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English - Or. English
24 July 2023

ENVIRONMENT DIRECTORATECHEMICALS AND BIOTECHNOLOGY COMMITTEE

Epidermal Sensitisation Assay (EpiSensA) Peer review Report
Series on Testing and Assessment
No. 383

JT03523614
OFDE

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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OECD Environment, Health and Safety Publications
Series on Testing & Assessment
No. 383

Epidermal Sensitisation Assay (EpiSensA) Peer review Report

Environment Directorate
ORGANISATION FOR ECONOMIC COOPERATION AND DEVELOPMENT
Paris 2023

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ENV/CBC/MONO(2023)19  5This publication is available electronically, at no charge.

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FOREWORD

This document contains the Peer review report of the Epidermal Sensitisation Assay (EpiSensA),
an in vitro method for identifying the skin sensitisation potential of chemicals.
The scientific validity of the EpiSensA was assessed by an international Peer Review Panelbetween June and November 2022. The peer review was led by the Japanese Center for the
Validation of Alternative Methods (JaCVAM) and supported by the Japanese Society for
Alternatives to Animal Experiments (JSAAE).
The peer review report supports the development of the corresponding test method for inclusion
in Test Guideline 442D for in vitro skin sensitisation assays, which assesses the Adverse
Outcome Pathway Key Event on Keratinocyte activation. The Peer review report and the
Validation report of the EpiSensA were circulated for comments to the Working Party of the
National Coordinators of the Test Guidelines Programme (WNT) in December 2022. The WNTendorsed the peer review report at its 35th meeting in April 2023. The WNT also endorsed the
Validation report of the EpiSensA.
This report is published under the responsibility of the Chemicals and Biotechnology Committee.

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EpiSensA
Report of the Peer Review Panelon

a JaCVAM coordinated study program addressing the validation
status of the EpiSensA for prospective identification of skinsensitizing chemicals

Report completed by the Peer Review Panel on November 3, 2022. The
report was revised in March 2023 with the inclusion of additional
clarifications to address the comments from the WNTEPIDERMAL SENSITISATION ASSAY (EPISENSA) PEER REVIEW REPORT
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Table of Contents

FOREWORD

6

Executive Summary

9

Peer Review Panel Composition

10

Background11

Evaluation Criterion

13

Evaluation Criterion 1: A rationale for the test method should be available, including a description of thehuman health effect, a clear statement of scientific need, and regulatory application.
Evaluation Criterion 2: The toxicological mechanisms and the relationship between the test method
endpoint(s) with the biological effect and the toxicity of interest should be addressed, describing thelimitations of the test method.
Evaluation Criterion 3: A detailed test method protocol should be available. Transferability to other labs
should be demonstrated.
Evaluation Criterion 4: The within and between laboratory reproducibility of the test method should bedemonstrated.
Evaluation Criterion 5: Demonstration of the test method’s performance should be based on testing adiversity of chemicals, preferably coded reference chemicals.
Evaluation Criterion 6: Predictive capacity should be demonstrated using representative chemicals.
Evaluation Criterion 7: All data from the validation study supporting the validity of a test method should
be obtained in accordance with the principles of Good Laboratory Practice (GLP).
Evaluation Criterion 8: The applicability domain of the test method should be defined.
Evaluation Criterion 9: Proficiency chemicals should be set up in the proposed protocol.
Evaluation Criterion 10: Performance standards should be set up with the proposed protocol.
Evaluation Criterion 11: Advantages in terms of time, cost, and animal welfare should be described.
Evaluation Criterion 12: Completeness of all data and documents supporting the assessment of thevalidity of the test method.
Evaluation Criterion 13: Validation Study should be managed and conducted adequately.

