EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)ECVAMSCIENTIFIC
ADVISORYCOMMITTEE(ESAC)ESAC OPINION
based on the ESAC Scientific Peer Review of the ECVAM Eye
Irritation Validation Study (EIVS) and a related Cosmetics Europe
study on HPLC/UPLC-photometry as an alternative endpoint
detection system for formazan reaction product
ESAC Opinion Nr.
Relevant ESAC request Nr.
Date of opinion

2014-032013-0317.11.2014

TABLE OF CONTENTS
SUMMARY OF THE ESAC OPINION .........................................................................................................................2
1.

MANDATE OF THE ESAC ................................................................................................................................42.

DETAILED OPINION OF THE ESAC ..................................................................................................................4
2.1 BACKGROUND, REGULATORY AND SCIENTIFIC RATIONALE ................................................................................................. 4

3.

2.2

DESIGN AND CONDUCT OF THE STUDY ...................................................................................................................... 6

2.3

STUDY RESULTS AND CONCLUSIONS ......................................................................................................................... 8

2.4

POTENTIAL REGULATORY USE OF THE TEST METHOD .................................................................................................. 12

2.5

RECOMMENDATIONS .......................................................................................................................................... 12

REFERENCES ................................................................................................................................................14

ANNEX 1ESAC AND ESAC WORKING GROUP CHARGED WITH THE SCIENTIFIC REVIEW ....................................15

ESAC OPINION

ESAC WG report on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 1 of 15

EUROPEAN COMMISSIONJOINT RESEARCH CENTRE
Institute for Health and Consumer Protection
European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)

17 November 2014

Summary of the ESAC opinion
The ESAC was requested (see Annex 2) to conduct a scientific peer review of and provide an ESAC
opinion on(1) the EURL ECVAM Eye Irritation Study (EIVS); co-sponsored by EURL ECVAM and Cosmetics
Europe) evaluating the EpiOcular Eye Irritation Test (EIT) and SkinEthic Human Corneal
Epithelium (HCE), both based on Reconstructed human Tissue (RhT); with the ESAC review
addressing the study design, results and conclusions. The review examined the scientificbasis of the assays, their reproducibility and transferability as assessed during the study, as
well as their predictive capacity. The latter was examined in the context of the test methods'
envisaged use within an in vitro testing strategy (Scott et al., 2010) based on strategic
combinations of alternative test methods to replace the Draize eye test, either as the initial
step in a Bottom-Up Approach or a second or subsequent step in a Top-Down Approach todiscriminate chemicals (substances and chemical mixtures) not requiring classification forserious eye damage/eye irritancy (No Category) from chemicals requiring classification andlabelling as Category 1 and Category 2 (UN 2011; EU 2008). The term "strategic
combinations" here refers to the placement of methods in a sequential manner in view ofeither identifying first negatives (high sensitivity: bottom-up approach) or first positives (highspecificity: top-down approach).
(2) The usefulness, based on a study conducted by Cosmetics Europe and submitted to EURLECVAM, of HPLC/UPLC-photometry as an alternative endpoint detection system in particularfor highly coloured chemicals interfering with the conventional formazan endpoint
measurement by OD-photometry.
EIVS
The ESAC concludes that the study was generally well planned and executed. Briefly, reproducibility,transferability and predictive capacity of both test methods were assessed in a three laboratory ringtrial testing 104 chemicals plus an additional 8 solids for assessing the optimised EpiOcular™ EIT
protocol. The chemicals represented a wide range of irritancy scores and chemical and use classes.
The aims of the study were well described and clear target criteria for test method performance had
been predefined. Using appropriate provisions for retesting, the study generated almost complete
data sets as a solid basis for evaluating test method performance. However, the ESAC does not agreewith the approach chosen by the Validation Management Team (VMT) to calculate the predictive
capacity based on the individual test runs: the ESAC believes that confidence limits based on thenumber of test chemicals are more appropriate. The ESAC believes that the approach chosen by the
Validation Management Team (VMT), over-estimated the sample size, produced overly narrowconfidence limits, and resulted in a seemingly high degree of certainty with respect to the point
estimates of sensitivity and specificity. In addition rather than relying only on single test runs to
calculate accuracy, the ESAC believes that, in case single runs are used for analysing predictive

ESAC OPINION

ESAC WG report on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 2 of 15

capacity, reliance is better placed on bootstrapping or worst-case/best-case scenarios of predictive
capacity.
EpiOcular™ EIT
The EpiOcular™ EIT test method was found to be highly reproducible within laboratories, irrespectiveof the protocol used. The ESAC considers that the VMG predictive capacity criteria are deemed tohave been met with the 60% cut-off value using protocol V8.0. This version was the result of
optimisation phase during the validation study aiming to improve results for solids while maintaining
the accuracy obtained for liquid test materials.
The ESAC concludes that the EIVS study findings with respect to transferability, reproducibility andpredictive capacity, taking account of the supplementary analysis undertaken by the ESAC WG, justify
the EpiOcular™ EIT test method (SOP V 8.0) being considered for use within a test strategy todetermine the eye irritation potential of chemicals, specifically to detect non-irritants as part of a
Top-Down or Bottom-Up approach (Scott et al, 2010).
However, the ESAC believes that confidence in the test method would be further increased if
supplementary studies confirm that the test method correctly classifies a small but representative
sample of labelled products or mixtures as defined by REACH and from various sectorial use classes:
for products containing mixtures of substances, such as plant protection products or cosmetic
products, the testing of the mixture is considered more representative of the expected exposure
situation in actual practice than testing its constituent substances separately.

