Organisation for Economic Co-operation and Development

ENV/JM/MONO(2019)15
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28 August 2019

ENVIRONMENT DIRECTORATEJOINT MEETING OF THE CHEMICALS COMMITTEE AND THE WORKING PARTYON CHEMICALS, PESTICIDES AND BIOTECHNOLOGYREPORTS OF THE PEER REVIEWS OF THE epiCS AND SKIN+ TESTMETHODS IN VIEW OF THEIR INCLUSION IN TEST GUIDELINE 439 ON IN
VITRO SKIN IRRITATION
Series on Testing and Assessment
No. 299

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JT03450439

This document, as well as any data and map included herein, are without prejudice to the status of or sovereignty over any territory, to thedelimitation of international frontiers and boundaries and to the name of any territory, city or area.

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ENV/JM/MONO(2019)15  3

SERIES ON TESTING AND ASSESSMENT
NO. 299

REPORTS OF THE PEER REVIEWS OF THE epiCS AND SKIN+ TEST METHODS IN
VIEW OF THEIR INCLUSION IN TEST GUIDELINE 439 ON IN VITRO SKIN
IRRITATION

Environment Directorate
ORGANISATION FOR ECONOMIC COOPERATION AND DEVELOPMENT
Paris 2019

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ENV/JM/MONO(2019)15  5This publication is available electronically, at no charge.
For this and many other Environment,
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FOREWORD

This document contains two reports related to the Peer Reviews of the Epics and Skin+
Test Methods contained in Test Guideline 439:
Annex 1: epiCS Skin Irritation Test Methods ( CellSystems);

Annex 2: Skin+ Reconstructed human Epidermis assay ( Sterlab)

This document includes the peer-review reports of the validation of epiCS® and Skin+®
test methods. The two methods were validated following the Performance Standards for
similar methods or modified in vitro Reconstructed Human Epidermis (RHE) test methods
for skin irritation (No. 219 in the Series on Testing and Assessment). In 2018, Germany
and France jointly proposed the project to include these test methods in the Test Guideline439 on in vitro skin irritation (TG 439). Two independent peer-reviews were organised in
March and September 2018 for epiCS® and Skin+®, respectively. The supporting data foreach test method and the draft updated TG 439 were circulated for review and commentsin October. Comments received were addressed at the meeting of the dedicated Expert
Group in November 2018. The elements supporting the validation of the test methods are
currently available in different files available as individual annexes, upon request to theOECD Secretariat.
In April 2019, the Working Group of the National Coordinators of the Test Guidelines
Programme approved the updated TG 439, now including epiCS® and Skin+®, andendorsed the peer-review reports presented in this document.

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Annex 1: epiCs Skin Irritation Test Methods(Cellsystems)

1

PRP Evaluation

epiCS® SIT (CellSystems)

21 March 2018

epiCS® Skin Irritation Test method (CellSystems)
Expedite Peer-Review of the Performance Standard (PS)- based validation study according
to OECD GD 220 for similar or modified in vitro Reconstructed human Epidermis (RhE) test
methods for skin irritation testing as described in TG 439
Date: 21 March 2018

Summary
The epiCS® RhE model produced by CellSystems® (Germany) is proposed as a similar assay to thevalidated reference models falling within the OECD TG 439, and has undergone a performancestandard (PS)-based validation study for skin irritation testing according to OECD GD 220. The
assay underwent a first independent peer-review by the EURL-ECVAM Scientific Advisory
Committee (ESAC) which published its opinion and recommendations in June 2016. Based on the
ESAC recommendations, CellSystems® gathered additional information (in particular on technicalissues encountered during the PS-based validation study and on trans-continental tissuetransportation). Following availability of these additional information, a further expedited
independent peer-review took place. The peer-review was coordinated by SeCAM and seh
consulting+services, in close collaboration with the National Coordinators representing France andGermany at the OECD Working Group of the National Coordinators of the Test Guidelines
Programme (WNT). The similarity of the two me-too assays to the validated reference method, as
well as their scientific validity were assessed by the international peer-review panel (PRP)
composed of:- Dave Allen (ILS, Contractor supporting NICEATM, USA)- Hajime Kojima (JaCVAM, NIHS, Japan)- Ilyoung Ahn (KoCVAM, NIFDS, Republic of Korea)- Uwe Pfannenbecker (Beiersdorf, Germany)- John Harbell (JHarbell Consulting LLC, USA)The criteria for peer-review evaluation were prepared by SeCAM and by the National Coordinatorsfrom France and Germany and were revised by the PRP members. The PRP members external to
the OECD WNT provided with a declaration of interest that was made available to the WNT. The
peer-review took place from November 2017 to February 2018, during which one initialteleconference took place between the PRP and CellSystems (where additional information was
requested to CellSystems), and two additional teleconferences took place in the absence of
CellSystems.
Based on its evaluation (see detailed criteria and evaluation below), the PRP is of the opinion
that the information made available to them do support the scientific similarity of the epiCS®
RhE assay to the validated reference method both in terms of the essential test method
components and of assay performance regarding its reproducibility and predictive capacity
as described within the GD 220. 

