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Description of key information

Skin irritation (in-vitro). Key Study. Test method according to OECD TG 439, GLP study. Under test conditions, the mean percent viability of the treated tissues was 94.8% versus 1.8% with the positive control item (5% SDS). Therefore, the test item is non-irritating to the skin.


Skin corrosion (in-vitro). Key Study. Test method according to OECD TG 431, GLP study. Under test conditions, the mean percent viabilities of the treated tissues were 85.9% and 93.6% for 3 and 60 min exposure, versus 10.4% and 0.3% with the positive control item, respectively. Therefore, the test item is non-corrosive to the skin.


Eye irritation (in-vitro). Key Study. Test method according to OECD TG 438, GLP study. Under the test conditions, the combination of the 3 endpoints was 3 x I. Therefore, the test item is not irritating to the eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 August 2021 - 02 September 2021
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)
Version / remarks:
2021
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
SkinEthic RHE®
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Justification for test system used:
The SkinEthic RHE® model has been validated for irritation testing (Validation study based on the original ECVAM Performance Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE®
- Tissue batch number(s): 21-RHE-138 (living) and 21-RHE-104 (killed)
- Shipping date: 31 August 2021 and 22 June 2021
- Delivery date: 31 August 2021 and 01 September 2021 (defreezing).
- Expiration date: 6 September 2021
- Date of initiation of testing: 01 September 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS (Dutscher - Batch No. 7380521)
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

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 and 02 minutes at 37°C, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
- Filter: no
- Linear OD range of spectrophotometer: not specified.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.3 (CV = 6.4%), specification OD > 0.7
- Barrier function: > 10.0h (pecification 4.0 ≤ ET50 ≤ 10.0h); ET50 value was out of specification but declared satisfactory by dispensation. Quality of the RHE batch is good according to 2 further QC tests (normal values), especially histology showing good morphology.
- Morphology: 6.5 cell layers (specification ≥ 4); multi-layered, highly differentiated epidermis consisting of organized basal, spinous and granular layers, and a multilayered stratum corneum.
- Contamination: no, on the blood of the donors and the cells from the donors, absence of contamination was verified.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- 2 killed tissues
- Procedure used to prepare the killed tissues (if applicable): 2 killed Human skin model surfaces were treated in the same manner as the living tissues in order to generate non-specific MTT reduction.
- N. of replicates: 2
- Method of calculation used: True viability % = [(living tissues OD exposed to test item - killed tissues OD exposed to test item)/living tissues OD exposed to negative control] x 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin (or corrosive) if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure and 42 hours of post-treatment incubation is greater than 50%.
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 (32 mg/cm2) (+10 μL distilled water)
- Vehicle: no

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL of distilled water (ADL Prochilab, Batch No. 201117)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL of 5% SDS.
- Concentration (if solution): 5% SDS solution was prepared by weighing 0.5000 g of SDS (SIGMA Batch No. STBJ3028) in a 10 mL volumetric flask q.s. 10 mL of distilled water
Duration of treatment / exposure:
42 min
Duration of post-treatment incubation (if applicable):
41 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean (42 min exposure)
Value:
94.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% viability (distilled water)
Positive controls validity:
valid
Remarks:
1.8% viability (5% SDS)
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes (relevant control added)
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was
performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model.
Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. Mean OD of the tissue replicates treated with
the negative control was in the range ≥ 0.8 and ≤ 3.0.
- Acceptance criteria met for positive control: yes. Mean viability of the tissue replicates treated
with the positive control (SDS 5%), expressed as % of the negative control was < 40%
- Acceptance criteria met for variability between replicate measurements: yes. Standard deviation
between tissue replicates was SD ≤ 18%

Table 1. Summary of results.









































































































 



Well ID



OD



Mean OD /        disc (#)



  Mean OD / product



Viability


%



Mean


viability


%



SD


viability



Conclusion



Negative control



SPL 1



0.922


0.941


0.926



0.929



0.902



103.0


100.7



100.0



3.4



 