13

13
14
15
15
16
17
17
18
18
19
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Recommendations and Further Considerations

20

Conclusion

22

Acknowledgments

23

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ENV/CBC/MONO(2023)19  9Executive Summary
The EpiSensA has been proposed as an in vitro skin sensitization alternative method, providing
information on key event 2 (KE2) in the adverse outcome pathway. The EpiSensA Peer Review
Panel (PRP) believes the validation study led by the Japanese Center for the Validation of
Alternative Methods (JaCVAM) with the cooperation of test developers was successful. The
assay is a reconstructed human epidermis (RhE) model that demonstrates advantages for testingof lipophilic compounds and pre- and pro-haptens. The validation study conducted for the
EpiSensA demonstrated the assay's applicability, transferability, and inter- and intra-laboratory
reproducibility. Both within- and between-laboratory reproducibility easily met the pre-specified
85% and 80% thresholds, respectively. The proficiency chemicals and individual assay
performance measures have been provided along with a detailed Standard Operating Procedure(SOP). The EpiSensA provides similar predictivity as other validated methods for the same
endpoint with increased capacity to correctly identify pre/pro-haptens and lipophilic compounds.

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Peer Review Panel Composition
G. Frank Gerberick, PhD (PRP Chair) – GF3 Consultancy, USA
Emily N. Reinke, PhD – Integrated Laboratory Systems, LLC an Inotiv Company, Contractor
supporting the NTP Interagency Center for the Evaluation of Alternative
Toxicological Methods (NICEATM), USA
Emanuela Corsini, PhD – Università degli Studi di Milano, Italy
Atsushi Ono, PhD – Okayama University, Japan
Tomoki Fukuyama, DVM, PhD – Azabu University, Japan
Support from Takao Ashikaga, PhD and Hajime Kojima, PhD – JaCVAM, Japan

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Background1.
Significant progress has been achieved in developing New Approach Methods (NAM) for
assessing the skin sensitization potential of chemicals over the last two decades. The OECDdeveloped the adverse outcome pathway for skin sensitization divided into mechanistic key
events (OECD 2014). Three OECD guidelines have been published that cover these mechanistic
events (covalent binding to protein, keratinocyte activation, and dendritic cell activation). Eight
non-animal test methods are approved in OECD TG 442C, 442D, and 442E (OECD 2018a;
2018b; 2021b). However, each of the test methods has some limitations, especially with testing
challenging test materials such as highly lipophilic compounds.

2.
The current focus is to find ways to combine in vitro, in chemico, and in silico assessmentswith read-across predictions from similar chemicals to generate integrated approaches to testing
and assessment (IATA) or Defined Approach (DA). The OECD has published a new guideline(No. 497) that describes three simple DAs for assessing skin sensitization (OECD 2021a).
Combining NAM data will enhance the vigor of skin sensitization hazard identification calls on
chemicals and further address the critical need for predicting a chemical’s potency. Potency
prediction is needed for the UN Global Harmonized System (GHS) subclassification of sensitizers
into 1A (strong sensitizers) and 1B (other sensitizers). For the specific need of GHS
subclassification into sub-category 1A, the kDPRA assay is accepted as a standalone assay(Natsch et al. 2020; OECD 2021b). However, assessing potency is also required for conducting
Next Generation Risk Assessments on new chemical entities where only non-animal information
is available (Api et al. 2020; Bernauer et al. 2021; Dent et al. 2018; Gilmour et al. 2020).

3.
Another focus area in NAM development is to have reconstructed human epidermis (RhE)models available for testing materials that are incompatible with solution-based systems or may
require metabolic activation of the test materials under evaluation. The RhE EpiSensA assay
permits the ability to evaluate more lipophilic compounds. The model also has some metabolic
capacity, so pre- and pro-haptens can be assessed. The assay addresses KE2 of the skinsensitization AOP (OECD 2014).

4.
Against this background, the Japanese company Kao Corporation undertook to addresscertain limitations of current methods to develop a RhE model consisting of normal humanderived epidermal keratinocytes. The EpiSensA evaluates the gene expression of four relevantmarkers, including (i) the encoding activating transcription factor 3 (ATF3), (ii) the glutamatecysteine ligase, modifier subunit (GCLM), (iii) the DnaJ (Hsp40) homolog, subfamily B, member
4 (DNAJB4), and (iv) interleukin-8 (IL-8). The expression of these genes reflects the keratinocyte
response to the early phase of skin sensitization, including (i) the induction of cyto-protective
gene pathways (e.g., the antioxidant response element (ARE)-dependent pathways) and (ii) the
inflammatory response (e.g., the induction of inflammatory cytokines) (OECD 2014)(VMT Report,

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May 2022). The EpiSensA method, like any new method under evaluation, must be judged
independently, in terms of its relevance and reliability via the process of validation

5.
The PRP held its kick-off meeting on June 1, 2022 and continued to interact throughNovember 1, 2022. A face-to-face meeting was held in Tokyo, Japan, on September 8-9, 2022.
The PRP focused on reviewing the detailed report on the EpiSensA prepared by the Validation
Management Team (VMT). Following the commentary on this work by the PRP, the VMT clarified
some minor details in the protocol and made some adjustments to their validation report. The
PRP engaged in assessing the assay's validation status, leading to the generation of this
EpiSensA Validation Report.

