Human Eye Model Test

Human Eye Model Test: In Vitro Eye Irritation

(EpiOcular™)

The prediction and classification of eye irritation potential of substances can be performed by the assessment of the effect on the EpiOcular™ model.

The human skin model test is validated by the EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing) and is performed in accordance with the OECD guidance OECD 492 at Eurofins BioPharma Product Testing Munich GmbH¹ with chemicals, cosmetics or personal care products and pharmaceuticals.

Assessment of Eye Irritation Potential

Eye irritation accordingly refers to the production of changes in the eye which are fully reversible within this time range (i.e., UN GHS “Category 2”)².
This test uses the three-dimensional RhCE EpiOcular™ model (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium.
To determine eye irritation potential substances are applied directly on the eye tissue surface and the cell viability is measured by the MTT assay.
The EpiOcular™ human eye model test can be used as a reliable in vitro method to identify substances as non-classified ("No Category"). However, this test cannot be used to classify substances into UN GHS "Category 1” or “Category 2"¹. A positive result will further require e.g. an eye hazard test (OECD 492B) for a final classification into UN GHS "Category 1” or “Category 2".

Procedure

Principle of the Human Eye Model Test

Protocol
Model	EpiOcular
Supplier	MatTek
Analysis	Eye irritation potential: cytotoxicity measurement with MTT (mean tissue viability compared to negative control)
Test chemical concentrations	Liquids: 50 µL (undiluted) Solids: 50 mg
Exposure time and incubation periods	Two different protocols: Liquids: 30 min incubation with dose groups 12 min post-soak after application 120 min post-treatment Solids: 6 h incubation with dose groups 25 min post-soak after application 18 h post-treatment
Quality controls	Positive control: Methyl acetate Negative control: Aqua dest.
Pre-tests	To determine if additional controls are needed: *NSMTT: mixing of test item with MTT medium to determine if test item alone can reduce MTT à blue colouring: in main experiment two killed tissues treated with test item and two untreated killed tissues are added as controls NSC_living#: mixing of test item with H₂O or isopropanol to determine if strong colour of test item can discolour at contact with this liquids à optical discolouring (measuring of spectrum): in main experiment two living tissues without incubation with MTT medium are added as controls NSC_killed§: if the two other controls were determined à in main experiment two killed tissues without incubation with MTT medium are added as controls**
Application	Direct topical application of chemicals on eye tissues Two tissue replicates per dose group
Data delivery	Optical density (OD) value with microplate spectrophotometer at 570 nm
Prediction model	Mean tissue viability (% negative control): ≤ 60%: Irritant (I): No prediction can be made > 60%: Non-Irritant (NI): UN GHS "No Category"

*NSMTT: non-specific MTT reduction

#NSC_living: non-specific colouring of living tissues

$NSC_killed: non-specific colouring of killed tissues

Data

Eurofins Data for demonstration technical proficiency of the Human Eye Model Test (EpiOcular™)

Chemical	Physical State	UN GHS category	EF category for EpiOcular™
*In vivo* Category 1
Methylthioglycolate	Liquid	No prediction can be made	No prediction can be made
Hydroxyethyl acrylate	Liquid	No prediction can be made	No prediction can be made
2,5-Dimethyl-2,5- hexanediol	Solid	No prediction can be made	No prediction can be made
Sodium oxalate	Solid	No prediction can be made	No prediction can be made
*In vivo* Category 2A
2,4,11,13- Tetraazatetradecane-diimidamide, N,N''- bis(4-chlorophenyl)- 3,12-diimino-, di-D-gluconate (20%, aqueous)	Liquid	No prediction can be made	No prediction can be made
Sodium benzoate	Solid	No prediction can be made	No prediction can be made
*In vivo* Category 2B
Diethyl toluamide	Liquid	No prediction can be made	No prediction can be made
2,2-Dimethyl-3- methylenebicyclo [2.2.1] heptane	Solid	No prediction can be made	No prediction can be made
*In vivo* No Category
1-Ethyl-3- methylimidazolium ethylsulphate	Liquid	No Category	No Category
Dicaprylyl ether	Liquid	No Category	No Category
Piperonyl butoxide	Liquid	No Category	No Category
Polyethylene glycol (PEG-40) hydrogenated castor oil	Viscous	No Category	No Category
1-(4-Chlorophenyl)-3- (3,4-dichlorophenyl) urea	Solid	No Category	No Category
2,2'-Methylene-bis-(6- (2H-benzotriazol-2- yl)-4-(1,1,3,3- tetramethylbutyl)- phenol)	Solid	No Category	No Category
Potassium tetrafluoroborate	Solid	No Category	No Category

EF = Eurofins Munich GmbH
No prediction can be made: “Category 1 or Category 2”

Table 1: Eurofins data of in vitro eye irritation with EpiOcular™ of fifteen tested proficiency chemicals compared to the data of the OECD guideline 492¹.