SPL 2



0.851 0.873


0.884



0.869



SPL 3



0.870 0.934


0.920



0.908



Positive control



SPL 4



0.018 0.016


0.018



0.017



0.016



1.9


1.8



1.8



0.1



Irritant



SPL 5



0.016 0.015


0.015



0.015



SPL 6



0.016 0.016


0.016



0.016



Test item PH- 21/0781



SPL 7



0.969 0.996


0.917



0.960



0.862



106.4


87.8



95.5



9.7



 



SPL 8



0.861 0.836


0.803



0.833



SPL 9



0.824 0.773


0.781



0.792



Test item


PH- 21/0781


NSMTT



SPL 10



0.007 0.008


0.008



0.007



0.007



0.8


0.7



0.7



0.1



SPL 11



0.006 0.007


0.007



0.006



Test item


PH- 21/0781 Corrected



 



 



 



 



94.8



 



Non irritant


Interpretation of results:
GHS criteria not met
Remarks:
Non-irritant to skin, no category (CLP Regulation EC no. 1272/2008)
Conclusions:
Under test conditions, the mean percent viability of the treated tissues was 94.8% versus 1.8% with the positive control item (5% SDS). Therefore, the test item is not irritant to the skin.
Executive summary:

An in vitro skin irritation test was performed with the test item in a reconstructed human epidermis SkinEthic RHE® model, according to OECD TG 439, under GLP conditions.


Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. Exposure to the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol for 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Distilled water was used as a negative control and 5% SDS solution as positive control; two killed control tissue models were added to the study which underwent the entire testing procedure to generate a non-specific MTT reduction control.


Under test conditions, the mean percent viability of the treated tissues was 94.8% versus 1.8% with the positive control item (5% SDS). Therefore, the test item must be considered as non-irritant to skin.

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 August 2021 - 03 September 2021
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:
2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
epiCS®
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Justification for test system used:
The use of the epiCS® model is recommended by the relevant OECD guideline; therefore, it was considered to be suitable for this study. Further, the model has been validated for corrosion testing (Validation study based on the statement issued by the ECVAM Scientific Advisory Committee (ESAC30) on 12 June 2009).
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS®
- Tissue batch number(s): 21-31 (living) and 21-11 (killed)
- Production date: August 2021 (living) and 19 March 2021 (killed)
- Shipping date: 02 September 2021 (living) and 12 April 2021 (frozen 19 April 2021)
- Delivery date: 02 September 2021 (delivery/defreezing)
- Date of initiation of testing: 02 September 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 3 min exposure and 37°C ± 1ºC for 1 hour exposure.
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 20 x 1 mL of DPBS (Dutscher, Batch No. 7380521).
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

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 at 37°C, 5% CO2
- Spectrophotometer: Elx800 absorbance microplate reader (BioTek).
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: OD between 0 and 2.0

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 0.8 ≤ OD ≤ 2.8. Historical negative control mean OD range (measured after 1:3 dilution in isopropanol) = 0.719-1.374 (3 min exposure) and 0.735-1.395 (1 hour exposure).
- Barrier function: 2h 47m (specification 2.0h < ET50< 5.0h)
- Morphology: multi-layered human epidermis-like structure (sufficient nº of cornified layers), absence of significant histological abnormalities, well differentiated epidermis.
- Contamination: no
- Reproducibility: yes

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- 4 killed tissues.
- Procedure used to prepare the killed tissues: four control tissues (two for 3-minute treatment and two for 1-hour treatment) were incubated with culture medium instead of MTT solution during the MTT incubation step to generate a non-specific colour control
- N. of replicates : 2
- Method of calculation used: True viability % = [(living tissues OD exposed to test item - killed tissues OD exposed to test item)/living tissues OD exposed to negative control]x100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
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): 25 mg (41.7 mg/cm2)
- Vehicle: no