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Evaluation Criterion

Evaluation Criterion 1: A rationale for the test method should be available, including adescription of the human health effect, a clear statement of scientific need, and regulatory
application.
6.
The EpiSensA is an in vitro test method designed to be part of a non-animal test battery
or an integrated testing strategy for assessing the skin sensitization potential of chemicals(Mizumachi et al. 2021; Mizumachi et al. 2018; Saito et al. 2013; Saito et al. 2017). The validation
report does provide a rationale for the test method as well as the published references by thetest developer. The report states that while there are already validated alternative methodologies
for Key Events 1-3 of the skin sensitization adverse outcome pathway, each has defined
limitations, especially concerning low solubility of lipophilic compounds and limited metabolic
capabilities to detect pre- and pro-haptens. The EpiSensA assay highlights that the use ofreconstructed human epidermis (RhE) allows the ability to assess more lipophilic compounds.
As the model also has some metabolic capacity, pre- and pro-haptens can also be evaluated.
The assay is designed to address KE2 in the AOP, and the current validation effort was designed
to assess technical transferability and reliability for GHS hazard classification in the labelling ofskin sensitization and as part of an ongoing effort for it to be used as part of the already existing
Defined Approaches for skin sensitization. There are immediate applications and improvements
in using the 3D epidermal EpiSensA model and it will be of great interest to regulators worldwide.

Evaluation Criterion 2: The toxicological mechanisms and the relationship between thetest method endpoint(s) with the biological effect and the toxicity of interest should be
addressed, describing the limitations of the test method.
7.
The EpiSensA prediction model evaluates the gene expression of four relevant markers,
including (i) the encoding activating transcription factor 3 (ATF3), (ii) the glutamate-cysteine
ligase, modifier subunit (GCLM), (iii) the DnaJ (Hsp40) homolog, subfamily B, member 4(DNAJB4), and (iv) interleukin-8 (IL-8). The expression of these genes reflects the keratinocyte
response to the early phase of skin sensitization, including (i) the induction of cyto-protective
gene pathways (e.g., the antioxidant response element (ARE)-dependent pathways) and (ii) the
inflammatory response (e.g., the induction of inflammatory cytokines) (OECD 2014)(VMT Report,
May 2022). Thus, the EpiSensA test method addresses a biological mechanism: inflammatory
and cytoprotection-related gene expression of keratinocytes, which is known to be associatedwith underlying mechanisms of skin sensitization defined in the AOP (Sullivan et al. 2017).

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8.
Analyses showed that the expression of the four genes (GCLM, DNAJB4, IL-8, ATF3)
provides reliable and predictive biomarkers for the discrimination of sensitizers from nonsensitizers (Saito et al. 2013; Saito et al. 2017). In an early study, 16 chemicals (8 sensitizers, 4
non-sensitizers, and 4 pre-/pro-haptens) were used to assess the predictive performance of the
final four marker gene candidates. Based on testing 72 test chemicals containing 54 sensitizers
and 18 non-sensitizers, the cut-off values of the respective marker genes were maximized. In theend, the cut-off values were set (ATF3: 15-fold, GCLM: 2-fold, DNAJB4: 2-fold, and IL-8: 4-fold).
The cut-offs were found to be appropriate when analyzing the data from 136 chemicals.