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PRP Evaluation

epiCS® SIT (CellSystems)

21 March 2018

Evaluation criterion 1: Rationale for the test method, including a description of theadvantages of the similar or modified test method in terms of i) mechanistic advantages,
applicability, predictive capacity, technical advances, reduction in hazardous reagents, ii) IP
rights, geographical availability and animal welfare, iii) costs, analysis time, sample amount,competitiveness, iv) others.
An additional model can favour market competitiveness and ensure the availability of models e.g., incase of cease of production or regional unavailability of a model. The epiCS® RhE assay offers thesame mechanistic, technical and animal welfare advantages as the currently adopted test methods
falling within TG 439. Based on the information provided by the tissue developers, its costs are also
within the range of costs from the currently adopted RhE models falling within TG 439.
Evaluation criterion 2: A detailed protocol for the similar or modified test method should beavailable.
A detailed protocol of the similar test method is available and was considered to be adequate by thePeer Review Panel (PRP).


Evaluation criterion 3: Adherence to the essential test method components as described inparagraphs 6 to 23 of GD 220 should be demonstrated for the similar or modified test
methods regarding i.e., the general conditions, the functional conditions and the procedural
conditions. Note: For specific parameters or modified procedures, adequate values or proceduresshould be provided for the proposed similar or modified test method (e.g. Tables 1 and 2 of GD
220).
The PRP considered the similarity of the epiCS® RhE assay in terms of essential test method
components, to be adequate and sufficient and recommends the tissue developer to continue toassess, as a tissue batch production release criteria, the barrier function of the epiCS® RhE tissue
batches based on at least two time-points (one above and one beneath the ET50 value), as
implemented in January 2018 and reported in the certificate of analysis provided to the PRP(Annexes 1 and 2).
Evaluation criterion 4: In addition, for modified test methods, the toxicological mechanisms
and the relationship between the test method endpoint(s) with the biological effect as well as
the toxicity of interest should be addressed, describing limitations of the test method.
The criterion is not applicable as the epiCS® RhE model does not represent a modified test method,
but a functionally and mechanistically similar assay.
Evaluation criterion 5: At least the 20 recommended reference chemicals within GD 220
should be tested with the similar or modified test method according to recommendations ofparagraphs 24, 25, 26 and 31 of GD 220, to demonstrate reliability and accuracy. I.e., testingshould be conducted in at least three independent laboratories if the similar or modified test method
is proposed to be used in several laboratories. In each laboratory, each chemical should be tested
in three independent runs performed with different tissue batches and at sufficiently spaced time
points. Each run should consist of at least three concurrently tested tissue replicates. Furthermore,

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PRP Evaluation

epiCS® SIT (CellSystems)

