Human Skin Model Test

Human Skin Model Test: In Vitro Skin Irritation

(EpiDerm™ and SkinEthic™ RHE)

The prediction and classification of skin irritation potential of substances can be performed by the assessment of the effect on EpiDerm™ and SkinEthic™ RHE (Reconstructed Human Epidermis) models.

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 439 at Eurofins BioPharma Product Testing Munich GmbH¹ with chemicals, cosmetics or personal care products and pharmaceuticals.

Assessment of Skin Irritation Potential

Acute irritation characterises a local, reversible inflammatory response of normal living skin to direct injury after application of irritant substances².
The EpiDerm™ and SkinEthic™ RHE represent a reconstituted three-dimensional human epidermis (RhE) model which consists of human epidermal keratinocytes. This in vitromodel mimics biochemical and physiological properties of the upper human skin.
To determine skin irritation potential substances are applied directly on the skin tissue surface and the cell viability is measured by the MTT assay.
The EpiDerm™ and SkinEthic™ RHE human skin model test can be used as a reliable in vitromethod to identify substances as non-classified ("No Category") or they can be classified into at least UN GHS "Category 2". A positive result will further require a skin corrosion test (OECD 431) for a final classification into "Category 1"¹.
The skin irritation potential of medical device extracts can also be determined by using a modified EpiDerm™ / SkinEthic™ RHEhuman skin model test.

Procedure

Principles of the Human Skin Model Test

Protocol
Model	EpiDerm™	SkinEthic™ RHE
Supplier	MatTek	SkinEthic
Analysis	Skin irritation potential: cytotoxicity measurement with MTT (mean tissue viability compared to negative control)
Test chemical concentrations	Liquids: 30 µL (undiluted) Solids: 25 mg + 25 µL DPBS	Liquids: 16 µL (undiluted) Solids: 16 mg + 10 µL Aqua dest.
Exposure time and incubation periods	60 min incubation with dose groups 24 h post-incubation and further 18 h incubation in media	42 min incubation with dose groups 42 h post-incubation
Quality controls	Positive control: 5% SDS 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 (EpiDerm™) or three (SkinEthic™ RHE) killed tissues treated with test item and two or three 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 (EpiDerm™) or three (SkinEthic™ RHE) 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 (EpiDerm™) or three (SkinEthic™ RHE) killed tissues without incubation with MTT medium are added as controls
Application	Direct topical application of chemicals on skin tissues Three tissue replicates per dose group
Data delivery	Optical density (OD) value with microplate spectrophotometer at 570 nm
Prediction model	Mean tissue viability (% negative control): ≤ 50%: Irritant (I): UN GHS at least "Category 2" (IL-1α > 60 pg/mL) > 50%: Non-Irritant (NI): UN GHS "No Category" (IL-1α ≤ 60 pg/mL)

^*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 Skin Model Test (EpiDerm™ and SkinEthic™ RHE)

Chemical	UN GHS category	EF category for EpiDerm™	EF category for SkinEthic™ RHE
Non-Classified Chemicals (UN GHS "No Category")
Naphthalene acetic acid	No Category	No Category	No Category
Isopropanol	No Category	No Category	No Category
Methyl stearate	No Category	No Category	No Category
Heptyl butyrate	No Category	No Category	No Category
Hexyl salicylate	No Category	No Category	No Category
Classified Chemicals (UN GHS "Category 2")
Cyclamen aldehyde	Category 2	Category 2	Category 2
1-Bromohexane	Category 2	Category 2	Category 2
Potassium hydroxide (5% KOH)	Category 2	Category 2	Category 2
1-Methyl-3-phenyl-1-piperazine	Category 2	Category 2	Category 2
Heptanal	Category 2	Category 2	Category 2

EF = Eurofins Munich GmbH

Table 1: Eurofins data of in vitro skin irritation with EpiDerm™ and SkinEthic™ RHE of ten tested proficiency chemicals compared to the data of the OECD guideline 439¹.

In Table 1 the obtained data from the in vitro skin irritation with EpiDerm™ and SkinEthic™ RHE of five non-classified and five classified ("Category 2") chemicals are shown. The prediction of all tested chemicals was correct in comparison to the classification of the OECD guideline 439.

References

OECD (2021). OECD Guideline for the Testing of Chemicals No. 439: In VitroSkin Irritation: Reconstructed Human Epidermis Test Methods. 14 June 2021
UN (2023). United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS), tenth revised edition, UN New York and Geneva

Human Skin Model Test: in vitro Skin Corrosion EpiDerm™

The prediction and classification of skin corrositivity potential of substances can be performed by the assessment of the effect on EpiDermmodels.