9.
Importantly, limitations of the assay have been highlighted and include: (i) if the chemical
is not soluble or does not form a stable dispersion at 0.0122% in any vehicle, the chemical is notapplicable for testing with EpiSensA; (ii) chemicals that affect the expression of GAPDH might
not be suitable for testing; (iii) Chemicals that directly interfere with both LDH and MTT; and (iv)chemicals that affect RNA isolation. It is also noted that increases in the gene expression of ATF3
and IL-8 are also observed, along with a decrease in cell viability when some surfactants (suchas sodium lauryl sulfate and benzalkonium chloride) are tested on EpiSensA. In addition, the
EpiSensA assay has an inherent limitation in detecting some weak sensitizers, the same as theother threshold-based prediction models.

Evaluation Criterion 3: A detailed test method protocol should be available.
Transferability to other labs should be demonstrated.
10.
A detailed test method is available, written in such a way that a naïve facility should be
able to easily pick up the test method for their use. The validation report highlighted that themethod was transferable from the lead laboratory to three supporting facilities with some minor
adjustments to the protocol, which were captured in subsequent versions. Participating
laboratories underwent training to learn the method and returned to their home facilities to test 4
chemicals for the potential to cause skin sensitization. All test chemicals were appropriately
classified by the testing facilities, with one test facility conducting testing with two independent
technicians, both ultimately successful. However, one technician did require more tests to meetthe criteria. The technology transfer portion of this validation study also allowed for additional
refinement of the SOP to alter the positive control test chemicals due to a variety of factors, adjust
the viability acceptance criteria, and add endogenous control gene (GAPDH) Ct criteria.

11.
Specific changes to the SOP included changing the positive control from 6.25% w/v
bisphenol A diglycidyl ether (BADGE) to 0.78 w/v% clotrimazole and 0.10% w/v 4nitrobenzylbromide (4NBB) because of the poor stability and the inaccurate concentration of thecommercially available BADGE. The acceptance criteria for the mean viability of the vehicle
control have changed from 80% to 90%. The new acceptance criterion (requiring a ≥95% cell
viability in each vehicle control well) was set based on the statistical analysis of the cell viability
distribution of the vehicle control obtained by the lead laboratory.

12.
If there is a scientific rationale with supporting experimental data, other housekeeping
genes or RNA extraction isolation techniques could be used. It is recommended that such

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ENV/CBC/MONO(2023)19  15changes to the SOP will require an assessment of defined proficiency chemicals andperformance controls.

Evaluation Criterion 4: The within and between laboratory reproducibility of the test
method should be demonstrated.
13.
Both within and between laboratory reproducibility have been demonstrated in thevalidation project for EpiSensA, as measured using concordance of classification. Phase I was
conducted with 15 chemicals tested in three independent experiments, and the concordance
ranged between 86.7 to 93.3% for the participating laboratories. Combining these results withthe 12 Phase II chemicals, tested once, resulted in a between-laboratory reproducibility of 88.9%.
These are acceptable levels of concordance for both within and between laboratory
reproducibility and exceed the targeted 85% for within-laboratory and 80% for betweenlaboratory reproducibility.

14.
Following some adjustments in the acceptance criteria, the results support the EpiSensA
to be reproducible within and between laboratories, satisfying the performance criteria for the
EpiSensA.

Evaluation Criterion 5: Demonstration of the test method’s performance should be basedon testing a diversity of chemicals, preferably coded reference chemicals.
15.
JaCVAM oversaw test chemical distribution and coding. Coded chemicals were
distributed to participating laboratories and pertinent safety information was provided to achemical master at each facility. At the conclusion of the validation studies, the code was notbroken by any test facility. The validation management team selected chemicals by utilizing
published in vitro skin sensitization studies and previously conducted validation studies for in vitro
skin sensitization. Chemicals needed to meet a range of criteria for inclusion, such as backgroundinformation on mode or site of action, quality of the reference data, coverage of relevant chemicaland product classes, a range of potencies, including non-sensitizers, information on pre-and prohapten potential, physical and chemical properties, commercial availability, and high purity.
Therefore, chemical coding, distribution, and handling were conducted correctly and supervised
by JaCVAM.

16.
The EpiSensA performed well with a diversity of chemicals, including those with different
reactivity domains and physiochemical properties. As stated previously, the assay provides
advantages over other alternative methods in that it applies to a broader range of lipophilic
chemicals and more pre/pro-haptens. The performance of the EpiSensA against LLNA reference
data using 69 lipophilic compounds (LogKow >3.5) was better than 3 other in vitro assays (DPRA,
KertinoSens, h-CLAT) with sensitivity, specificity, and accuracy values of 83%, 65%, and 78%,
respectively. The predictivity of 37 pre/pro-haptens was even better, with a sensitivity of 95%.