21 March 2018

additional chemicals representing other chemical classes and for which adequate in vivo reference
data are available may be tested in addition to the minimum list of reference chemicals.
Note: the use of the reference chemicals for development/optimization of the proposed similar or
modified test method should be avoided to the extent possible.
Based on the information provided to the PRP, a total of 44 test chemicals were used for thedevelopment of the epiCS® RhE assay. The list provided to the PRP encompassed 19 reference
chemicals (9 Cat. 2, 3 Cat. 3 and 7 No Cat. according to the UN GHS classification scheme), and25 additional chemicals distributed as follows:- 4 UN GHS Cat. 2 chemicals- 10 UN GHS Cat. 3 chemicals (EU CLP No Cat.)- 7 UN GHS No Cat. chemicals- 4 chemicals with unclear identification and/or in vivo UN GHS classification
From these 25 chemicals, 22 corresponded to chemicals used in the validation, optimization andfollow-up studies of the test methods adopted within TG 439 1,2,3 whereas 3 of the 4 chemicals
having unclear identification and/or in vivo classification came from other sources. Out of the test
chemicals having a clear identification and in vivo classification (19 reference chemicals and 21
additional chemicals), a total of 7 were solid test chemicals (4 of which are reference chemicals)
representing circa 18% (7 out of 40). For comparison, solids represent 20% of the reference
chemicals (4 out of 20) and 25% of the chemicals used in the skin irritation validation study (25 outof 60). Finally, although based on a different dataset, the predictive capacity obtained with the 40
chemicals having a clear identification and in vivo classification was of the same order of magnitude
as the currently adopted RhE models (based on the dataset collected from literature).
Based on the above information and the testing of 21 chemicals having clear identification and invivo classification in addition to the reference chemicals, the PRP considers the information
provided as sufficient. It would have been ideal however, to have a number of chemicals, different
from the chemicals used to develop the model, tested in a blind manner.
Evaluation criterion 6: The reliability obtained with the reference chemicals should be equal
to or better than the defined minimum target values for the similar or modified test method
as specified in paragraphs 28 and 29 of GD 220, and the analyses of reliability should be
conducted according to the specifications described in paragraph 27 of GD 220.
In its evaluation, ESAC considered that the epiCS did not meet the criteria for within-laboratory
reproducibility in one of the participating laboratory situated across ocean (89% (16/18) versus 90%
as required by the performance standards) whereas the within-laboratory reproducibility in the twoother laboratories and the between-laboratory reproducibility were considered appropriate. This wasdue to the fact that GD 220 allows for a maximum of 5 test runs for each chemical, whereas in thelaboratory in question, two chemicals had more than 5 test runs. As a consequence, ESAC did notaccount for the additional runs, and considered the two chemicals to have incomplete test
ϭƐŬĞƐ͕ŽůĞd͕,ŽĨĨŵĂŶŶ^͕tŽƌƚŚ͕ŽĐŬƐŚŽƚƚ͕'ĞƌŶĞƌ/͕ƵĂŶŐs;ϮϬϬϳͿ͘dŚĞsDŝŶƚĞƌŶĂƚŝŽŶĂůǀĂůŝĚĂƚŝŽŶƐƚƵĚǇŽŶŝŶǀŝƚƌŽƚĞƐƚƐĨŽƌĂĐƵƚĞƐŬŝŶŝƌƌŝƚĂƚŝŽŶ͗ƐĞůĞĐƚŝŽŶŽĨƚĞƐƚĐŚĞŵŝĐĂůƐ͘d>ϯϱ͕ϲϬϯͲϲϭϵ͘Ϯ<ĂŶĚĄƌŽǀĄ,͕,ĂǇĚĞŶW͕<ůĂƵƐŶĞƌD͕<ƵďŝůƵƐ:͕<ĞĂƌŶĞǇW͕͘^ŚĞĂƐŐƌĞĞŶ:͘;ϮϬϬϵͿ͘/ŶsŝƚƌŽ^ŬŝŶ/ƌƌŝƚĂƚŝŽŶdĞƐƚŝŶŐ͗/ŵƉƌŽǀŝŶŐƚŚĞ^ĞŶƐŝƚŝǀŝƚǇŽĨƚŚĞƉŝĞƌŵ^ŬŝŶ/ƌƌŝƚĂƚŝŽŶdĞƐƚWƌŽƚŽĐŽů͘d>ϯϳ͗ϲϳϭʹϲϴϵ͘ϯŽƚŽǀŝŽ:͕͘'ƌĂŶĚŝĚŝĞƌD͘Ͳ,͕͘WŽƌƚĞƐW͕͘ZŽŐƵĞƚZ͕͘ZƵďŝŶƐƚĞŶŶ'͘;ϮϬϬϱͿ͘dŚĞŝŶǀŝƚƌŽĂĐƵƚĞƐŬŝŶŝƌƌŝƚĂƚŝŽŶŽĨĐŚĞŵŝĐĂůƐ͗ŽƉƚŝŵŝƐĂƚŝŽŶŽĨƚŚĞW/^</EƉƌĞĚŝĐƚŝŽŶŵŽĚĞůǁŝƚŚŝŶƚŚĞĨƌĂŵĞǁŽƌŬŽĨƚŚĞsDǀĂůŝĚĂƚŝŽŶƉƌŽĐĞƐƐ͘d>ϯϯ͕ϯϮϵͲϯϰϵ͘
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epiCS® SIT (CellSystems)

PRP Evaluation

21 March 2018

sequences (Annex 3). However, the trans-continental laboratory was able to demonstrate that theadditional runs were performed due to technical errors (e.g., contamination and non-exposure to thepositive control [Annexes 4 and 5]).
The present PRP is of the opinion that technical issues (such as contamination) are not a reflectionof the performance of the RhE model, so that it should not be taken into account when comparing
the scientific similarity between assays. Furthermore, as suggested within the originally proposed
performance standards developed by EURL-ECVAM from 2009, a margin of tolerance shall be
accepted when comparing the performance of similar assays especially when scientifically justified.
When the test runs having technical issues are not considered for the comparison of similarity, thewithin-laboratory reproducibility of the trans-continental laboratory and between-laboratory
reproducibility do fall within the requirements of the GD 220 (see table below as extracted fromAnnex 6). Furthermore, following the ESAC peer-review, the tissue developer has optimized theconditions for trans-continental transportation, leading to controlled temperature conditions, andsimulation experiments indicated appropriate cell viability with 1% Triton X-100, used to assessbarrier function (Annex 7).
Based on the above considerations, the present PRP considers that the reliability of the epiCS®
RhE assay obtained with the reference chemicals (summarized below) to be sufficient. Furthermore,
it suggests that in case future trans-continental transportation is conducted, that the barrier functionof the tissues is assessed by measuring viability to 1% Triton X-100 for at least two time-point (i.e.,
one above and one beneath the ET50) in order to confirm the barrier properties of the tissues
transported across continents.
ĞƉŝ^®ZŚ