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 431) at Eurofins BioPharma Product Testing Munich GmbH¹ with chemicals, cosmetics or personal care products and pharmaceuticals.

Assessment of skin corrosion potential

Corrosion represents irreversible tissue damage of the skin (visible necrosis) after application of a substance. The underlying mechanisms are either the destruction of the skin penetration barrier or the rapid penetration of highly cytotoxic chemicals through the skin without its destruction.
The EpiDerm™ represents a reconstituted three-dimensional human epidermis (RhE) model which consists of human epidermal keratinocytes. This in vitro model mimics biochemical and physiological properties of the upper human skin.
To determine skin corrosion potential chemicals are applied directly on the skin tissue surface and the cell viability is measured by MTT assay.
The EpiDerm™ human skin model test can be used as a reliable in vitro method to distinguish between corrosive and non-corrosive chemicals. Additionally, sub-categorisation in "Category 1A" or a combination of sub-categories "1B" and "1C" is possible with this test².
The skin corrosion test can be performed subsequently to a positive skin irritation testfor a final classification. If the skin corrosion test is performed at first, a negative result can be followed by an irritation test to conclude if the chemical has irritant potential or not.

Corrosion represents irreversible tissue damage of the skin (visible necrosis) after application of chemicals. The underlying mechanisms are either the destruction of the skin penetration barrier or the rapid penetration of highly cytotoxic chemicals through the skin without its destruction.

Procedure

Principles of the Human Skin Model Test

Protocol
Model	EpiDerm
Supplier	MatTek
Analysis	Corrosivity potential: cytotoxicity measurement with MTT (mean tissue viability compared to negative control)
Test chemical concentrations	Liquids: 50 µL (undiluted) Solids: 25 mg + 25 µL H₂O
Exposure time	3 min and 60 min incubation with dose groups
Quality controls	Positive control: 8 N (KOH) Negative control: H₂O
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 were added as controls NSC_living^#: mixing of test item with H₂O or isopropanol to determine if strong own 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 were added as controls NSC_killed*^§: if the two other controls were determined à in main experiment two killed tissues without incubation with MTT medium were added as controls
Application	Direct topical application of chemicals on skin tissue Two tissue replicates per dose group
Data delivery	Optical density (OD) value with microplate spectrophotometer at 570 nm

*NSMTT: non-specific MTT reduction

^#NSC_living: non-specific colouring of living tissues

^$NSC_killed: non-specific colouring of killed tissues

Prediction

Prediction Model of the Human Skin Model Test

Prediction EpiDerm™
Mean tissue viability (% negative control)	Prediction optional sub-categorisation
Step 1
< 50% after 3 min exp.	Corrosive
³ 50% after 3 min exp. and < 15% after 60 min exp.	Corrosive A combination of optional sub-categories 1B and 1C
³ 50% after 3 min exp. and ³ 15% after 60 min exp.	Non-Corrosive
Step 2
< 25% after 3 min exp.	Optional sub-category 1A
≥ 25% after 3 min exp.	A combination of optional sub-categories 1B and 1C

Exp. = exposure

Table 1: Prediction model of the corrosivity potential of the EpiDerm^TM tissues dependent on the mean tissue viability.¹

Based on the mean tissue viability, chemicals can be classified with the skin corrosion test into corrosive and non-corrosive and can be furthermore sub-categorised.

Data

Eurofins Data for demonstration technical proficiency of the Human Skin Model Test (EpiDerm^TM)

Chemical	UN GHS category	EF category for EpiDerm™
Corrosive chemicals (sub-category 1A)
Bromoacetic acid	1A	1A
Boron trifluoride dihydrate	1A	1A
Phenol	1A	1A
Dichloroacetyl chloride	1A	1A
Corrosive chemicals (combination of sub-categories 1B and 1C)
Glyoxylic acid monohydrate	1B and 1C	1B and 1C
Lactic acid	1B and 1C	1B and 1C
Ethanolamine	1B and 1C	1B and 1C
Hydrochloric acid (14.4%)	1B and 1C	1B and 1C
Non-corrosive chemicals
Phenethyl bromide	NC	NC
4-Amino-1,2,4-triazole	NC	NC
4-(Methylthio)-benzaldehyde	NC	NC
Lauric acid	NC	NC

EF = Eurofins Munich GmbH NC = Non-Corrosive

Table 2: Eurofins data of in vitro skin corrosion with EpiDerm™ of twelve tested proficiency chemicals compared to the data of the OECD guideline 431 ¹.