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Evaluation Criterion 6: Predictive capacity should be demonstrated using representative
chemicals.
17.
Predictive capacity was demonstrated with representative chemicals across a range ofchemical classes, including lipophilic and pre-/pro-haptens, and was comparable to otherchemical sets utilized in other validation studies. Regarding the actual results for the assay, 136
chemicals were tested by the lead laboratory using EpiSensA. As stated in the validation report,
the sensitivity, specificity, and accuracy were 88.1%, 65.7%, and 82.4%, respectively, comparedto the LLNA. These were comparable to existing in vitro methods. Additionally, with the ability totest pre/pro-haptens, the EpiSensA had a 97.3% sensitivity for predicting these chemicals. The
cumulative sensitivity, specificity, and accuracy for the three participating labs were 92.6%, 63%,
and 82.7%, respectively, compared to the LLNA. These are similar to the larger dataset assessedby the lead lab. Against human data, the cumulative sensitivity, specificity, and accuracy were
92.2%, 61.9%, and 83.3%.

18.
EpiSensA performed adequately when used to predict potency (GHS 1A/1B). While thiswas not a primary endpoint in the validation study, the assay demonstrated a sensitivity of 92.6%,
a specificity of 63.0%, and an accuracy of 82.7% compared to LLNA results for the threeparticipating laboratories. This is comparable to the predictive performance found following thelead laboratory’s assessment of 136 chemicals (88.1%, 65.7%, and 82.4% for sensitivity,
specificity, and accuracy). However, when the accuracy is broken down a bit further, there issome concern that the model may be missing 1A chemicals, with 29% of chemicals defined as
1B and 6% as NC from the set of 1A chemicals. Approximately 15% of the 1B chemicals were
under classified as NC in the assay as well. While overall accuracy compared with human data,i.e., GHS category, is not high (64.4%), this is the same accuracy for LLNA as compared withhuman data. However, there is some concern for the chemicals underpredicted when comparedwith 1A/1B/NC class by LLNA. Overall underprediction values were reported at 16.1%.
Compared to the kDPRA, the EpiSensA results seem comparable, with 31% of Cat. 1A chemicals
underpredicted compared to 36% for the kDPRA (Natsch et al. 2020). A comparison of thepredictive capacity of the LLNA GHS classification of 1A chemicals between EpiSensA andkDPRA for the same set of chemicals (72) demonstrated similar accuracy values of 88.9% and91.7%, respectively. A comparison of the predictive capacity of the strong human sensitizers
between the EpiSensA and kDPRA for the same set of chemicals (57) also showed similar
accuracy values of 82.5% and 77.2%, respectively.

19.
EpiSensA has enough predictive capacity in numerical terms compared with othermethods such as LLNA, human tests, in vitro and in vivo studies. But there is a slight concern
about the predictability of 1A and 1B classification. The aim of this validation is not the evaluationof the potency predictability of this method; therefore, discussion of applying EpiSensA to in vitro
potency prediction methods of skin sensitizers should be considered as a next step.

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Evaluation Criterion 7: All data from the validation study supporting the validity of a test
method should be obtained in accordance with the principles of Good Laboratory Practice(GLP).
20.
Studies were carried out in non-GLP laboratories in accordance with the principles of
GLP, with quality controls in place. The lead lab created data reporting templates and record
sheets and distributed them to the participating labs. The record sheets included important
information such as start/end time of operation, lot number, and manufacturer of reagents;
amount of reagents used; plate design and measurement conditions; and operator/technician
signature and performance dates. Statistical analysis of the data was conducted utilizing
standard Microsoft Excel formulas to determine fold change for each endpoint. All the records(data sheets and record sheets) from the participating laboratories were carefully checked by QC
experts at the lead lab who are proficient in the principles of GLP. JaCVAM also reviewed therecords, which are available on the JaCVAM website for review.

