[No public or meaningful name is available]

Endpoint summary

Description of key information

Skin irritation: In vitro skin irritation (OECD 439, GLP)

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after exposure to the test article. The protocol was based upon the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439).

According to the prediction model presented in OECD TG 439, the test article was predicted to be an
irritant since the treated tissues resulted in a mean tissue viability of 2.88%. Additional testing would be needed to determine if the test article was a GHS Category 1 (Severe/Corrosive) or GHS Category 2 (Irritant) for skin irritation.

 

Skin corrosion: In vitro skin corrosion (OECD 431, GLP)

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential skin corrosivity of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after test article exposure. The protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”. According to the prediction model presented above, the test article was considered to be non-corrosive.

 

Eye irritation: In vitro eye irritation (OECD 437, GLP)

 

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test article to isolated bovine corneas. Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test article. An In Vitro Score was determined for the test article based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas. The methods and procedures used in this assay were consistent with OECD Test Guideline 437: Bovine Corneal Opacity and Permeability Test Method for
Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2020).

 

The In Vitro Score of the test article was 11.3. According to the prediction model presented in OECD TG 437, a prediction for eye irritation potential could not be determined. Additional testing would be warranted for definitive classification of eye irritation potential for labeling purposes.

Eye irritation: In vitro eye irritation (OECD 492, GLP)

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

The test article resulted in a viability of 64.03% relative to the negative control. According to the prediction model presented in OECD 492, the test article is predicted to not require classification or labelling for ocular irritation (GHS No Category).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 July to 04 November 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

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:

The EpiDerm Model (EPI-200)

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The EpiDerm™ Model incorporates several features which make it advantageous in the study of potential dermal corrosivity. First, the test system uses a serum-free medium which eliminates the possibility of serum protein and test article interaction (Shopsis and Eng, 1988}. Secondly, the target cells are epithelial, derived from human skin (Cannon et al., 1994}. Third, since the tissue has a functional stratum corneum, the test materials are applied directly to the tissue surface, at air interface, so that undiluted and/or end use dilutions can be tested directly (Harbell et al., 1994). Prior to use, each 6-, 24- and 96-well plate will be uniquely identified with a number written in permanent
marker with the test substance identification or control treatment group.

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDerm Model (EPI-200) (MatTek Corporation, Ashland, USA) consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipud layers arranged in patterns analogous to those found in vivo.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was used as supplied, no vehicle used.

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

3 hours in an MTT dye solution to determine the degree of cytotoxicity (cell death) caused by exposure to the test material.

Number of replicates:

The test and control articles will be tested by treating 4 EpiDerm tissues per material.
Two tissues will be used to assess viability after the 3-minute exposure and two will be used to assess viability after the 60-minute exposure.

Details on study design:

The test and control substances will be tested by treating 4 EpiDerm™ tissues per material. Two tissues will be used to assess viability after the 3-minute exposure and two will be used to assess viability after the 60-minute exposure. Fifty microliters (50 μl) of control or liquid test substances or approximately 25 mg of solid test substances (measured using a 25 mg sharp spoon) will be applied to the apical side of the EpiDerm™ tissue. Each EpiDerm™ tissue treated with a solid test substance will receive 25 μl of sterile, deionized water applied directly onto the test substance. The 3-minute exposure time will begin as soon as the test substance is spread
onto the tissue (either directly or with the dosing device). The 3-minute exposure groups will be held at room temperature during the treatment incubation, while the 60-minute exposure groups will be placed in the incubator at standard culture conditions during treatment.
A 10X stock of MTT prepared in PBS (filtered at time of batch preparation) will be thawed and diluted in warm MTT Addition Medium to produce a 1.0 mg/ml solution and used within 2 hours. Three hundred microliters of the MTT solution will be added to each designated well of a labelled 24-well plate.
At the end of the test substance incubation period, each tissue will be rinsed and transferred to the MTT solution. Rinsing will be accomplished using warmed (approximately 37°C sterile CMF-DPBS delivered from a plastic squeeze bottle. The tip of the bottle shall be cut so as to enlarge the orifice to at least 3 mm (interior diameter) that will allow a gentle stream of CMF-OPBS to be delivered to the surface of the tissue.
After the appropriate exposure time, each EpiDerm™ tissue will be thoroughly rinsed on both sides of the tissue insert with CMF-DPBS. The CMF-DPBS will be decanted and the cell culture insert will be blotted on sterile paper towels. The EpiDerm™ tissues will be
transferred to the appropriate wells on the labelled 24-well MTT plate after rinsing. The 24-well plates will be incubated at standard culture conditions for 3 ± 0.1 hours. After 3 ± 0.1 hours, the cell culture insert will be removed from the MTT solution, the bottom blotted on sterile paper towels, cleared of excess liquid, and the cell culture insert transferred to a labelled 24-well plate containing 2.0 ml of isopropanol in each designated well. The plates will be covered with paraffin film and stored in the refrigerator (2 to 8 °C) until all of the tissues have been placed into the isopropanol. If necessary, the plate may be sealed and stored overnight before the extraction step is begun.
To extract the reduced MTT, the plates will be shaken for 2 to 3 hours at room temperature. The shaking should be sufficiently vigorous to move the isopropanol around the cell culture insert. At the end of the extraction period, the liquid within the cell culture inserts will be decanted into the well from which the cell culture insert was taken. The extracted MTT solution will be mixed and 200 μL will be
transferred to the appropriate wells of a labelled 96-well plate. Two hundred microliters of isopropanol will be added to the 2 wells designated as blanks. The absorbance at 570 nm (OD570) of each well will be measured with a Molecular Devices VersaMax plate reader with the shaking function selected.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

After 3 minutes

Value:

118.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

After 3 minutes: 9.58% +/- 13.55%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

After 60 minutes

Value:

88.88

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

After 60 minutes: 6.28% +/- 0.90%

Other effects / acceptance of results:

The assay was accepted since:
1. the positive control resulted in a corrosive classification (i.e., < 50% cell viability compared to negative controls, after a 3‐minute exposure and/or < 15% cell viability compared to negative controls after a 60‐minute exposure)
2. the mean OD570 value of the negative control tissues was ≥ 0.8 and ≤ 2.8. The mean OD570 value of the tissues treated with the negative control at 3‐ and 60‐minutes was 2.216.
3. in the range of 20 to 100% viability, the Coefficient of variation (CV) between 2 identically treated replicates of the negative and positive control per each exposure time are ≤ 30%.

The results were evaluated according to the prediction model presented in OECD TG 431 (2019) in the table below. A test article was determined to be Corrosive or Non-Corrosive using the 3- and/or 60-minute exposures; and when corrosive, an optional sub-classification may be determined using the % relative viability after the 3-minute exposure as presented below:

Viability after exposure times (3 & 60 minutes)	Prediction (OECD TG 431)	Viability after exposure time (3 minutes)	Optional Sub-Classification
< 50% after 3-minutes	Corrosive	<25% after 3-minutes	Sub-category 1A*
≥ 50% after 3-minutesAND < 15% after 60-minutes		≥25% after 3-minutes	Combination of Sub-categories 1B & 1C
≥ 50% after 3-minutesAND ≥ 15% after 60-minutes	Non-corrosive	Not Applicable	Not Applicable

 

Summary of results for the skin corrosion assay:

Test Article Number	Sponsor’s Designation	Conc.	Exposure Time	% Viability	*Corrosive?	Subcategory	pH
22AA19	GR-89-1114 (Rosabloom)	Neat	60 minutes 3 minutes	88.88 +/- 1.06 118.30 +/- 10.95	Non-corrosive	NA	4.5
Positive Control	8N KOH	Neat	60 minutes 3 minutes	6.28 +/- 0.90 9.58 +/- 13.55	Corrosive	1A

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

According to the prediction model presented above, the test article was considered to be non-corrosive.