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of distilled water (ADL Prochilab, Batch No. 201117)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of 8N KOH (Fisher Scientific, Batch No. A0412420)
- Concentration (if solution): 8N
Duration of treatment / exposure:
3 min and 1 hour
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean (3 min exposure)
Value:
85.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% viability (distilled water)
Positive controls validity:
valid
Remarks:
10.40% viability (8N KOH)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean (60 min exposure)
Value:
93.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100% viability (distilled water)
Positive controls validity:
valid
Remarks:
0.3% viability (8N KOH)
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: yes (relevant control added)
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A demonstration of proficiency was performed for the EpiCS® model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. Mean OD of the tissue replicates should be ≥ 0.8 and ≤ 2.8 for epiCS® model, for every exposure time. As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control. The mean OD of negative control tissues for treatment time 3 min and 1 hour were 0.599 and 0.498.
- Acceptance criteria met for positive control: yes. Mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, should be < 20% for epiCS model. It was 10.4%.
- Acceptance criteria met for variability between replicate measurements: yes. In the range 20-100% viability and for ODs ≥ 0.3, difference of viability between the two tissue replicates should not exceed 30%. Viability difference between replicates % was below 9% for all replicates.

Table 1. Summary of results.































































































 



Well ID



OD



Mean OD /     disc (#)



 Mean OD / product



Viability


%



Mean


viability


%



SD


viability



Viability difference between replicates %



Negative control



SPL 1



0.530


0.618


0.642



0.596



0.599



99.5



100.0



0.7



1.0



SPL 2



0.617 0.618


0.571



0.602



100.5



Positive control



SPL 3



0.040 0.035


0.034



0.036



0.062



6.0



10.4



6.1



8.7



SPL 4



0.076 0.099


0.089



0.088



14.7



Test item PH-21/0781



SPL 5



0.483 0.522


0.523



0.509



0.521



85.0



86.9



2.7



3.8



SPL 6



0.484 0.526


0.588



0.532



88.8



PH-21/0781 NSMTT



SPL 7



0.007 0.007


0.006



0.006



0.006



1.0



1.0



0.0



0.0



SPL 8



0.006 0.006


0.007



0.006



1.0



PH-21/0781 corrected



 



 



 



 



85.9



 



 



 


Table 2. Summary of results































































































 



Well ID



OD



Mean OD /     disc (#)



 Mean OD / product



Viability


%



Mean


viability


%



SD


viability



Viability difference between replicates


%



Negative control



SPL 9



0.492


0.505


0.506



0.501



0.498



100.6



100.0



0.9



1.2



SPL 10



0.495 0.499


0.493



0.495



99.4



Positive control



SPL 11



0.000 0.001


0.001



0.000



0.002



0.0



0.3



0.4



0.6



SPL 12



0.003 0.003


0.003



0.003



0.6



Test item PH-21/0781



SPL 13



0.474 0.481


0.434



0.463



0.473



93.0



94.9



2.7



3.8



SPL 14



0.467 0.532


0.447



0.482



96.8



PH-21/0781 NSMTT



SPL 15



0.006 0.007


0.006



0.006



0.007



1.2



1.3



0.1



0.2



SPL 16



0.007 0.007


0.008



0.007



1.4



PH-21/0781 corrected



 



 



 



 



93.6



 



 



 


Table 3. Assessment.





























 



 



Mean viability (%)



 



3 min exposure



1 hours exposure



Conclusion



Positive control



10.4



0.3



Category 1A



PH-21/0781



85.9



93.6



Non Corrosive


Interpretation of results:
study cannot be used for classification
Remarks:
not classified as corrosive (Cat 1) (CLP Regulation EC no. 1272/2008)
Conclusions:
Under test conditions, the mean percent viabilities of the treated tissues were 85.9% and 93.6% for 3 and 60 min exposure, versus 10.4% and 0.3% with the positive control item, respectively. Therefore, the test item is not corrosive to the skin.
Executive summary:

An in vitro skin corrosion test was performed with the test item in a reconstructed human epidermis EpiCS® model, according to OECD TG 431, under GLP conditions.