Evaluation Criterion 8: The applicability domain of the test method should be defined.
21.
The chemical applicability domain is similar in makeup to the chemical set utilized in the
Defined Approach for Skin Sensitization (TG497) for both reactivity domains andphysicochemical properties.

22.
The EpiSensA dataset contains many pre/pro-haptens and lipophilic chemicals, which
can be considered more challenging for skin sensitization NAMs. The EpiSensA showed goodpredictive performance, with only a few limitations described regarding applicability domains. For
example, if the chemical is not soluble or does not form a stable dispersion at 0.0122% in any
specified vehicle, the chemical is not applicable for testing with EpiSensA. However, anothervehicle outside of the protocol specified vehicles could be employed if sufficient scientificrationale can be provided. In addition, testing chemicals at high concentrations may lead to highosmotic stress conditions and lead to a false positive response.

23.
The misclassification of lauryl gallate, a known pre/pro-hapten, suggests that chemical
exposure time may not be optimal for some chemicals requiring time for autooxidation or
metabolism. Therefore, if an unknown chemical is predicted in silico to be metabolized to apotential skin sensitizer and negative EpiSensA results are obtained, it may be necessary toincrease incubation time for these challenging materials. The Lead Lab shared that a positive
response was observed with lauryl gallate when 6 hr treated cultures were allowed to incubate
for a total of 24 hours. However, it is important to point out again that EpiSensA correctly identified
35 out of 37 (95%) pre/pro-haptens.

24.
Chemicals that affect the expression of GAPDH or interfere with viability assays, LDH,
and MTT will be problematic for the assay. Limitation of the chemicals that may influence theRNA extraction and qPCR performance might be a future concern to spreading this method
widely. Like most available alternative assays, the EpiSensA has an inherent limitation in

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detecting very weak sensitizers. Analysis of the DASS human set1 showed that most false
predicted chemicals have weak sensitization potential.

Evaluation Criterion 9: Proficiency chemicals should be set up in the proposed protocol.
25.
The Proficiency chemicals have been shared by the test developers and were adaptedfrom chemicals used in the JaCVAM coordinated validation study to demonstrate the successful
implementation of the protocol. The chemicals contained in the list are commercially available
and provide a range of responses. Many chemicals are similar to the proficiency chemicals used
for other skin sensitization NAMs.

26.
The chemicals are intended for method optimization in a lab performing the assay using
the EpiSensA LabCyte RhE model described in the most current version of the SOP, Version:
2.6. Successful implementation of the assay in a new laboratory is demonstrated by correctly
identifying 8 or more of the 10 chemicals. This success criterion is identical to that set for OECD
accepted skin sensitization NAMs.

Evaluation Criterion 10: Performance standards should be set up with the proposed
protocol.
27.
The same criteria as stated under criterion 2 would apply here as well. Performancestandards that refer to the acceptability of an individual test run or set of runs, also referred to as
acceptance criteria, should be available for the test method. Clotrimazole and 4-nitrobenzyl
bromide are used as positive controls. Clotrimazole dissolved to a final concentration of 0.78
w/v% in AOO, and 4-nitrobenzyl bromide (4NBB) dissolved to a final concentration of 0.10 w/v%.
There is no negative control chemical tested in each run. The vehicle control is used as thenegative control.

28.
The SOP (Appendix 7) does provide requirements for qualified testing and data
acceptance that are inclusive of cell viability limits, concordance in controls, minimums for
positive control gene expression, and requirements for endogenous control gene variability. The
mean cell viability of both positive controls should be equal to or greater than 80%. For
clotrimazole, the mean values of fold induction for ATF3 and IL-8 should exceed the cut-off
values. The mean values of fold induction for GCLM and DNAJB4 should exceed the cut-off
values for 4-Nitrobenzyl bromide.

1

The DASS human set is a high quality, highly curated reference set of chemicals made up of Human Predictive
Patch Tests that was used to evaluate performance of the Defined Approaches for Skin Sensitization and can be used
to evaluate performance in other assays against well characterized human skin sensitizers (OECD 2021c).