In Table 1 the obtained data from the in vitro eye irritation with EpiOcular™ of seven non-classified (“No Category”) and eight chemicals without prediction (“No prediction can be made”) are shown. The prediction of all tested chemicals was correct in comparison to the classification of the OECD guideline 492.

References

OECD (2024). OECD Guideline for the Testing of Chemicals. No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage
UN (2023), United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS), Tenth revised edition, UN New York and Geneva

Human Eye Model Test: In Vitro Eye Irritation/Corrosion

(SkinEthic™ HCE TTT)

The prediction and classification of eye irritation/corrosion potential of substances can be performed by the assessment of the effect on the SkinEthic™ HCE TTT model.

The human skin model test is validated by the EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing) and is performed in accordance with the OECD guidance OECD 492B at Eurofins BioPharma Product Testing Munich GmbH¹ with chemicals, cosmetics or personal care products and pharmaceuticals.

Assessment of Eye Irritation Potential

Serious eye damage refers to the production of tissue damage in the eye, or serious physical decay of vision, following application of a test chemical to the anterior surface of the eye, which is not fully reversible within 21 days of application as defined by the UN GHS (i.e., “Category 1”). Eye irritation accordingly refers to the production of changes in the eye which are fully reversible within this time range (i.e., UN GHS “Category 2”).
This test uses the three-dimensional RhCE SkinEthic™ HCE TTT model (EpiSkin). It consists of human immortalised corneal epithelial cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in the human cornea. The SkinEthic™ HCE tissue construct consists of at least 4 viable layers of cells including columnar basal cells, transitional wing cells and superficial squamous cells similar to that of the normal human corneal epithelium
To determine eye irritation/corrosion potential substances are applied directly on the eye tissue surface and the cell viability is measured by the MTT assay.
The SkinEthic™ HCE TTT human eye model test can be used as a reliable in vitro method to identify chemicals that do not require classification (“No Category”), do require classification for eye irritation (“Category 2”) and do require classification for serious eye damage (“Category 1”) according to the UN GHS ocular hazard categories.

Procedure

Principle of the Human Eye Model Test

Protocol
Model	SkinEthic™ HCE TTT
Supplier	EpiSkin
Analysis	Eye irritation potential: cytotoxicity measurement with MTT (mean tissue viability compared to negative control)
Test chemical concentrations	Liquids: 80 µL (undiluted) + 10 µL DPBS (5 min application period) 80 µL (20% w/v in Aqua dest.) + 10 µL DPBS (16 min application period) 80 µL (20% w/v in Aqua dest.) + 10 µL DPBS (120 min application period) Solids: 80 mg + 80 µL Aqua dest. (30 min and 120 min application period)
Exposure time and incubation periods	Two different protocols: Liquids: 5 min incubation with dose groups and undiluted sample 16 min incubation with dose groups and diluted sample 120 min incubation with dose groups and diluted sample 10 min post-soak after application (for all three time periods) Solids: 30 min incubation with dose groups 120 min incubation with dose groups 30 min post-soak after application (for both time periods)
Quality controls	Positive control: Methyl acetate (using the liquid test protocol, also the PC is diluted accordingly to the sample) Negative control: DPBS
Pre-tests	To determine if additional controls are needed: *NSMTT: mixing of test item with MTT medium to determine if test item alone can reduce MTT à blue colouring: in main experiment two killed tissues treated with test item and two untreated killed tissues are added as controls NSC_living#: mixing of test item with H₂O or isopropanol to determine if strong colour of test item can discolour at contact with this liquids à optical discolouring (measuring of spectrum): in main experiment two living tissues without incubation with MTT medium are added as controls NSC_killed§: if the two other controls were determined à in main experiment two killed tissues without incubation with MTT medium are added as controls**
Application	Direct topical application of chemicals on eye tissues Two tissue replicates per dose group
Data delivery	Optical density (OD) value with microplate spectrophotometer at 570 nm
Prediction model	Mean tissue viability (% negative control):	Prediction:
	≤ 40% after 30 minutes and ≤ 60% after 120 minutes	UN GHS Category 1 (serious eye damage)
	Any other combination of values	UN GHS Category 2 (eye irritation)
	> 40% after 30 minutes and > 60% after 120 minutes	UN GHS No Category (not classified)

