Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irriation/ corrosion


Key Study: Method according to OECD 439, GLP Study. The test item has to be considered as Non-irritant to skin corresponding to UN GHS No Category.


 


Eye Irritation:


Key study: Method according to OECD 438, GLP conditions. The test item was determined to not cause severe damage or irrritation for eyes in the ICE test.

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:
25 August 2020 to 15 October 2020
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
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
Episkin SA, RHE/S/17 Batch No. 20-RHE-133
Source species:
human
Cell type:
other: reconstructed epidermis of normal human keratinocytes
Cell source:
other: Foreskin, number of donors not specified
Source strain:
other: BATCH 20-RHE-133
Justification for test system used:
EpiSkin (VRM) is specified in the OECD Guidelines 439 and has undergone a validation study from 2003 to 2007 and was used to develop the original PS. Thus it was considered a adequate in vitro test system
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE/ Human Epidermis RHE/S/17
- Tissue batch number(s): 20-RHE-133
- Production date: 13 October 2020
- Shipping date: 13 October 2020
- Delivery date: 13 October 2020
- Date of initiation of testing: 13 October 2020

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

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

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: 3hours and 4 minutes
- Spectrophotometer: ELx800 absorbance microplate reader BioTek, Software Gen5 ELISA V1.05.11, BioTek.
- Wavelength: 570nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: mean viability OD 1.3 (specification O.D. > 0.7)
- Barrier function: 7.4 h (specification 4.0h≤ET50 ≤ 10.0h)
- Morphology: 6.5, basal, spinous, granular layers and a multilayerd stratum corneum
- Contamination: no
- Reproducibility: Yes

NUMBER OF REPLICATE TISSUES: 3

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 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” OR if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.
- The test substance is considered to be non-corrosive to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
other: NSC living control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg


NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% sodium dodecyl sulfate

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
41 hours and 50 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
91.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Mean viabilityh 100%
Positive controls validity:
valid
Remarks:
Mean viability: 1.9% Conclusion: Irritant
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: In 2019 the lab conducted more than 60 studies with different test items according to OECD 439 guideline and the same test system, prooving their technical proficiency

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, OD value of the 3 replicates in the range ≥ 0.8 and ≤ 3.0. The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control. Mean OD of negative control is 0.948
- Acceptance criteria met for positive control: Yes Mean Viability < 40%, and SD value of the % viability ≤ 18%. Viability of positive control is 1.9%
- Acceptance criteria met for variability between replicate measurements: Yes, SD ≤ 18% SD of negative 8,8, positive control 0.3 and test item 14.3
- Range of historical values if different from the ones specified in the test guideline: The values were in range of historical data of the facility







































































































 



Well ID



OD



Mean OD /   disc (#)



Mean OD / product



Viability


%



Mean


viability


%



SD



Conclusion



Negative control



SPL 1



0.862 0.863


0.867



0.864



0.948



91.2


108.7


100.1



100.0



8.8



 



SPL 2



1.052 1.030


1.010



1.030



SPL 3



1.041 0.944


0.862



0.949



Positive control



SPL 4



0.017 0.017


0.020



0.018



0.018



1.9


1.7


2.2



1.9



0.3



Irritant



SPL 5



0.016 0.017


0.016



0.016



SPL 6



0.022 0.021


0.021



0.021



Test item


PH-20/0589



SPL 10



1.136 1.010


0.920



1.022



0.871



107.8


80.3


87.5



91.9



14.3



 



SPL 11



0.834 0.718


0.733



0.761



SPL 12



0.902 0.724


0.862



0.829



Test item


 PH-20/0589


NSC living control



SPL 18



0.003 0.003


0.004



0.003



0.003



0.3


0.3



0.3



0.0



SPL 19



0.004 0.004


0.003



0.003



Test item


 PH-20/0589 corrected



 



 



 



 



91.6



 



Non irritant


Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Remarks:
not irritating according to EU criteria
Conclusions:
The test item is not irritant to skin
Executive summary:

The skin irritating properties of the test item were determined according to OECD 439 under GLP conditions. The test item was directly applied to EpiSkin SkinEthic RHE/Human Epidermis RHE/S/17 consisting of 6.5 cell layers (basal, spinous and granular layers and a multilayered stratum corneum). After initial exposure of three replicates with the test item of 42 minutes the test system was washed 25 times with 1ml DPBS and subseqently incubated for 41 hours and 50 minutes @ 37C and 5% CO2. Following contact with MTT solution the cell viability was assessed by extracting the formazan crystal from the tissue and its optical density measured. Preliminary testing showed that the test item causes colour interference and thus, next to a positive control (SDS 5%) and a negative control (Aq.dest) a NSC living control with the test item underwent the entire test procedure but not incubated with MTT to generate a non-specific colour control. The positive and negative control all fullfilled the acceptability criteria. The SD of all samples fell into the range of ≤ 18% and thus fullfilled the acceptability criteria. The test item has to be considered as Non-irritant to skin corresponding to UN GHS No Category.

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:
31 August 2020
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:
2 hrs 20 minutes start of enucleation-selection instead of 2 hours. As the positive and negative control conformed within expectation, this deviation is considered to not impact the result.
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Etablissement Brun, 33820
Etauliers, France) where they are killed for human consumption
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): approx 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): Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 31 August 2020 at 8:15 am. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated at Laboratoire ICARE –Site de Martillac on 31 August 2020 at 9:50 am.
- Time interval prior to initiating testing: 2 hours 20 minutes
- Indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: not reported
- Selection and preparation of corneas: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 at 32.0°C.
- Quality check of the isolated 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 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
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): 30mg


Duration of treatment / exposure:
10 seconds
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 at 32.0°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 between 45 and 61 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
physiological saline – Dutscher Batch No. 3014011

POSITIVE CONTROL USED
sodium hydroxide – Fisher Scientific, Batch No. 1550248

APPLICATION DOSE AND EXPOSURE TIME
30mg for 10 seconds

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: rinsed twice with 10ml of physiological saline at ambient temperature
- Indicate any deviation from test procedure in the Guideline: The start of the enucleation-selection was carried out 2 hours and 20 minutes after the collection of the heads instead of 2 hours at the maximum, as initially scheduled.As the results obtained with the eyes treated with the negative control and the positive control were conformed to what was expected, this deviation is considered as without impact on the results of the study.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was 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: The 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; 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 thickness at time = 0)/corneal thickness at time = 0] x 100
The mean percentage of corneal swelling for all test eyes was calculated for all observation time
points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.
- Macroscopic morphological damage to the surface: Morphological effects 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. The classification of these findings is subjective according to the interpretation of the investigator.
- Others (e.g, histopathology):

SCORING SYSTEM:
- Mean corneal swelling [(corneal thickness at time t - corneal thickness at time = 0)/corneal thickness at time = 0] x 100
The mean percentage of corneal swelling for all test eyes was calculated for all observation time
points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item.

- Mean maximum opacity score
See table 1
- Mean fluorescein retention score at 30 minutes post-treatment
See table 2


DECISION CRITERIA: Decision criteria was used as indicated in the TG
Irritation parameter:
cornea opacity score
Run / experiment:
Maximal mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class 1
Irritation parameter:
fluorescein retention score
Run / experiment:
mean score
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class I
Irritation parameter:
percent corneal swelling
Run / experiment:
maximal mean
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class I
Irritation parameter:
morphological effects
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, historical lab data records

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 hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

Table 7





































































































 



 



 



 



Time (min)



 



 



Endpoint measured



Eye No.



0



30



75



120



180



240



Corneal opacity



13



0.0



0.0



0.0                       0.0



0.0



0.0



ICE class



 



 



I



 



 



Fluorescein retention



13



0.5



0.5



-                            -



-



-



ICE class



 



I



-                            -



-



-



Corneal thickness



13



0.50



0.50



0.50                    0.50



0.50



0.50



Corneal swelling (%)



13



-



0



0                           0



0



0



ICE class



 



 



I



 



 



Combination of the 3 Endpoints



 



 



3 x I



 



 



CLASSIFICATION



 



 



No category



 



 



Table 8


































































































 



 



 



Time (min)



 



Endpoint measured



Eye No.