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Evaluation Criterion 11: Advantages in terms of time, cost, and animal welfare should be
described.
29.
The animal welfare advantages are straightforward and similar to other NAMs that havebeen developed for evaluating the skin sensitization potential of chemicals. The benefits in termsof cost and time are not addressed here since a comparison would need to be made by contract
laboratories or individual laboratories that include the cost of personnel time for conducting theassay, not just materials. The expectation would be that the cost and time would be reasonable
and comparable to other NAMs, and the cost being easily justified based on the advantages the
EpiSensA provides. It is important to note that the test developers have begun to assess costcomparisons of EpiSensA compared to both in vivo and in vitro test methods.

Evaluation Criterion 12: Completeness of all data and documents supporting theassessment of the validity of the test method.
30.
This validation study aimed to assess the transferability, the within-laboratory
reproducibility, and the between-laboratory reproducibility of EpiSensA. The study was
coordinated by the JaCVAM in consultation with the VMT regarding the study design, the test
chemical selection, and the SOP. The study was undertaken using coded test chemicals,
enabling the VMT to assess the scientific validity and appropriateness of EpiSensA in anticipationof the development and issue of an OECD test method guideline for its use.

31.
Raw data sheets, GLP notations sheets, and other supporting documents for thevalidation of EpiSensA were made available as part of the validation report. They did appear to
be complete and in support of the validity of the test method.

Evaluation Criterion 13: Validation Study should be managed and conducted adequately.
32.
The primary goals of this validation study were to assess the test definition, transferability,
and within- and between-laboratory reproducibility while additionally using the data produced toevaluate predictive capacity and the applicability domain compared to the LLNA. The
management and conduct of the validation study were adequate.

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Recommendations and Further
Considerations
33.
EpiSensA Considerations for Longer Incubations - The EpiSensA was demonstrated bytest developers to show remarkable predictivity of skin sensitization with respect to pre-/prohaptens, with 35/37 chemicals accurately predicted. The two missed chemicals werebenzo[a]pyrene and lauryl gallate. The latter tested negative in the validation study. Further
assessment by the test developers regarding the incorrect prediction of lauryl gallate showedthat lower concentrations, as tested by participating laboratories, as well as longer incubation
times, resulted in positive predictions for the chemical. Several factors appear to affect the abilityof EpiSensA to predict lauryl gallate, inclusive of the lipophilicity of the compound (high Log KOW)
and the rapidity with which the chemical may undergo metabolism or auto-oxidation. With lauryl
gallate, the test developers showed that a 6-hour exposure, followed by an additional 18-hour
post-wash incubation period, resulted in a strong response and a positive skin sensitization
prediction in the EpiSensA. With these factors in mind, it is recommended that the test developers
address the potential for additional follow-on testing options to assess chemicals that are
predicted to be pre-/pro-haptens and require longer incubation times to activate and/or also havephysical-chemical properties that could indicate longer incubation times are necessary, such asa high log KOW, which may affect absorption into the metabolically active layers of the model.
34.
Development of ‘Me Too’ Methods Based on EpiSensA - Prior to the routine use of the
EpiSensA test method, laboratories should demonstrate technical proficiency, using the ten
Proficiency Chemicals suggested by the test developer. These chemicals can be used bylaboratories to demonstrate technical competence with a standardized test method or todemonstrate the reproducible performance of the test method over time. Proficiency chemicals
represent a subset of the reference chemicals included in Performance Standards as defined by
the OECD, which include essential test method components, a list of reference substances andaccuracy and reliability performance values, which are distinct from the performance standards
referred to in Evaluation Criterion 10 above. Test developers should determine an expanded setof reference chemicals to allow the method to be transferred to another model of reconstituted
epidermis (colloquially referred to as “me-too” test methods). This is important for a greater
distribution of the method worldwide, where accessibility of the different epidermal models isvaried. Performance standards must be available to facilitate the validation of new or modified
EpiSensA test methods similar to the original EpiSensA, which will allow for timely amendmentof the Test Guideline for their inclusion, which is crucial for the Mutual Acceptance of Data.