W^ƚĂƌŐĞƚǀĂůƵĞƐtŝƚŚŝŶͲůĂďŽƌĂƚŽƌǇƌĞƉƌŽĚƵĐŝďŝůŝƚǇϵϱй;ϭϴͬϭϵͿ͕ϭϬϬй;ϮϬͬϮϬͿ͕ϵϬй;ϭϴͬϮϬͿшϵϬй

ĞƚǁĞĞŶͲůĂďŽƌĂƚŽƌǇƌĞƉƌŽĚƵĐŝďŝůŝƚǇϴϰй;ϭϲͬϭϵͿшϴϬй

йŽĨĐŽŵƉůĞƚĞƌƵŶƐĞƋƵĞŶĐĞƐͬůĂď͘ϵϱй͕ϭϬϬй͕ϭϬϬйшϴϱйϵϴйшϵϬй

йŽĨĐŽŵƉůĞƚĞƌƵŶƐĞƋƵĞŶĐĞƐŽǀĞƌƚŚĞϯůĂďƐ

Evaluation criterion 7: The predictive capacity obtained with the reference chemicals shouldbe equal to or better than the defined minimum target values for the similar or modified test
method as specified in paragraph 30 of GD 220, and the analyses of predictive capacity
should be conducted according to the specifications described in paragraph 27 of GD 220.
The PRP considered the predictive capacity of the epiCS® RhE assay obtained with the reference
chemicals (summarized below) to be adequate and sufficient.
ĞƉŝ^®ZŚ
;ĂǀĞƌĂŐĞϯůĂďƐΎͿ

W^ƚĂƌŐĞƚǀĂůƵĞƐ^ĞŶƐŝƚŝǀŝƚǇ;ŶсϭϬͿϴϳйшϴϬй

^ƉĞĐŝĨŝĐŝƚǇ;ŶсϭϬͿϴϬйшϳϬй

ĐĐƵƌĂĐǇ;ŶсϮϬͿϴϯйшϳϱй

ΎĂƐĐĂůĐƵůĂƚĞĚǁŝƚŚŝŶƚŚĞhZ>ͲsDƚĞŵƉůĂƚĞ;ŶŶĞǆϲͿ

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PRP Evaluation

epiCS® SIT (CellSystems)

21 March 2018

Evaluation criterion 8: The applicability domain of the new or modified test method should
be defined.
A total of 21 test chemicals with clear identification and in vivo classification have been tested inaddition to the 20 reference chemicals, providing information on the applicability domain of theepiCS® RhE similar assay, so that the provided information was considered to be sufficient by the
PRP.
Evaluation criterion 9: All data from the PS-based validation study supporting the validity ofthe similar or modified test method should be obtained in accordance with the principles ofGood Laboratory Practice (GLP).
According to the information provided to the PRP, the study was conducted according to GLP
principles.
Evaluation criterion 10: Completeness of all data and documents supporting the assessmentof the validity of the similar or modified test method.
The information provided by the test method developer was considered to be sufficient for theassessment of the validity of the epiCS® RhE assay.
Evaluation criterion 11: PS-based validation study management and conduct.
The PRP considered the information provided on the study management conduct to be adequate
and sufficient.
Evaluation criterion 12: Other considerations: A) quality control procedures for lot release;B) audit of tissue production.
A) The PRP considered the quality control procedures for lot release sufficient provided that thetissue developer continues to assess the barrier function of the epiCS® RhE tissue batches, basedon at least two time-points (i.e., one above and one below the ET50 value), similarly to the certificate
of analysis provided to the PRP from the 8 and 22 January 2018 (Annexes 1 and 2).
B) Based on the information provided to the PRP, the production of the epiCS® RhE tissues has
been independently audited within the framework of an ISO 9001:2008 certification (Annex 8).
Evaluation criterion 13: All data should adequately support the peer review assessment thatthe proposed test method is structurally and mechanistically similar to the validated
reference method, and demonstrate sufficient reliability and relevance for the proposed
specific testing purpose i.e., that the proposed similar or modified test method isscientifically valid.
The data assessed by the peer-review panel supports the scientific similarity of the epiCS® RhE
assay to the validated reference method both in terms of the essential test method components andthe performances of the assay in terms of reproducibility and predictive capacity as described within
the GD 220.