Two epidermis units were treated with 25 mg test item for 3 minutes at room temperature and for 1 hour, at 37°C, 5% CO2. Exposure to the test item was terminated by rinsing with 20 x 1 mL of DPBS. The viability of each disk was assessed by incubating the tissues with MTT for 3 hours, extracting the precipitated formazan crystals using isopropanol during 16.5 hours under agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:3 in isopropanol.


Under test conditions, the mean percent viabilities of the treated tissues were 85.9% (for 3 min exposure) and 93.6% (for 60 min exposure) versus 10.4% and 0.3%, respectively, with the positive control item (potassium hydroxide 8N). Therefore, the test item must be considered as non-corrosive to 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:
09 August 2021
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)
Version / remarks:
2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: licensed slaughterhouse, Etablissement Brun, 33820 Etauliers, France.
- Characteristics of donor animals (e.g. age, sex, weight): healthy approximately 7-week-old chickens used for human consumption; their body weights ranging from 1.5 - 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): intact heads were transported to the laboratory at ambient temperature, in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The heads were collected on 09 August 2021 at 8:14 am and they were enucleated at Laboratoire ICARE – Site de Martillac on 09 August 2021 at 10:36 am.
- Indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: no
- Selection and preparation of corneas: 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 > 0.5; (ii) corneal opacity > 0.5; or, (iii) any additional signs of damage were replaced. For eyes that were 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 were rejected.
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 of test item
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
No post-treatment incubation was performed.
Number of animals or in vitro replicates:
3
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 31.8°C and 31.9°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
Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated in the superfusion apparatus between 45 and 65 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

NEGATIVE CONTROL USED: 30 μL physiological saline (Dutscher Batch No. C0543A01).

POSITIVE CONTROL USED: 30 mg sodium hydroxide (Fisher Scientific, Batch No. 0000080257).

APPLICATION DOSE AND EXPOSURE TIME: 30 mg test item were applied for 10 seconds.

OBSERVATION PERIOD: Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.
- Indicate any deviation from test procedure in the Guideline: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.
- Damage to epithelium based on fluorescein retention: mean fluorescein retention value for all test eyes was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.
- Swelling: measured with optical pachymeter on a slit-lamp microscope (Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I); slit-width setting: at 9½, equaling 0.095 mm.
- Macroscopic morphological damage to the surface: The aim of this evaluation was to determine whether any “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea were visible.These findings can vary in severity and may occur simultaneously.

SCORING SYSTEM:
- Mean corneal swelling (%) = [(corneal thickness measurement at time t - corneal thickness at time=0) / corneal thickness at time =0] x 100

- Mean maximum opacity score:
0 - No opacity,
0.5 - Very faint opacity
1 - Scattered or diffuse areas; details of the iris clearly visible
2 - Easily discernible translucent area; details of the ris are slightly obscured,
3 - Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely
discernible
4 - Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment
0 - No fluorescein retention,
0.5 - Very minor single cell staining,
1 - Single cell staining scattered throughout the treated area of the cornea,
2 - Focal or confluent dense single cell staining,
3 - Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: The decision criteria indicated in the TG was used.
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
maximum
Value:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
morphological effects
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no. No morphological effects were noted, whatever the examination time.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. 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.
- Acceptance criteria met for positive control: yes. The combination of the three endpoints for the positive control, Sodium Hydroxyde, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

Table 1. Results for test item


























































































































































































Endpoint measured



Eye No.