35.
Performance standards are typically based on a scientifically valid and accepted test
method and can be used to evaluate the reliability and relevance of other analogous test methods
that are based on similar scientific principles and measure or predict the same biological or toxic
effect. Based on previous experience (moving from EpiDerm to LabCyte EPI-MODEL 24), theEPIDERMAL SENSITISATION ASSAY (EPISENSA) PEER REVIEW REPORT
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test developer is encouraged to provide the performance standards, together with the criteria fora limited assessment of reliability, sensitivity, specificity, and relevance without needing toundergo a formal validation. A similar approach used by the KeratinoSens, DPRA, and LLNA ‘metoo’ methods should be adopted, where 20 Reference Substances have been identified, alongwith other factors to determine the performance of the proposed new method.
36.
Borderline Responses – The test developer and participating laboratories demonstrated
that the EpiSensA test method has enough sensitivity, specificity, and accuracy to predict typicalstrong skin sensitizers compared to the LLNA and other in vitro methods (e.g., EpiSensA andkDPRA for the same set of chemicals of GHS1A demonstrated similar accuracy values of 88.9%
and 91.7%, respectively). On the other hand, there is a slight concern about the ability of the test
to discriminate between 1A and 1B, and 1B and NC. Approximately 15% of the 1B chemicals
were underclassified as NC with the EpiSensA test method. While the accuracy of classificationof GHS 1A/1B/NC class by EpiSensA is comparable with the kDPRA, there are concerns for thechemicals underpredicted when compared with 1A/1B/NC class by LLNA. The test developers
did provide some additional analysis of setting borderline thresholds of the cut-off value ±10%,which resulted in 3 false negative chemicals (3%, 3/101) in the original dataset being classified
as borderline under these criteria. However, the developers do state that the EpiSensA has fewer
borderline results than the TG497 methods KeratinoSens and h-CLAT (5.9%; 8/135 and 6.7%,
9/134 respectively), demonstrating that the method already has adequate retesting criteria toprevent false negative judgement. Regardless, while discerning borderline responses is not theaim of the validation, discussion of how borderline responses should be classified/handled with
EpiSensA should be undertaken by the test developers if inclusion into a defined approach suchas TG497 should be considered.

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Conclusion
37.
The peer review panel (PRP) for the EpiSensA validation study has assessed the method
for appropriate rationale, mechanistic applicability, scientific need, test method description,transferability, within- and between-laboratory reproducibility, predictive capacity based on arelevant set of reference chemicals, the defined domain of applicability, and overall conduct of
the study (GLP, completeness of protocol, etc). The PRP has found that the EpiSensA test
method is robust, meets specified criteria for validity, and additionally fills in testing gaps with theability to correctly assess pre-/pro-haptens and lipophilic chemicals. The PRP has made someminor recommendations related to development of me-too test method performance standards,
further assessment of the feasibility of using the test method for potency predictions, how toaddress borderline responses, and potential follow-on confirmatory testing when predicted pre/pro-haptens or highly lipophilic compounds test as negative (e.g., longer incubation time). The
recommendations are meant to help strengthen the assay but are not limiting factors in the use
and application of the assay as it is currently designed.

38.
Overall, the assay performed well against pre-specified assessment criteria, easily
exceeding the 85% and 80% thresholds for within- and between-laboratory reproducibility(>86.7% and 88.9% respectively). Transferability between labs was also successful, with a welldefined SOP for conduct of the assay utilizing the LabCyte EPI-MODEL 24 skin model. The
assay’s sensitivity, specificity, and accuracy for a hazard classification were comparable to otherexisting skin sensitization assays, inclusive of other OECD guideline-based in vitro test methods.
The assay shows promise to be applied for potency prediction, but further assessment isnecessary. Regarding the applicability domain of the assay, its ability to better predict lipophiliccompounds and pre-/pro-haptens is a distinct advantage over other existing in vitro assays.

39.
Overall, the PRP agrees that this model has performed well in the validation study andcan be applied to predict skin sensitization hazard, and potentially potency, with the intent toeventually apply it into a defined approach for skin sensitization.

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Acknowledgments
The PRP is grateful to the members of the VMT for their hard work and excellent responsivenessto our questions. Thanks, are also due to JaCVAM for their support in setting up and hosting ourmeeting in Japan, as well as for setting up each of our telephone conferences. This work was
supported by the Japanese Society for Alternatives to Animal Experiments (JSAAE). Finally, a
special thanks to Tamaki Yoshikawa for her outstanding administrative support in helping us with
our visas during these challenging times.

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