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PRP Evaluation

epiCS® SIT (CellSystems)

21 March 2018

List of Annexes
Annex 1: Certificate of Analyses of the epiCS® RhE from 8 January 2018
Annex 2: Certificate of Analyses of the epiCS® RhE from 22 January 2018
Annex 3: ESAC opinion on the epiCS® RhE performance-based validation study for in vitro skinirritation testing from 24 June 2016 (with relevant text highlighted in yellow)
Annex 4: Letter from US-based laboratory explaining technical issues encountered during testingfrom 1st September 2016
Annex 5: Pictures of the invalid runs (December 2017)
Annex 6: epiCS® RhE results according to the EURL-ECVAM reporting template (from October
2015)
Annex 7: CellSystems’ responses to the PRP questions regarding transportation issues & qualityof the model from November 2017
Annex 8: ISO 9001:2008 certificate including ‘production of research products’

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

Annex 2: Skin+ Reconstructed human Epidermis
assay (Sterlab)

1

Skin+® Reconstructed human Epidermis assay (Sterlab)
Expedite Peer-Review of the Performance Standard (PS)- based validation study according
to OECD GD 220 for similar or modified in vitro Reconstructed human Epidermis (RhE) test
methods for skin irritation testing as described in TG 439
Date: 13 September 2018

Summary
The Skin+® Reconstructed human Epidermis (RhE) model produced by Sterlab (France) is
proposed as a similar assay to the validated reference models falling within the OECD TG 439, andhas undergone a performance standard (PS)-based validation study for skin irritation testingaccording to OECD GD 220. The PS-based validation study was submitted in June 2015 to EURLECVAM, which provided an assessment report of the test submission in December 2015 (Annex 1).
In this report a number of questions were raised including the need to have a statistical report of thePS-based study, to explain the discrepancies observed in data entry and data analyses and theneed to clarify which chemicals were used to define the final version of the SOP. Based on theserecommendations, Sterlab prepared a reply (Annex 2), and contracted out an independent
biostatistician that went over the PS-based validation study results, to check and correct for any
data discrepancy, and to provide an independent biostatistical report (Annex 3).
Following availability of this additional information, the present expedite independent peer-review
took place. The peer-review was coordinated by SeCAM and seh consulting+services, in close
collaboration with the National Coordinators representing France and Germany at the OECD
Working Group of the National Coordinators of the Test Guidelines Programme (WNT). The
similarity of the me-too assay to the validated reference method, as well as its scientific validity wasassessed by the international Peer Review Panel (PRP) composed of:- Dave Allen (ILS, Contractor supporting NICEATM, USA)- Hajime Kojima (JaCVAM, NIHS, Japan)- Ilyoung Ahn (KoCVAM, NIFDS, Republic of Korea)- Uwe Pfannenbecker (Beiersdorf, Germany)- John Harbell (JHarbell Consulting LLC, USA)The criteria for peer-review evaluation were prepared by SeCAM and by the National Coordinatorsfrom France and Germany and were revised by the PRP members. The PRP members external to
the OECD WNT provided with a declaration of interest that was made available to the WNT. The
peer-review took place from November 2017 to July 2018, during which two teleconferences took
place between the PRP and Sterlab (where additional information was requested to Sterlab), andthree additional teleconferences took place in the absence of Sterlab.
Based on its evaluation (see detailed criteria and evaluation below), the PRP is of the opinion
that the information made available for peer-review support the scientific similarity of the
Skin+® RhE assay, when conducted within Europe, to the validated reference method both interms of the essential test method components and of assay performance regarding its
reproducibility and predictive capacity as described within the GD 220. In case transcontinental transportation is foreseen, it is recommended that the barrier function of the tissues is

PRP Evaluation

Skin+® RhE model (Sterlab)

13 September 2018
assessed after the trans-continental transportation in order to confirm the barrier properties of thetissues transported across continents1.

Evaluation criterion 1: Rationale for the test method, including a description of theadvantages of the similar or modified test method in terms of i) mechanistic advantages,
applicability, predictive capacity, technical advances, reduction in hazardous reagents, ii) IP
rights, geographical availability and animal welfare, iii) costs, analysis time, sample amount,competitiveness, iv) others.
An additional model can favour market competitiveness and ensure the availability of models e.g., incase of cease of production or regional unavailability of a model. The Skin+® RhE assay offers thesame mechanistic, technical and animal welfare advantages as the currently adopted test methods
falling within TG 439. The Peer Review Panel (PRP) could not make an economical comparison as
it had no access to the costs of the Skin+® RhE assay. Furthermore, regarding the geographicalavailability of the tissue, the PRP had no access to information regarding the functional conditions of
the tissues after trans-continental shipment1. It is therefore recommended that in case transcontinental transportation is foreseen, that the barrier function of the tissues is assessed bymeasuring viability to 1% Triton X-100 for at least two time-points (i.e., one above and one beneaththe ET50) after the trans-continental transportation (as described under evaluation criterion 12.A) inorder to confirm the barrier properties of the tissues transported across continents1.
Evaluation criterion 2: A detailed protocol for the similar or modified test method should beavailable.
A detailed protocol of the similar test method is available and was considered to be adequate by the
PRP (Annex 4).