Time (min)



-45



30



75



120



180



240



Corneal opacity



13



0



0



0.5



1



1



1



14



0



0



0



0



0



0



15



0



0



0



0.5



0.5



0.5



Mean



0.0



0.0



  0.2



0.5



0.5



0.5



ICE class



I



Fluorescein retention



13



0.5



0.5



-



-



-



-



14



0.5



0.5



-



-



-



-



15



0.5



0.5



-



-



-



-



Mean



0.5



0.5



-



-



-



-



ICE class



 



I



-



-



-



-



Corneal thickness



13



0.52



0.54



0.54



0.54



0.54



0.54



14



0.53



0.54



0.54



0.55



0.55



0.55



15



0.54



0.56



0.58



0.58



0.58



0.58



Corneal swelling (%)



13



-



4



4



4



4



4



14



-



2



2



4



4



4



15



-



4



7



7



7



7



Mean



-



3



4


5

5



5



ICE class



 



I



Combination of the 3 Endpoints



 



3 x I



CLASSIFICATION



 



No category



 


Table 2. Results for the positive control. 






























































































































































































Endpoint measured



Eye No.



Time (min)



 



 



-45



30



75



120



180



240



Corneal opacity



1



0



4



4



4



4



4



2



0



4



4



4



4



4



3



0



4



4



4



4



4



Mean



0.0


4.04.04.04.0

4.0



ICE class



-



IV



 



Fluorescein retention



1



0.5



3



-



-



-



-



2



0.5



3



-



-



-



-



3



0.5



3



-



-



-



-



Mean



0.5



3.0



-



-



-



-



ICE class



-



IV



-



-



-



-



Corneal thickness



1



0.49



0.70



0.72



0.82



0.85



0.85



2



0.52



0.66



0.84



0.96



0.96



0.96



3



0.50



0.72



0.82



0.96



0.97



0.97



Corneal swelling (%)



1



( - )



43



47



67



73



73



2



( - )



27



62



85



85



85



3



( - )



44



64



92



94



94



Mean



-


38578184

84



ICE class



-



IV



Combination of the 3 Endpoints



 



3 x IV



CLASSIFICATION



 



Category 1 : "Corrosive/Severe Irritant"



Table 3. Results for the negative control.


















































































































Endpoint measured



Eye No.



Time (min)



-45



30



75 



120



180



240



Corneal opacity



16



0



0



0



0



0



0



Mean



0.0



0.0



 0.0



0.0



0.0



0.0



ICE class



I



Fluorescein retention



16



0.5



0.5



 



-



-



-



Mean



0.5



0.5



-



-



-



-



ICE class



 



I



-



-



-



-



Corneal thickness



16



0.52



0.52



0.52



0.52



0.52



0.52



Corneal swelling (%)



16



-



0



0



0



0



0



Mean



-



0



  0 



0



0



0



ICE class



 



I



Combination of the 3 Endpoints



 



3 x I



CLASSIFICATION



 



No Category


Interpretation of results:
GHS criteria not met
Remarks:
Not irritant (CLP Regulation EC no. 1272/2008)
Conclusions:
Based on this in vitro eye irritation study, the combination of the 3 endpoints was 3 x I. Therefore, the test item is not eye-irritant.
Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye-damaging effects of the test item according to the OECD 438 (Isolated Chicken Eye), under GLP conditions.


Eyeballs were isolated from chickens killed for human consumption, the test system was equilibrated and zero reference measurements were taken. Three groups of three eyeballs each were exposed to either 30 mg test item, 30 mg sodium hydroxide (positive control), or 30 μL physiological saline (negative control). After 10 seconds of exposure, all eyeballs were rinsed with 20 mL of physiological saline at ambient temperature and examined. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 45, 30, 75, 120, 180, and 240 minutes post-dose to assign an ICE class.


The results for the test item treated eyes showed mean fluorescein retention of 0.5 (ICE class I), maximum mean corneal opacity of 0.5 (ICE class I), and a maximal mean corneal swelling of 5.0 % (ICE class I). Concurrent positive and negative control values fell within the acceptable ranges for the method. Gross examinations did not exhibit any changes of the treated test item corneal surface.


Based on these results, the combination of the 3 endpoints was 3 x I. Therefore, the test item does not have any negative effects on the chicken cornea and is deemed not irritating.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available data, the substance is not classified as irritating according to CLP Regulation no. 1272/2008.