*NSMTT: non-specific MTT reduction

#NSC_living: non-specific colouring of living tissues

$NSC_killed: non-specific colouring of killed tissues

Data

Eurofins Data for demonstration technical proficiency of the Human Eye Model Test (SkinEthic™ HCE TTT) for Liquids

Chemical	Physical State	UN GHS category	EF category for SkinEthic™ HCE TTT
UN GHS Category 1
2-(Diethylamino)-ethanol	Liquid	Category 1	Category 1
Acetic Acid (10%)	Liquid	Category 1	Category 1
UN GHS Category 2
2-Butanon	Liquid	Category 2	Category 2
Acetone	Liquid	Category 2	Category 2
Hexadecyltrimethyl-ammoniumchloride (2%)	Liquid	Category 2	Category 2
UN GHS No Category
1.3-Diisopropyl-benzene	Liquid	No Category	No Category
Dodecane	Liquid	No Category	No Category

EF = Eurofins Munich GmbH

Table 1: Eurofins data of in vitro eye irritation/corrosion with SkinEthic™ HCE TTT of seven tested liquid proficiency chemicals compared to the data of the OECD guideline 492B¹.

In Table 1 the obtained data from the in vitro eye irritation/corrosion with SkinEthic™ HCE TTT of two non-classified (“No Category”), three irritant (“Category 2”) and two corrosive (“Category 1”) chemicals are shown. The prediction of all tested chemicals was correct in comparison to the classification of the OECD guideline 492B.

Eurofins Data for demonstration technical proficiency of the Human Eye Model Test (SkinEthic™ HCE TTT) for Solids

Chemical	Physical State	UN GHS category	EF category for SkinEthic™ HCE TTT
UN GHS Category 1
1-Naphthalene acetic acid sodium salt (alpha-)	Solid	Category 1	Category 1
1,2-Benzisothiazol-3(2H)-one	Solid	Category 1	Category 1
UN GHS Category 2
4-Carboxybenz-aldehyde	Solid	Category 2	Category 2
2-Hydroxy-1,4-naphthoquinone	Solid	Category 2	Category 2
Ammonium nitrate	Solid	Category 2	Category 2
UN GHS No Category
Magnesium carbonate hydroxide pentahydrate, light	Solid	No Category	No Category
Anthracene	Solid	No Category	No Category

EF = Eurofins Munich GmbH

Table 2: Eurofins data of in vitro eye irritation/corrosion with SkinEthic™ HCE TTT of seven tested solid proficiency chemicals compared to the data of the OECD guideline 492B¹.

In Table 2 the obtained data from the in vitro eye irritation/corrosion with SkinEthic™ HCE TTT of two non-classified (“No Category”), three irritant (“Category 2”) and two corrosive (“Category 1”) chemicals are shown. The prediction of all tested chemicals was correct in comparison to the classification of the OECD guideline 492B.

References

OECD (2024). OECD Guideline for the Testing of Chemicals. No. 492B: Reconstructed Human Cornea-like Epithelium (RHCE) Test Method for Eye Hazard Identification
UN (2023), United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS), Tenth revised edition, UN New York and Geneva

Human Eye Model Test: In Vitro Eye Irritation (OECD 492)

Human Eye Model Test: In Vitro Eye Irritation

(EpiOcular™)

Assessment of Eye Irritation Potential

Procedure

Protocol

Data

References

Human Eye Model Test: In Vitro Eye Irritation/Corrosion (OECD 492B)

Human Eye Model Test: In Vitro Eye Irritation/Corrosion

(SkinEthic™ HCE TTT)

Assessment of Eye Irritation Potential

Procedure

Protocol

Data

References