In Table 2 the obtained data from the in vitro skin corrosion with EpiDerm™ of eight corrosive and four non-corrosive chemicals are shown. The prediction of all tested chemicals was correct in comparison to the classification of the OECD guideline 431.

References

OECD (2019). OECD Guideline for the Testing of Chemicals. No. 431: In VitroSkin Corrosion: Reconstructed Human Epidermis (RHE) Test Method, 18 June 2019
UN (2023), United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS), Tenth revised edition, UN New York and Geneva

In vitro Membrane Barrier Test Method for Skin Corrosion

(Corrositex®)

The prediction and classification of skin corrositivity potential of substances can be performed by the assessment of the effect on the Corrositex®Test Method.

The In vitro Membrane Barrier Test Method is validated by the ICCVAM (Interagency Coordinating Committee on the Validation of Alternative Methods) and is performed in accordance with the OECD guidance (OECD 435) at Eurofins BioPharma Product Testing Munich GmbH¹ with chemicals, cosmetics or personal care products and pharmaceuticals.

Assessment of skin corrosion potential

Corrosion represents irreversible tissue damage of the skin (visible necrosis) after application of a substance. The underlying mechanisms are either the destruction of the skin penetration barrier or the rapid penetration of highly cytotoxic chemicals through the skin without its destruction.
The Corrositex®Test Method is composed of a synthetic macromolecular bio-barrier that is placed on top of a chemical detection system (CDS). Corrosive materials are identified by their ability to pass through the bio-barrier membrane and elicit a colour change or a structure change in the underlying CDS. The time it takes for a test chemical to penetrate through the barrier is measured in the CORROSITEX™ Assay.
To determine skin corrosion potential chemicals are applied directly on the synthetic macromolecular bio-barrier and the breakthrough time to active the CDS is recorded.
The Corrositex®Test Method can be used as a reliable in vitro method to distinguish between corrosive and non-corrosive chemicals. Additionally, sub-categorisation in "Category 1A" " Category 1B" or " Category 1C" is possible with this test².
The skin corrosion test can be performed subsequently to a positive skin irritation testfor a final classification. If the skin corrosion test is performed at first, a negative result can be followed by an irritation test to conclude if the chemical has irritant potential or not.

Procedure

Principles of the Corrositex®Test Method

Protocol
Model	Corrositex®Test Method
Supplier	InVitro International
Analysis	Corrosivity potential: Time of a sample to break through a synthetic macromolecular bio-barrier to active the underlying CDS* is measured and category is read using a Prediction Table.
Test chemical concentrations	Liquids: 500 µL (undiluted) Solids: 500 mg (undiluted)
Exposure time	Depends on the Categorisation Test: up to 240 min (Category 1) up to 60 min (Category 2)
Quality controls	Positive control: Phosphoric Acid (85%) Negative control: Citric Acid (10%) in Aqua dest.
Steps of the Test	Qualification Test: This step ensures that the sample is compatible with the Corrositex® system. 100 mg or 150 µL of the sample are added to Qualify test tube (CDS). If the colour or consistency of the CDS changes at the sample/testing fluid interface, the test item is qualified for the assay. If no reaction is observed within five minutes, the sample is not qualified for the Corrositex® Assay. Preparation of Biobarrier: The synthetic macromolecular bio-barrier is prepared, pipetted into membrane discs and kept cool until start of the test. Categorisation Test: This step establishs the category of cut-off times (up to 240 min or up to 60 min) for the sample. 100 mg or 150 µL of the sample are added to different tubes and checked for a colour change that is matched to the corresponding colour charts of the Corrositex® Testing Protocol Poster. Classification Test: This step determins the appropriate Packing Group for the test sample. 500 mg or 500 µL of the sample are added to the synthetic macromolecular bio-barrier that is placed onto the CDS* vial. As soon as a reaction is observed in the CDS*, the time is recorded.
Application	Direct topical application of chemicals on a synthetic macromolecular bio-barrier with underlying CDS* Four replicates per dose group
Data delivery	The mean time of four replicates to active the CDS* is calculated. The appropriate Packing Group Designation, Hazard Statement and UN GHS Subcategory Prediction by sample category is read from the Corrositex^® Table (see below).