Executive summary:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential skin corrosivity of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after test article exposure. The protocol is consistent with the OECD Test Guideline 431 “In Vitro Skin Corrosion: Human Skin Model Test”. According to the prediction model presented above, the test article was considered to be non-corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 28 January to 30 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See 'Any other information on materials and methods incl. tables' section

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiDerm'M Model (EPl-200) (MatTek Corporation, Ashland, USA) consists of normal, humanderived epidermal keratinocytes which have been cultured to form a multilayered, highly
differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm'M Model incorporates several features which make it advantageous in the study of potential dermal toxicity. First, the test system uses a serum-free medium which eliminates the possibility of serum protein and test article interaction (Shopsis and Eng, 1988). Secondly, the target cells are epithelial, derived from human skin. Third, since the tissue has a functional stratum corneum, the test materials are applied directly to the tissue surface, at air interface, so that undiluted and/or end use dilutions can be tested directly. Prior to use, each 6-, 24- and 96-well plate will be uniquely identified with a number written in permanent marker with the test article identification or control treatment group.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Negative control: Neat (undiluted)
Positive control: 5%
Test item: Neat (undiluted)

Duration of treatment / exposure:

60 +/- 1 minutes

Duration of post-treatment incubation (if applicable):

42 +/- 2 hours post-exposure incubation period

Number of replicates:

Negative control: 3
Positive control: 3
Test item: 3

Irritation / corrosion parameter:

% tissue viability

Value:

2.88

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The assay was accepted since the following criteria were met:
1) the positive control (5% SDS) resulted in a mean tissue viability <20% (2.63%);
2) the mean OD570 value of the negative control tissues (1.637) was >= 0.8 and <= 2.8;
and 3) the standard deviation of the positive and negative control calculated from individual percent tissue viabilities of the three identically treated replicates were <= 18% (6.63% for the negative control and 0.19% for the positive control).

The skin irritancy potential of the test article was evaluated as described in OECD TG 439:

In VitroResult	GHS Category
mean tissue viability ≤ 50%	Category 1 or 2*
mean tissue viability > 50%	No Category

* - Additional testing would be warranted to resolve between GHS Category 1 (Severe/Corrosive) and GHS Category 2 (Irritant)

 

Test results:

Test Article Number	Sponsor’s Designation	Conc. (w/v)	pH	Mean Viability (%)	Skin Irritation Prediction
22AA20	GR-87-6331 (Scentaurus Melrose)	Neat	5.0	2.88 +/- 0.28	Irritant
Positive Control	SDS	5%	NA	2.63 +/- 0.19	Irritant

Interpretation of results:

study cannot be used for classification

Conclusions:

According to the prediction model presented in OECD TG 439, the test article was predicted to be an
irritant since the treated tissues resulted in a mean tissue viability of 2.88%. Additional testing would be needed to determine if the test article was a GHS Category 1 (Severe/Corrosive) or GHS Category 2 (Irritant) for skin irritation.

Executive summary:

The EpiDerm™ Skin Model (MatTek Corporation, MA, USA) was used to assess the potential dermal irritation of the test article. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to a blue formazan precipitate, was used to assess cellular metabolism after exposure to the test article. The protocol was based upon the OECD guideline, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method” (TG 439).

 

According to the prediction model presented in OECD TG 439, the test article was predicted to be an
irritant since the treated tissues resulted in a mean tissue viability of 2.88%. Additional testing would be needed to determine if the test article was a GHS Category 1 (Severe/Corrosive) or GHS Category 2 (Irritant) for skin irritation.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 01 April to 27 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular™ human cell construct, provided by the MatTek Corporation, will be used in this study. The use of EpiOcular"' cultures offers features appropriate for a model for ocular irritation. First, the model is composed of stratified human keratinocytes in a three-dimensional structure.
Secondly, test materials can be applied topically to the model so that water insoluble materials may be tested. Prior to use, each plate (6-, 12-, and 24-well) and its cover will be uniquely identified in permanent marker with a plate number, the test article number, and the test phase.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL
- Concentration: Undiluted

Duration of treatment / exposure:

30±2 minutes

Duration of post- treatment incubation (in vitro):

- Post-treatment immersion period: 12±2 minutes
- Post-treatment incubation period: 120±15 minutes

Number of animals or in vitro replicates:

Each test article and control were tested by treating at least 2 tissues.

