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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 2017-05-24 to 2017-06-09
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
Cell source:
other: Episkin SA, RHE/S/17 (batches 17-RHE-064, Episkin SA, RHE/S/17 and 17-RHE-059)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin SA, RHE/S/17

- Tissue batch number(s): 17-RHE-064 and 17-RHE-059
- Delivery date: 2017-06-07


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





CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: 2 living and 2 killed Human skin model surfaces
- Procedure used to prepare the killed tissues : The 2 additional killed Human skin model surfaces (Episkin SA, RHE/S/17 Batch
No. 17-RHE-059) were defrozen the day of the treatment. 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 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
- 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



PREDICTION MODEL / DECISION CRITERIA
Following the guideline TG 439
Control samples:
yes, concurrent negative control
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

POSITIVE CONTROL
- Concentration (if solution): 5% SDS prepared by weighing 0.5 g of SDS on a 10 mL flask and filled up to 10 mL with distillated water
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
Reconstructed human epidermis: 3 replicates for test item, positive and negative controls

Living human skin model surfaces (Non specific colour controls or NSC): 2 replicates
killed human skin model surfaces (Non specific colour controls or NSC): 2 replicates
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 84.5
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:

The test item is considered to be non irritant to skin:
- if the viability after 42 minutes of exposure and 42 hours of post-treatment incubation is > 50%.

The test item is considered to be irritant to skin:
- if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50%.
In accordance with the Regulation EC No. 1272/2008, the item is to be classified in Category 2 “Irritant”.
The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.

  number of replicates Mean OD / product mean viability (%) SD Conclusion
Negative control 3 0.895 100.0 6.7  
Positive control 3 0.013 1.5 0.3 Irritant
Test item 3 0.814 91.0 5,1  
Test item NSC Control living tissues 2 0,06 6,6 0,4  
Test item NSC Control killed tissues 2 0,036 4 3,8  
Test item killed tissues MTT control 2 0,35 3,9 1,6  
Test item corrected     84,5   non irritant 
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 mg, to 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes. The application was followed by a rinse with 25 mL of DPBS and a 42 hours and 05 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 were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific living and killed colour controls. Moreover, 2 living and 2 killed Human skin model surfaces were treated in the same manner but they were incubated in culture growth medium instead of MTT solution in order to generate non-specific living and killed colour 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 84.5%, versus 1.5% 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 2017-05-17 to 2017-05-17
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:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
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
Species:
other: eye of chicken
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Etablissement Brun, 33820 Etauliers, France). Chicken are 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, 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: 1h43
* Tranport temperature: ambient temperature
* transport conditions : in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: 1h43 for the enucleation
45 and 56 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 positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 minutes
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 criter ia, 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 45 and 56 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
- Test item : 3 replicates
- Concurrent negative control: 1 replicate
- Concurrent positive control: 3 replicates

NEGATIVE CONTROL USED
Physiological saline


POSITIVE CONTROL USED
Sodium hydroxide

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 or positive control for 10 seconds evenly covering the surface of the cornea.

OBSERVATION PERIOD
240 minutes +/- 5 min (observation post-treatment)

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:eyes were rinsed twice with 10 mL ofphysiological saline


METHODS FOR MEASURED ENDPOINTS:
All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm.
Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

- Corneal opacity: calculated by using the area of the cornea that was most densely opacified for scoring.

- Damage to epithelium based on fluorescein retention: calculated for the 30-minute observation time point only

- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Corneal swelling was determined from corneal thickness measurements made with an optical pachymeter on a slit-lamp microscope. It was expressed as a percentage and was calculated from corneal thickness
measurements according to the following formula:
[(corneal thickness at time t - corneal thicknes at time t=0)/(corneal thickness at time t=0)] x 100
The mean percentage of corneal swelling for all test eyes was calculated for all observation time points.


- Macroscopic morphological damage to the surface: include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea. These findings can vary in severity and may occur simultaneously.


SCORING SYSTEM:
- Mean corneal swelling (%): scoring system as indicated in the TG
- Mean maximum opacity score: scoring system as indicated in the TG
- Mean fluorescein retention score at 30 minutes post-treatment: scoring system as indicated in the TG

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



Irritation parameter:
cornea opacity score
Value:
ca. 2
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Classe III
Irritation parameter:
fluorescein retention score
Value:
ca. 3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class IV
Irritation parameter:
percent corneal swelling
Value:
ca. 9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class III
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.
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: 2.0, corresponding to ICE class III;

- mean score of fluorescein retention: 3.0, corresponding to ICE class IV;

- maximal mean corneal swelling: 9%, corresponding to ICE class II.

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

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 3 x I. 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. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

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 corresponding to the ICE Class III (out of IV) and corneal swelling with a score of 9, corresponding to the ICE Class III (out of IV). The fluorescein retention also show effects with a score of 3, corresponding to the maximum ICE class IV.

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.