Evaluation criterion 3: Adherence to the essential test method components as described inparagraphs 6 to 23 of GD 220 should be demonstrated for the similar or modified test
methods regarding i.e., the general conditions, the functional conditions and the procedural
conditions. Note: For specific parameters or modified procedures, adequate values or proceduresshould be provided for the proposed similar or modified test method (e.g. Tables 1 and 2 of GD
220).
The PRP considered the similarity of the Skin+® RhE assay in terms of essential test method
components, to be adequate and sufficient.
Evaluation criterion 4: In addition, for modified test methods, the toxicological mechanisms
and the relationship between the test method endpoint(s) with the biological effect as well as
the toxicity of interest should be addressed, describing limitations of the test method.
The criterion is not applicable as the Skin+® RhE assay does not represent a modified test method,
but a similar assay.

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WŽƐƚͲĞǀĂůƵĂƚŝŽŶŶŽƚĞ͗ƚŚĞWZWǁĂƐŝŶĨŽƌŵĞĚƚŚĂƚĂƐƚƵĚǇŽŶƚƌĂŶƐĂƚůĂŶƚŝĐƐŚŝƉŵĞŶƚŽĨƚŚĞƚŝƐƐƵĞƐŝƐďĞŝŶŐĐŽŶĚƵĐƚĞĚ͘
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PRP Evaluation

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13 September 2018


Evaluation criterion 5: At least the 20 recommended reference chemicals within GD 220
should be tested with the similar or modified test method according to recommendations ofparagraphs 24, 25, 26 and 31 of GD 220, to demonstrate reliability and accuracy. I.e., testingshould be conducted in at least three independent laboratories if the similar or modified test method
is proposed to be used in several laboratories. In each laboratory, each chemical should be tested
in three independent runs performed with different tissue batches and at sufficiently spaced time
points. Each run should consist of at least three concurrently tested tissue replicates. Furthermore,
additional chemicals representing other chemical classes and for which adequate in vivo reference
data are available may be tested in addition to the minimum list of reference chemicals. Note: theuse of the reference chemicals for development/optimization of the proposed similar or modified test
method should be avoided to the extent possible.
The test method developer states in its answers to PRP questions from 21 November 2017, that the
20 reference chemicals listed in the OECD GD 220 have been used to develop the RhE test
method, and that no additional chemicals to these 20 reference chemicals have been tested withthe model. GD 220 states however that “the exclusive use of the reference chemicals fordevelopment/optimization of the proposed similar or modified test method should be avoided to theextent possible”. Furthermore, TG 439 (both versions from 2013 and 2010) states that “TheReference Chemicals represent the minimum number of chemicals that should be used to evaluate
the accuracy and reliability of a proposed similar or modified test method, but should not be used forthe development of new test methods.”
Based on the above considerations, the PRP recommended in February 2018 that additional
chemicals are tested with the Skin+® RhE assay such as the test chemicals used during thevalidation, optimization and follow-up studies of the test methods adopted within TG 4392,3,4 in atleast one laboratory and preferentially in a blind manner, in order to gain further information on thepredictive capacity and applicability domain of the assay.
Following this recommendation, Sterlab proceeded with the testing of additional 17 test chemicals
that were selected by the PRP in collaboration with an independent biostatistician (Els Adriaens)and an independent test chemical distribution facility (VITO, Belgium). These test chemicals
encompassed 6 UN GHS Cat. 2 chemicals, 3 UN GHS Cat. 3 (EU CLP No Cat.) chemicals and 8
UN GHS No Cat. chemicals. The 17 additional chemicals were tested under blind conditions by
Sterlab and results were summarized by the independent biostatistician (Annex 5).
Based on the above information and the blind testing of the additional 17 chemicals having clear
identification and in vivo classification in addition to the reference chemicals, the PRP considered
the information provided as sufficient.