Details on study design:

Assessment of Direct Test Article Reduction of MTT:
Prior to conducting any assays with viable tissues, it is necessary to assess the ability of each test article to directly reduce MTT. A 1.0 mg/ml MTT solution will be prepared.
Approximately 50 μl (liquid test articles) or approximately 50 mg (using dosing spoon or pre-weighed) (solid test articles) will be added to 1.0 ml of the MTT solution and the mixture incubated in the dark at 37 ± 1 °C in a humidified atmosphere of 5 ± 1% CO2 in air (standard culture conditions) for 180 ± 10 minutes. A negative control (SO μL of sterile deionized water) will be run concurrently. If the MTT solution color turns blue/purple, the test article is presumed to have reduced the MTT. Water insoluble test materials may show direct reduction (darkening) only at the interface between the test article and the medium.
In cases where the test article is shown to reduce MTT, a functional check using freeze killed tissues is performed in at least l definitive assay to evaluate whether the test material is binding to the tissue and leading to a false MTT reduction signal.

Assessment of Colored or Staining Materials:
Prior to conducting any assays with viable tissues, it is necessary to assess if the test articles interfere with the photometric MTT measurement. Each test article should be checked for its colorant properties. Approximately 50 μl (liquid test articles) or approximately 50 mg (using one leveled dosing spoon or pre-weighed) (solid test articles) will be added to 2.0 ml isopropanol in 6-well plates and placed on an orbital plate shaker for two to three hours at room temperature. Two x 200 μL aliquots of the isopropanol solutions and 2 blank samples of isopropanol are transferred to a 96-well plate, and the absorbance is measured with a plate reader, with the "shake" function selected, at the MTT measurement wavelength .
Some test articles may precipitate or become cloudy after addition to the isopropanol and interfere with the optical density (OD) measurement. In such cases, after the two to three hour shaking period, the test article-isopropanol mixture may be transferred into centrifuge tubes and centrifuged (in a centrifuge with settings up to 15,000 rpm, for up to S minutes at room temperature) prior to transfer to the 96-well plates for the absorbance measurement. The same centrifugation procedure may be performed after the isopropanol extraction period in the definitive assay.
If, after subtraction of the OD for the blank isopropanol, the OD of the test article solution is > 0.08 the material has to be considered as possibly interfering with the MTT measurement.

pH Determination:
The pH of each liquid test article (and/or dosing dilution as appropriate) will be determined, if possible. The pH will be determined using pH paper (e.g., with a pH range of O to 14 to estimate, and/or a narrower pH range). The typical pH increments on the narrower range pH paper are approximately 0. 3 to 0.5 pH units. The maximum increment on the pH paper is 1.0 pH units.

Receipt of the EpiOcular model:
Upon receipt of the EpiOcular assay materials, the solutions will be stored as indicated by the manufacturer. The tissue will be stored at 2 to 8 °C until used. On the day of receipt, an appropriate volume of EpiOcular™ Assay Medium will be warmed to approximately 37 °C. One (1.0) milliliter of Assay Medium will be aliquoted into the appropriate wells of labelled 6-well plates. The 6-well plates will be labelled with the test article or control codes and exposure times. Each tissue will be inspected for air bubbles between the agarose gel and cell culture insert prior to opening the sealed
package. Cultures with air bubbles greater than 50% of the cell culture insert area will not be used . Each 24-well shipping container will be removed from its plastic bag and its surface disinfected by wiping with 70% ethanol-soaked tissue paper. The tissues will be allowed to come to room temperature for at least 15 minutes prior to transfer to the 6-well plates . An appropriate number of tissues will be transferred aseptically from the 24-well shipping containers into the 6-well plates. The EpiOcular tissues will be incubated at standard culture conditions for 60 ± 5 minutes. After the incubation, the tissues will either be refed, or transferred to new plates containing fresh warmed media and incubated at standard culture conditions overnight (16 to 24 hours) . Upon opening the bag, any unused tissues remaining on the shipping agar at the time of tissue transfer will be briefly gassed with an atmosphere of 5% CO2/95% air, and the bag will be sealed and stored at 2 to 8 °C for subsequent use.