*CDS: Chemical Detection System

Prediction

Prediction Model of the Corrositex® Test Method

Category	Time [min]	Time [min]	Time [min]	Time [min]
1	0 – 3	> 3 - 60	> 60 - 240	> 240
2	0 - 3	> 3 - 30	> 30 - 60	> 60
UN GHS Subcategory	1A	1B	1C	non corrosive
Packing Group	1	2	3	not applicable
Hazard Statement	H314	H314	H314	no label

Table 1: Prediction model of the corrosivity potential using the Corrositex® Test Method dependent on the mean breakthrough time.¹

Based on the mean breakthrough time, samples can be classified with the skin corrosion test into corrosive and non-corrosive and can be furthermore sub-categorised.

Data

Eurofins Data for demonstration technical proficiency of the In Vitro Membrane Barrier Test Method (Corrositex®)

Chemical	UN GHS category	EF category for Corrositex®
Corrosive chemicals (sub-category 1A)
Boron trifluoride dihydrate	1A	1A
Nitric acid	1A	1A
Phosphorus pentachloride	1A	1A
Corrosive chemicals (sub-category 1B)
Valeryl chloride	1B	1B
Sodium Hydroxide	1B	1B
1-(2-Aminoethyl) piperazine	1B	1B
Corrosive chemicals (sub-category 1C)
Benzenesulfonyl chloride	1C	1C
N,N-Dimethyl benzylamine	1C	1C
Tetraethylenepentamine	1C	1C
Non-corrosive chemicals
Eugenol	NC	NC
Sodium bicarbonate	NC	NC

EF = Eurofins Munich GmbH NC = Non-Corrosive

Table 2: Eurofins data of in vitro skin corrosion with Corrositex® of eleven tested proficiency chemicals compared to the data of the OECD guideline 435 ¹.

In Table 2 the obtained data from the In Vitro Membrane Barrier Test Method (Corrositex®) of nine corrosive and two non-corrosive chemicals are shown. The prediction of all tested chemicals was correct in comparison to the classification of the OECD guideline 435 ¹.

References

OECD (2015). OECD Guideline for the Testing of Chemicals. No. 435: In Vitro Membrane Barrier Test Method for Skin Corrosion, 28 July, 2015
UN (2023), United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS), Tenth revised edition, UN New York and Geneva

Human Skin Model Test with Medical Devices: In Vitro Skin Irritation
(EpiDerm™ or SkinEthic™ RHE)

At Eurofins Biopharma Product Testing Munich GmbH the Human Skin Model Test can be used for detection of skin irritation potential of medical devices. The prediction and classification can be done by the assessment of the effect on EpiDerm^TM model or SkinEthic^TM RHE model.

The modified Human Skin Model Test for medical devices is a reliable in vitro test method and is performed in accordance with the draft ISO guidelines 10993-12 and 10993-23 Part 10 at Eurofins¹.

Assessment of Skin Irritation Potential of Medical Devices

Acute irritation characterises a local, reversible inflammatory response of normal living skin to direct injury after application of irritant substances².
The EpiDerm^TMand the SkinEthic^TM RHE model represent a reconstituted three-dimensional human epidermis (RhE) model which consists of human epidermal keratinocytes. This in vitro model mimics biochemical and physiological properties of the upper human skin.
To determine skin irritation potential the test item is extracted and applied directly on the skin tissue surface. The cell viability is measured by MTT assay.
The Skin Irritation Test can be used as a reliable in vitrotest method to identify chemicals in extracts from medical devices as "irritant" or "non-irritant"³.

Acute irritation characterises a local, reversible inflammatory response of normal living skin to direct injury after application of irritant substances².

Procedure

Principles of the Human Skin Model Test with Medical Devices

Protocol
Model	EpiDerm^TM (Reconstructed human skin model) supplied from MatTek	SkinEthic^TM RHE (Reconstructed human skin model) supplied from EpiSkin
Analysis	skin irritation potential: cytotoxicity measurement with MTT (mean tissue viability compared to negative control tissues) optional: Interleukin-1α (IL-1α) release into the tissue culture medium
Test chemical concentrations	100 µL undiluted extract in 0.9% NaCl (polar) 100 µL undiluted extract in sesame oil (non-polar)
Extraction and absorption capacity	extraction at 37 ± 1 °C for 72 ± 2 h in 0.9% NaCl or sesame oil with continuous agitation/shaking determination of the absorption capacity of the test item in NaCl (polar) or in sesame oil (non-polar)
Exposure time	18 ± 0.5 h in the incubator	24 ± 2 h in the incubator
Quality controls	positive control: 1% SDS in NaCl and sesame oil negative control: DPBS vehicle controls: → 0.9% NaCl as solvent for polar extracts → sesame oil as solvent for non-polar extracts
Pre-tests	to determine if additional controls are needed: NSMTT: mixing of test item extract with MTT medium to determine if test item alone can reduce MTT → blue colouring: in main experiment two killed tissues treated with test item and two (EpiDerm™) or three (SkinEthic™ RHE) untreated killed tissues were added as controls NSC_living: mixing of test item extract with H₂O or isopropanol to determine if strong own colour of test item can discolour at contact with this liquids → optical discolouring (measuring of spectrum): in main experiment two (EpiDerm™) or three (SkinEthic™ RHE) living tissues without incubation with MTT medium were added as controls NSC_killed*: if the two other controls were determined → in main experiment two (EpiDerm™) or three (SkinEthic™ RHE) killed tissues without incubation with MTT medium were added as controls
Application	direct topical application of extracts on skin tissues three tissue replicates per dose group
Data delivery	optical density (OD) value with microplate spectrophotometer at 570 nm tissue viability of each dose group
Prediction model	mean tissue viability (% negative control): ≤ 50%: Irritant; (IL-1α > 60 pg/mL) > 50%: Non-Irritant; (IL-1α ≤ 60 pg/mL)