0



          30                   75



         120                 180



240



Corneal opacity



1


2


3



0


0


0



           4                     4                      4                     4


           4                     4                      4                     4


           4                     4                      4                     4



4


4


4



Mean



0.0



         4.0                  4.0                  4.0                  4.0



4.0



ICE class



 



IV



 



Fluorescein retention



1


2


3



0.5 0.5


0.5



           3                      -                      -                      -


           3                      -                      -                      -


           3                      -                      -                      -



-


-


-



Mean



0.5



3.0



           -                       -                      -



-



ICE class



 



          IV                     -                      -                      -



-



Corneal thickness



1


2


3



0.47 0.47


0.46



-                                  -           -              -


-                                  -           -              -


-                                  -           -              -



-


-


-



Corneal swelling (%)



1


2


3



( - )


( - )


( - )



         ( - )                 ( - )                 ( - )                 ( - )


         ( - )                 ( - )                 ( - )                 ( - )


         ( - )                 ( - )                 ( - )                 ( - )



( - )


( - )


( - )



Mean



-



           -                       -                      -                      -



-



ICE class



 



IV



 



Combination of the 3 Endpoints



 



3 x IV



 



CLASSIFICATION



 



category 1 : "Corrosive/severe irritant"



 



Table 9





























































































































 



 



 



 



Time (min)



 



 



Endpoint measured



Eye No.



0



30



75



120



180



240



Corneal opacity



7


8


9



0


0


0



0


0


0



            0                        0


            0                        0


            0                        0



0


0


0



0


0


0



Mean



0.0



0.0



           0.0                     0.0



0.0



0.0



ICE class



 



 



I



 



 



Fluorescein retention



7


8


9



0.5 0.5


0.5



0.5 0.5


0.5



-                                      -


-                                      -


-                                      -



-


-


-



-


-


-



Mean



0.5



0.5



            -                         -



-



-



ICE class



 



I



            -                         -



-



-



Corneal thickness



7


8


9



0.50 0.46


0.49



0.50 0.48


0.50



0.50               0.50 0.48      0.49


          0.50                   0.50



0.50 0.49


0.50



0.50 0.49


0.50



Corneal swelling


(%)



7


8


9



-


-


-



0


4


2



            0                        0


            4                        7


            2                        2



0


7


2



0


7


2



Mean



-



2



            2                        3



3



3



ICE class



 



 



I



 



 



Combination of the 3 Endpoints



 



 



3 x I



 



 



CLASSIFICATION



 



 



No category



 



 



 

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The test item was determined to not cause severe damage or irrritation for eyes in the ICE test.
Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effectsof the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hidroxide (positive control) or 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. According to CLP Regulation EC no. 1272/2008 the test item does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No category), since the combination of the 3 endpoints for the test item was 3x I. The test item was determined to not cause severe damage or irrritation for eyes in the ICE test.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation: The skin irritating properties of the test item were determined according to OECD 439 under GLP conditions. The test item was directly applied to EpiSkin SkinEthic RHE/Human Epidermis RHE/S/17 consisting of 6.5 cell layers (basal, spinous and granular layers and a multilayered stratum corneum). After initial exposure of three replicates with the test item of 42 minutes the test system was washed 25 times with 1ml DPBS and subseqently incubated for 41 hours and 50 minutes @ 37C and 5% CO2. Following contact with MTT solution the cell viability was assessed by extracting the formazan crystal from the tissue and its optical density measured. Preliminary testing showed that the test item causes colour interference and thus, next to a positive control (SDS 5%) and a negative control (Aq.dest) a NSC living control with the test item underwent the entire test procedure but not incubated with MTT to generate a non-specific colour control. The positive and negative control all fullfilled the acceptability criteria. The SD of all samples fell into the range of ≤ 18% and thus fullfilled the acceptability criteria. The test item has to be considered as Non-irritant to skin corresponding to UN GHS No Category.


 


Eye irritation:


An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effectsof the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hidroxide (positive control) or 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. According to CLP Regulation EC no. 1272/2008 the test item does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No category), since the combination of the 3 endpoints for the test item was 3x I. The test item was determined to not cause severe damage or irrritation for eyes in the ICE test.

Justification for classification or non-classification

Skin irritation: Based on the available information (91.9 % tissue viability), the test item is not classified for skin irritation/corrosion, in accordance with CLP Regulation (EU) No. 1272/2008.


Eye irritation: Based on available information (OECD 438 3x ICE class I) the substance does not cause irritation or serious eye damage according to CLP Regulation (EC) No. 1272/2008.

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