Definitive Assay-Treatment of Liquid Test Articles:
Each test article and control will be tested by treating at least 2 tissues.
An appropriate volume of EpiOcular™ Assay Medium will be warmed to room temperature for conducting the post-soak immersion incubation in 12-well plates and the post-treatment incubation in 6-well plates. After the overnight incubation, the tissues will be pre-treated with 20 μl of sterile Ca++ Mg++ Free-DPBS (CMF-DPBS). If the sterile CMF·DPBS does not spread across the tissues, the plate may be tapped to assure that the entire tissue surface is wetted. The tissues will be incubated at standard culture conditions for 30 ± 2 minutes.
After the 30 ± 2-minute sterile CMF-DPBS pre-treatment, each liquid test and control article will be tested by applying SO μL topically on the EpiOcular™ tissues. The tissues will be incubated at standard culture conditions for 30 ± 2 minutes. At the end of the 30 ± 2-minute treatment time, the test articles will be removed by extensively rinsing the tissues with sterile CMF-DPBS brought to room temperature, as described in detail below. If a dosing device is used, it will be removed and discarded.
Three clean beakers (glass or plastic with 100 ml capacity), containing ~100 ml each of sterile CMF-DPBS will be used per test article (8 oz/220 ml disposable specimen containers from Falcon, Cat# 354020 are suggested). Each test article utilizes a different set of 3 beakers. The cell culture insert containing the tissue will be lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. Use of curved forceps facilitates handling and decanting. To assure throughput, the tissues may be rinsed 2 at a time.
At the end of the exposure period, the test or control articles will be decanted from the tissue surface onto a clean absorbent material (paper towel, gauze, etc.) and the cultures dipped into the first beaker of sterile CMF-DPBS, swirled in a circular motion in the liquid for approximately two seconds, lifted out so that the cell culture inserts are mostly filled with sterile CMF-DPBS, and the liquid will be decanted back into the container. This process will be performed two additional times in the first beaker. The culture will then be rinsed in the second and third beakers of sterile CMF-DPBS 3 times each in the same fashion. Finally, any remaining liquid will be decanted onto the absorbent material. Decanting is most efficiently performed by rotating the cell culture insert to approximately a 45· angle (open end down) and touching the upper lip to the absorbent material (to break the surface tension).

After rinsing, the tissue will be immediately transferred to and immersed in 5 ml of previously-warmed Assay Medium in a labelled 12-well plate for a 12 +/- 2 minute immersion incubation (Post-Soak) at room temperature. This incubation in Assay Medium is intended to remove any test article absorbed into the tissue.
At the end of the Post-Soak immersion, each cell culture insert will be removed from the Assay Medium, the medium will be decanted off the tissue, the insert will be blotted on absorbent material, and transferred to the appropriate well of the labelled 6-well plate containing 1 ml of warm Assay Medium. The tissues will be incubated for 120 :I: 15 minutes at Standard Culture Conditions (Post-treatment Incubation).