*NSMTT: non-specific MTT reduction
^#NSC_living: non-specific colouring of living tissues
^§NSC_killed: non-specific colouring of killed tissues

References

ISO 10993-23, 2021, “Biological evaluation of medical devices - Part 23: Tests for irritation
UN (2023). United Nations Globally Harmonised System of Classification and Labelling of Chemicals (GHS), Tenth revised edition, UN New York and Geneva
DeJong W., Hoffmann S., Lee M., Kandárova H., Pellevoisin, C., Haishima Y., Rollins B., Zdawczyk A., Willoughby J., Bachelor M., Schatz T., Skoog S., Parker S., Sawyer A., Pescio P., Fant K., Kim, KM., Kwon JS., Gehrke H., Hofman-Hüther H., Meloni, M., Julius C., Briotet D., Letasiova S., Kato R., Miyajima A., De La Fonteyne L., Videau C. Tornier C., Turley A.P., Christiano N., Rollins T.S. and Coleman K.P., (2018) Round robin study to evaluate the reconstructed human epidermis (RhE) model as an in vitro skin irritation test for detection of irritant activity in medical device extracts. Toxicology in Vitro.

Human Skin Model Test: In Vitro Skin Irritation (OECD 439)

Human Skin Model Test: In Vitro Skin Irritation

(EpiDerm™ and SkinEthic™ RHE)

Assessment of Skin Irritation Potential

Procedure

Protocol

Data

References

Human Skin Model Test: In Vitro Skin Corrosion (OECD 431)

Human Skin Model Test: in vitro Skin Corrosion EpiDerm™

Assessment of skin corrosion potential

Procedure

Protocol

Prediction

Prediction EpiDerm™

Data

References

In vitro Membrane Barrier Test Method for Skin Corrosion (OECD 435)

In vitro Membrane Barrier Test Method for Skin Corrosion

(Corrositex®)

Assessment of skin corrosion potential

Procedure

Protocol

Prediction

Data

References

Human Skin Model Test with Medical Devices: In Vitro Skin Irritation (ISO 10993-23)

Human Skin Model Test with Medical Devices: In Vitro Skin Irritation
(EpiDerm™ or SkinEthic™ RHE)

Assessment of Skin Irritation Potential of Medical Devices

Procedure

Protocol

References

Human Skin Model Test

Human Skin Model Test: In Vitro Skin Irritation (OECD 439)

Human Skin Model Test: In Vitro Skin Irritation

(EpiDerm™ and SkinEthic™ RHE)

Assessment of Skin Irritation Potential

Procedure

Protocol

Data

References

Human Skin Model Test: In Vitro Skin Corrosion (OECD 431)

Human Skin Model Test: in vitro Skin Corrosion EpiDerm™

Assessment of skin corrosion potential

Procedure

Protocol

Prediction

Prediction EpiDerm™

Data

References

In vitro Membrane Barrier Test Method for Skin Corrosion (OECD 435)

In vitro Membrane Barrier Test Method for Skin Corrosion

(Corrositex®)

Assessment of skin corrosion potential

Procedure

Protocol

Prediction

Data

References

Human Skin Model Test with Medical Devices: In Vitro Skin Irritation (ISO 10993-23)

Human Skin Model Test with Medical Devices: In Vitro Skin Irritation(EpiDerm™ or SkinEthic™ RHE)

Assessment of Skin Irritation Potential of Medical Devices

Procedure

Protocol

References

Human Skin Model Test with Medical Devices: In Vitro Skin Irritation
(EpiDerm™ or SkinEthic™ RHE)