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ϮƐŬĞƐ͕ŽůĞd͕,ŽĨĨŵĂŶŶ^͕tŽƌƚŚ͕ŽĐŬƐŚŽƚƚ͕'ĞƌŶĞƌ/͕ƵĂŶŐs;ϮϬϬϳͿ͘dŚĞsDŝŶƚĞƌŶĂƚŝŽŶĂůǀĂůŝĚĂƚŝŽŶƐƚƵĚǇŽŶŝŶǀŝƚƌŽƚĞƐƚƐĨŽƌĂĐƵƚĞƐŬŝŶŝƌƌŝƚĂƚŝŽŶ͗ƐĞůĞĐƚŝŽŶŽĨƚĞƐƚĐŚĞŵŝĐĂůƐ͘d>ϯϱ͕ϲϬϯͲϲϭϵ͘ϯ<ĂŶĚĄƌŽǀĄ,͕,ĂǇĚĞŶW͕<ůĂƵƐŶĞƌD͕<ƵďŝůƵƐ:͕<ĞĂƌŶĞǇW͕͘^ŚĞĂƐŐƌĞĞŶ:͘;ϮϬϬϵͿ͘/ŶsŝƚƌŽ^ŬŝŶ/ƌƌŝƚĂƚŝŽŶdĞƐƚŝŶŐ͗/ŵƉƌŽǀŝŶŐƚŚĞ^ĞŶƐŝƚŝǀŝƚǇŽĨƚŚĞƉŝĞƌŵ^ŬŝŶ/ƌƌŝƚĂƚŝŽŶdĞƐƚWƌŽƚŽĐŽů͘d>ϯϳ͗ϲϳϭʹϲϴϵ͘ϰŽƚŽǀŝŽ:͕͘'ƌĂŶĚŝĚŝĞƌD͘Ͳ,͕͘WŽƌƚĞƐW͕͘ZŽŐƵĞƚZ͕͘ZƵďŝŶƐƚĞŶŶ'͘;ϮϬϬϱͿ͘dŚĞŝŶǀŝƚƌŽĂĐƵƚĞƐŬŝŶŝƌƌŝƚĂƚŝŽŶŽĨĐŚĞŵŝĐĂůƐ͗ŽƉƚŝŵŝƐĂƚŝŽŶŽĨƚŚĞW/^</EƉƌĞĚŝĐƚŝŽŶŵŽĚĞůǁŝƚŚŝŶƚŚĞĨƌĂŵĞǁŽƌŬŽĨƚŚĞsDǀĂůŝĚĂƚŝŽŶƉƌŽĐĞƐƐ͘d>ϯϯ͕ϯϮϵͲϯϰϵ͘
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Skin+® RhE model (Sterlab)

PRP Evaluation

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Evaluation criterion 6: The reliability obtained with the reference chemicals should be equal
to or better than the defined minimum target values for the similar or modified test method
as specified in paragraphs 28 and 29 of GD 220, and the analyses of reliability should be
conducted according to the specifications described in paragraph 27 of GD 220.
The RPR considered the reliability of the Skin+® RhE assay obtained with the reference chemicals(summarized below) to be adequate and sufficient.
^ŬŝŶн®ZŚĂƐƐĂǇW^ƚĂƌŐĞƚǀĂůƵĞƐtŝƚŚŝŶͲůĂďŽƌĂƚŽƌǇƌĞƉƌŽĚƵĐŝďŝůŝƚǇϵϱй;ϭϵͬϮϬͿ͕ϵϱй;ϭϵͬϮϬͿ͕ϵϬй;ϭϴͬϮϬͿшϵϬй

ĞƚǁĞĞŶͲůĂďŽƌĂƚŽƌǇƌĞƉƌŽĚƵĐŝďŝůŝƚǇϭϬϬй;ϮϬͬϮϬͿшϴϬй

йŽĨĐŽŵƉůĞƚĞƌƵŶƐĞƋƵĞŶĐĞƐͬůĂď͘ϭϬϬй͕ϭϬϬй͕ϭϬϬйшϴϱйϭϬϬйшϵϬй

йŽĨĐŽŵƉůĞƚĞƌƵŶƐĞƋƵĞŶĐĞƐŽǀĞƌƚŚĞϯůĂďƐ

Furthermore, when combining the results of reference chemicals with the additional 17 coded test
chemicals, a within-laboratory concordance of predictions of 91.7% (33/36) was found within the
Sterlab laboratory.
Evaluation criterion 7: The predictive capacity obtained with the reference chemicals shouldbe equal to or better than the defined minimum target values for the similar or modified test
method as specified in paragraph 30 of GD 220, and the analyses of predictive capacity
should be conducted according to the specifications described in paragraph 27 of GD 220.
The PRP considered the predictive capacity of the Skin+® RhE assay obtained with the reference
chemicals (summarized below) to be adequate and sufficient.
^ŬŝŶн®ZŚĂƐƐĂǇ;ĂǀĞƌĂŐĞϯůĂďƐͿ

W^ƚĂƌŐĞƚǀĂůƵĞƐ^ĞŶƐŝƚŝǀŝƚǇ;ŶсϭϬͿϭϬϬйшϴϬй

^ƉĞĐŝĨŝĐŝƚǇ;ŶсϭϬͿϳϬйшϳϬй

ĐĐƵƌĂĐǇ;ŶсϮϬͿϴϱйшϳϱйΎĂƐĐĂůĐƵůĂƚĞĚǁŝƚŚŝŶƚŚĞhZ>ͲsDƚĞŵƉůĂƚĞ

Furthermore, when combining the results of the reference chemicals with the additional 17
chemicals tested in a blind manner, a sensitivity of 93.8% (15/16), a specificity of 76.2% (16/21) andan accuracy of 83.8% (31/37) were found.
Evaluation criterion 8: The applicability domain of the new or modified test method should
be defined.
The PRP is of the opinion that, based on the data presented with the reference chemicals and on
the results obtained with the additional 17 test chemicals tested in a blind manner (see criterion 5, 6
and 7), there is no reason to consider the applicability domain of the Skin+® RhE to be different than
the applicability domain of the test methods adopted within TG 439.
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PRP Evaluation