MTT Assay:
A 1.0 mg/ml MTT solution will be prepared. Three hundred microliters of the MTT solution will be added to each designated well of a labelled 24-well plate. At the end of the Post-treatment Incubation, each cell culture insert will be removed from the 6-well plate and gently blotted on absorbent material. The tissues will be placed into the 24-well plate containing 0 .3 ml of MTT solution. The t issues will be incubated in the MTT solution for 180 ± 10 minutes at Standard Culture Conditions .
After 180 ± 10 minutes, each cell culture insert will be removed from the plate, the bottom of the insert blotted on absorbent material, and then transferred to a labelled 24-well plate containing 2.0 ml of isopropanol in each designated well. The plates will be sealed with parafilm (between the plate cover and upper edge of the wells) or using a standard plate sealer, and are either stored overnight at 2 to 8 °C in the dark or immediately extracted. To extract the MTT, the plates are placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature.
At the end of the extraction period, the liquid within each cell culture insert will be decanted into the well from which it was taken. The extract solution will be mixed and two x 200 μL aliquots will be transferred to the appropriate wells of a labelled 96-well plate(s). Two hundred microliters of isopropanol will be added to the wells designated as blanks. The absorbance at 550 nm (ODsso) (a range of 540 to 570 nm is acceptable but should be consistent within a laboratory's data set) of each well was measured with a Molecular Devices VersaMax plate reader with the "shake" function selected.

Killed Controls for Assessment of Residual Test Article Reduction of MTT:
In cases where the test article is shown to reduce MTT, only test articles that remain bound to the tissue after rinsing, resulting in a false MTT reduction signal, present a problem. To demonstrate that residual test article is not acting to directly reduce the MTT, a functional check is performed in the definitive assay to show if the test material is not binding to the tissue and leading to a false MTT reduction signal.
To determine whether residual test article is acting to directly reduce the MTT, at least one freeze-killed control tissue per test article is used. Freeze-killed tissue is prepared by placing untreated EpiOcular"' constructs in the -20 •c freezer at least overnight, thawing to room temperature, and then refreezing. Once frozen, the tissue may be stored indefinitely in the freezer. Freeze-killed tissues may be received already prepared from MatTek Corporation. To test for residual test article reduction, at least 1 killed tissue will be treated with the test article in the normal fashion . All assay procedures will be performed as for the viable tissue. A killed control treated with sterile deionized water (negative killed control) will be tested in parallel since a small amount of MTT reduction
is expected from the residual NADH and associated enzymes within the killed tissue. Additional steps must be taken to account for the chemical reduction or the test article may be considered untestable in this system.

Colorant Controls (CC) for Assessment of MTT Interaction:
In cases where a colored test article is shown to interact with the MTT measurement, colorant controls should be run alongside the live tissues. Ideally, colorant control tissues will be tested concurrently in the Definitive Assay; however, in some cases, follow-up testing of colorant controls may be warranted. To determine whether the test article is interfering with the MTT measurement, at least 1 live colorant control tissue per test article is used. The colorant control tissues are treated in the same way as the liquid or solid protocols, with the exception that in contrast to the normal viability test, no MTT assay is performed. The tissues are incubated in standard assay medium instead of MTT for the 180 ± 10-mlnute incubation.

Colorant Control-Killed Control experiment:
In the event that both a killed control experiment and a colorant control experiment are needed, an additional set of controls will be required to avoid a possible double correction for color interference. At least 1 killed control tissue will be treated with the test article using the same procedures as described for the colorant controls for assessment of MTT interaction. The ODsso values from the colorant controls will be analyzed as described below.

Presentation of Data:
The raw absorbance values will be captured, and the following calculations made:
The mean OD550 value of the blank control wells will be calculated. The corrected OD550 values of the negative control will be determined by subtracting the mean OD550 of the blank control from the negative control raw OD550 values. The mean corrected OD550 value of the negative control will be determined. The corrected OD550 values of the individual test article OD550 values and the positive control OD550 values are determined by subtracting the mean OD550 of the blank control from their raw OD550S. Generally, all calculations will be performed using Microsoft Excel. Although the algorithms discussed are performed to calculate the final endpoint analysis at the treatment group level, the same calculations can be applied to the individual replicates .

