCORING SYSTEM: scoring system as indicated in the TG was used.

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Irritation parameter:

cornea opacity score

Value:

ca. 3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class IV

Irritation parameter:

fluorescein retention score

Value:

ca. 1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE class II

Irritation parameter:

percent corneal swelling

Value:

ca. 3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The combination of the three endpoints for the negative control, physiological saline, was 2 x I, 1 x II. Therefore, the negative control is classified as “No Category”, as expected.
- Acceptance criteria met for positive control: The combination of the three endpoints for the positive control, Sodium Hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.
The test was considered to be valid.

Combination of the 3 Endpoints: 1 x IV, 1 x II, 1 x I.

Interpretation of results:

study cannot be used for classification

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method.

Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

The test item was applied, as supplied, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed four times with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose. The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 438 adopted 26 July 2013 and the test method B.48 – Commission Regulation (EU) No. 1152/2010 dated 08 December 2010 (EU Journal L324) - ATP Council regulation No. 440/2008 of 30 May 2008 (E.U. Journal L142).

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 3.0, corresponding to ICE class IV;

- mean score of fluorescein retention: 1.0, corresponding to ICE class II;

- maximal mean corneal swelling: 3%, corresponding to ICE class I.

The combination of the three endpoints for the test item was 1 x IV, 1 x II, 1 x I.

The combination of the three endpoints for the positive control, Sodium Hydroxide, was 3 x IV.

Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 2 x I, 1 x II.

Therefore, the negative control is classified as “No Category”, as expected.

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Eye irritation

Based on the results obtained under the experimental protocol performed in accordance with the OECD 438, no prediction could be made.

The ocular reactions observed in the treated eyes show corneal damage with a score of 3 (out of 4), corresponding to the ICE Class IV. The fluorescein retention also show effects with a score of 1.0, corresponding to ICE class II.

Additional testing would be required to establish a definitive classification.

However, considering the knowledge gained of the dyes portfolio for which there is in vitro and in vivo data and the results of the OECD 438, the substance is expected to cause serious eye damage (Category 1), and is therefore classified as such, without the need for additional data.