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Evaluation criterion 9: All data from the PS-based validation study supporting the validity ofthe similar or modified test method should be obtained in accordance with the principles ofGood Laboratory Practice (GLP).
Based on the information provided to the PRP, the study was conducted according to GLP
principles.

Evaluation criterion 10: Completeness of all data and documents supporting the assessmentof the validity of the similar or modified test method.
The information provided by the test method developer was considered to be sufficient for theassessment of the validity of the Skin+® RhE assay.
Evaluation criterion 11: PS-based validation study management and conduct.
The PS-based validation study of the Skin+® RhE assay attempted to follow the currently accepted
standards for validation study. The PRP considered the information provided on the study
management and conduct to be sufficient. However, it is noted that the coding of test chemicals
during the PS-based validation study could have been better randomized. In addition, the EURLECVAM evaluation from December 2015 stated that “a discrepancy of viability values” was found for
two of the three participating laboratories. EURL-ECVAM indicated however that this discrepancy
had “no influence on the predictions yielded” and that “the differences are mostly in the decimalrange...”, “...however too large to be explained as rounding error...”. Sterlab answered that such a
discrepancy may be due to the use of different softwares (Microsoft Excel and Libre Office). The
present PRP was informed that following the EURL-ECVAM evaluation, all data templates werereviewed, and the viability values were corrected when necessary by Sterlab’s technician.
Furthermore, an independent statistician analyzed the data and prepared a statistical report (Annex
3). During its evaluation, the PRP noticed that the data reported in the independent statistical report
did correspond to the data reported in the EURL-ECVAM template provided to the PRP by the testdeveloper. 
Evaluation criterion 12: Other considerations: A) quality control procedures for lot release;B) audit of tissue production.
A) The PRP considered the quality control procedures for lot release sufficient (Annexes 6a, 6b and6c) provided that the tissue developer continues to:- assess the barrier function of the Skin+® RhE tissue batches based on a sufficient number of time
points that cover the ET50 acceptability range (4.0  ET50 (hr)  9.0) such as for example 0, 2, 4 and9 hours;- calculates the ET50 based on one time-point just above and one time-point just below the ET50 andnot based on a linear correlation, since the correlation between time of exposure and cell viability
may not always follow a linear correlation.

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PRP Evaluation

Skin+® RhE model (Sterlab)

13 September 2018

B) Based on the information provided to the PRP, the production of the Skin+® RhE tissues has
been independently audited within the framework of an ISO 9001:2008 certification (Annexes 7 and8).
Evaluation criterion 13: All data should adequately support the peer review assessment thatthe proposed test method is structurally and mechanistically similar to the validated
reference method, and demonstrate sufficient reliability and relevance for the proposed
specific testing purpose i.e., that the proposed similar or modified test method isscientifically valid.
The data assessed by the peer-review panel supported the scientific similarity of the Skin+® RhE
assay, when the method is conducted within Europe, to the validated reference method both interms of the essential test method components and of assay performance regarding its
reproducibility and predictive capacity as described within the GD 220. In case trans-continental
transportation is foreseen, it is recommended that that the barrier function of the tissues is assessed
after the trans-continental transportation in order to confirm the barrier properties of the tissues
transported across continents5.

List of Annexes
Annex 1: EURL-ECVAM assessment report on the Skin® RhE PS-validation study from 17 Dec.
2015
Annex 2: Reply from Sterlab to EURL-ECVAM dated from May 2016
Annex 3: Independent statistical report on the Skin® RhE PS-validation study from 1st July 2016
Annex 4: Standard Operating Procedures (SOP) of the Sterlab’s Skin® RhE test method for skinirritation testingAnnex 5: Independent statistical report on the Skin® RhE testing of additional blind chemicals fromMay 2018
Annex 6a: Certificate of Analyses of the Skin® RhE model from 10 April 2018 and 19 March 2018
Annex 6b: ET50 calculations for the batch from 10 April 2018
Annex 6c: Calculations for the batch from 19 March 2018
Annex 7: ISO 9001:2008 certificate including ‘production of human or animal tissues, rebuilt in
vitro, in aim of tests’ from 6 January 2012
Annex 8: ISO 9001:2008 certificate including ‘production of human or animal tissues, rebuilt in
vitro, in aim of tests’ from 7 January 2015
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