Corrected Test Article 0D550 = Test Article 0D550 - Blank Mean 0D550

If killed controls (KC) are used, the following additional calculations will be performed to correct for the amount of MTT reduced directly by test article residues. The OD550 value
for the negative control killed control will be subtracted from the OD550 values for each of the test article-treated killed controls, to determine the net OD550 values of the test article-treated killed controls.

Net 0D550 for each test article KC = Mean Raw 0D550 Test Article KC - Mean Raw 0D550 Negative Control KC

The net OD550 value represents the amount of reduced MTT due to direct reduction by the test article residues. The net OD550 value will be subtracted from the corrected OD550 values of the viable test article-treated EpiOcular™ tissues, to obtain final corrected OD550 values . If the net OD550 results in a negative value, a correction for the viable tissue OD550 will not be required.

Final Corrected 0D550 = Corrected Test Article ODno (viable) - Net 0D550 Test Article (KC)

If the direct reduction of MTT by the test article is greater than 30% of the negative control, the test article may be considered untestable with MTT.
The following% of Control calculations will be made:
% of control = ((Corrected OD550 of each Test Article or Positive Control Exposure Time)/(Corrected Mean 0D550 of Negative Control))*100

If colorant controls (CC) are used, the following additional calculation will be performed to correct for the amount of interference by the colored test material. The OD550 value for the colorant control will be subtracted from the corrected OD550 value for the viable tissues.

Corrected 0D550 of the Test Article= Corrected Test Article 0D550 (viable) - CC 0D550

If colorant control-killed controls (CC-KC) are used, the OD550 value of the CC-KC will be added to corrected OD550 values of the viable treated tissues to obtain a final corrected OD550 value.

Final Corrected ODsso of the Test Article = Corrected Test Article 0D550 (viable) - Net KC 0D5550 - CC 0D550 + CC - KC 0D550

The following calculation will then be performed:

% of Viability = ((Mean Final Corrected 0D550 of Test Article or Control Tissue)/(Corrected Mean 0D550 of Negative Control)) x 100

CRITERIA FOR DETERMINATION OF A VALID TEST:
The assay will be accepted if the mean negative control OD550 > 0.8 and < 2.5, and the mean relative viability of the positive control is <= 50%.

EVALUATION OF TEST RESULTS:
The following Prediction Model has been endorsed by the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) for the prediction of ocular
irritation. A test article is predicted to not require classification or labelling for ocular irritation (GHS No Category) if the mean relative viability of the treated tissues is greater than 60% of the
mean viability of the negative control. A test article is predicted to potentially require classification and labelling according to UN GHS {Category 1 or 2) if the mean relative viability of the treated tissues is <= 60% of the mean viability of the negative control.

mean tissue viability <=60% -> GHS Category 1 or 2*
mean tissue viability > 60% -> No GHS Category
*Additional testing would be required to discriminate between a category 1 or 2 classification.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Main test

Value:

64.03

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:

Acceptability criteria:
The assay results were accepted when the corrected mean OD550 value for the negative control exposure time (1.828) was > 0.8 and < 2.5 and the mean relative viability of the positive control was ≤ 50%.

Interpretation of results:

GHS criteria not met

Conclusions:

The test article resulted in a viability of 64.03% relative to the negative control. According to the
prediction model presented in OECD 492, the test article is predicted to not require classification or
labelling for ocular irritation (GHS No Category).

Executive summary:

An in vitro eye irritation test using the Reconstructed human Cornea-like Epithelium (RhCE) (EpiOcular™ tissue) model was performed according to the OECD Guideline 492 and in compliance with GLP to predict the acute eye irritation potential of the test substance.

The test article resulted in a viability of 64.03% relative to the negative control. According to the prediction model presented in OECD 492, the test article is predicted to not require classification or labelling for ocular irritation (GHS No Category).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 25 January to 03 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See 'Any other information on materials and methods incl. tables' section

GLP compliance:

yes (incl. QA statement)

Species:

other: Isolated bovine cornea

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

Bovine eyes will be obtained from the abattoir of J.W. TREUTH & SONS, Inc., Baltimore, MD. The eyes will be excised by an abattoir employee (as soon after slaughter as possible) and held in HBSS on ice. Once the required number of eyes has been obtained, the eyes will be transported to IIVS. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas will be initiated .

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

100% for test material and controls

Duration of treatment / exposure:

10 minutes for test material and controls

Duration of post- treatment incubation (in vitro):

2 hours for test material and controls

Number of animals or in vitro replicates:

Test material: 3
Positive control: 3
Negative control: 3

Details on study design:

Three corneas were incubated in the presence of the test article at 32 ± 1 °C for 10 minutes. Three corneas were incubated in the presence of each control at 32 ± 1 °C for 10 minutes.
After the closed chamber rinse and refeed of the test article, it was observed that residual test article was present in the form of oily droplets. The chambers were rinsed again using the open chamber method, in which the anterior chamber windows are removed and the test article is directly rinsed
from the exposed corneas. Additionally, the residual test article was removed from the chamber walls and corneas with cotton tipped applicators soaked in MEM w/ phenol red.
After removal of the test or control article from the corneas, the corneas were returned to the incubator at 32 ± 1 °C for a post-exposure incubation period of 2 hours, after which a final opacity value was determined.

Fixation of Corneas and Histology evaluation:
After the medium was removed for the permeability determination, each cornea was carefully separated from its corneal holder and fixed in formalin. As instructed by the Sponsor, a histological evaluation was not performed. The fixed corneas will be stored at IIVS for up to 1 year.

Irritation parameter:

in vitro irritation score

Value:

11.3

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

The BCOP assay was accepted when the positive control, ethanol, produced an In Vitro Score that fell within 2 standard deviations of the historical mean. The current acceptance range for ethanol is 38.0 – 63.1.

Evaluation of Test Results for BCOP:

The following prediction models were used to evaluate the ocular irritancy potential as described by Sina et al. (1995)2 for non-regulatory purposes or as described in OECD TG 437 (2017).

The prediction model according to Sina et. al based on a wide range of materials (note: While this classification system provides a good initial guide to interpretation of these in vitro data, these specific ranges may not be applicable to all classes of materials or other exposure times. Whenever possible, results should be compared to “benchmark” materials tested under similar exposure conditions.):

 

In Vitro Score	Sina et al Prediction
≤ 25	Mild irritant
> 25; ≤ 55	Moderate irritant
> 55	Severe irritant

 

Prediction model outlined in OECD TG 437:

 

In Vitro Irritation Score (IVIS)	UN GHS
≤3	No Category
>3; ≤55	No prediction can be made*
>55	Category 1

* - Additional testing would be required for a definitive classification of ocular irritation potential according to GHS

Interpretation of results:

study cannot be used for classification

Conclusions:

The In Vitro Score of the test article was 11.3. According to the prediction model presented in OECD TG 437, a prediction for eye irritation potential could not be determined. Additional testing would be
warranted for definitive classification of eye irritation potential for labeling purposes.

Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was used to assess the potential ocular irritancy of the test article to isolated bovine corneas. Bovine corneas, obtained as a byproduct from freshly slaughtered animals, were mounted in special holders and exposed to the test article. An In Vitro Score was determined for the test article based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas. The methods and procedures used in this assay were consistent with OECD Test Guideline 437: Bovine Corneal Opacity and Permeability Test Method for
Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage (2020).

 

The In Vitro Score of the test article was 11.3. According to the prediction model presented in OECD TG 437, a prediction for eye irritation potential could not be determined. Additional testing would be warranted for definitive classification of eye irritation potential for labeling purposes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Based on skin irritation and corrosion study results, the test substance needs to be classified as skin irritant category 2 according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

According to the in vitro eye irritation studies and under the experimental conditions reported, the test item is not corrosive/irritating to the eye according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).