SkinEthic™ HCE
The ESAC agrees with the VMG's view that this test method does not meet the performance criteriathat would be required to consider it as part of a test strategy. ESAC also agrees that with the VMG
that there is potential for improvement but that the necessary work is beyond the scope of thevalidation study.
The Cosmetics Europe Report
The Cosmetics Europe Report is not a Validation Study Report (VSR) relating to a predictive
alternative test method, but is a report of a study which evaluated the performance of a bioanalytical endpoint measurement system that overcomes a limitation of the conventional ODphotometry measurements.
The ESAC believes the evidence presented tends to confirm that for chemicals compatible with these
RhT methods, the measurements obtained of formazan values by OD-photometry and the suitably
qualified HPLC/UPLC endpoint detection systems are comparable. ESAC appreciates that anadvantage of the HPLC/UPLC endpoint detection measurement is that it can also be used in the caseof strongly coloured chemicals incompatible with formazan measurement by OD-photometry (e.g.
colorants used in cosmetics and other consumer products). The report also provides plausible
arguments that this endpoint measurement system is suitable for use with other RhT test methods
relying on measurement of the same endpoint.
The ESAC concludes that the use of HPLC/UPLC would overcome the current limitation of RhT test
methods with respect to problems encountered with strongly-coloured chemicals which interferewith the measurement of formazan levels by OD-photometry.
ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 3 of 15

1.

Mandate of the ESACThe 38th ESAC plenary (19 June 2013) established an ESAC Working Group (WG)1 to undertake adetailed scientific review of an EURL ECVAM/Cosmetics Europe Eye Irritation Validation Study (EIVS)
evaluating two in vitro test methods based on Reconstructed human Tissue (RhT) models forassessing acute eye irritation caused by chemicals: the EpiOcular™ EIT and the SkinEthic™ HCE.
A revised EURL ECVAM Request for ESAC advice (Annex 2) was adopted at the 39th ESAC plenary(11/12 March 2014) extending the ESAC mandate to include a scientific review of a Cosmetics Europe
study of HPLC/UPLC-photometry as an alternative formazan endpoint measurement system.

2.

Detailed opinion of the ESACTaking into account (a) the report of the detailed review undertaken by the ESAC WG; (b) the
information made available to ESAC by EURL ECVAM (Including the EIVS VSR, the Cosmetics Europe
report, and other supporting documentation), and; (c) the ECVAM request for ESAC advice (Annex 2)- the ESAC has adopted the following opinion.

2.1 Background, regulatory and scientific rationale
Both the EIVS and the Cosmetics Europe reports describe the potential role of the RhT-based test
methods, and the endpoint measurement system, in the context of the regulatory testing.
EIVS
The ESAC considers that the VSR provides plausible arguments supporting the use of these RhT test
methods, when combined with other in vitro methods, within a tiered approach using strategic
combinations of alternative test methods. This tiered approach (Scott et al, 2010) is supposed to be
integrated into future more complete "Integrated Testing Strategies" (ITS) or "Integrated Approaches
to Testing and Assessement" (IATA) to replace the Draize eye test. The tiered approach is argued tohave the potential to be at least as relevant and reliable as the current in vivo test system for theidentification of non-irritants, either as the initial step in a Bottom-Up Approach or a second or
subsequent step in a Top-Down Approach test to discriminate chemicals not requiring classification
for serious eye damage/eye irritancy (No Category) from chemicals requiring classification andlabelling (UN GHS Category 1 and Category 2).
Taking account of the probably rather small prevalence of chemicals inducing serious eye damage(Adriaens et al, 2014) RhT test methods validated for this purpose would significantly reduce animal
testing by identifying the large number of chemicals not requiring classification.

1

The WG was established according to the ESAC Rules of Procedure and included experts nominated byinternational partner organisations of EURL ECVAM collaborating under the ICATM (International Collaboration
on Alternative Test Methods) framework.

ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 4 of 15

SkinEthic™ HCE
The tissue component of the test kit is manufactured by controlled culture of immortalised humancorneal epithelial cells to produce a multi-layered epithelium resembling in vivo corneal epithelium.
Although derived from cultured human corneal cells the cells used have been transformed andimmortalised. The ESAC offers no opinion on whether these cells are inherently more biologically
relevant in the context of eye irritation testing than other epithelial cells (e.g. RhT models derived
from normal untransformed human keratinocytes).
EpiOcular™ EIT
The EpiOcular™ EIT tissue construct is prepared from normal human-derived epidermal keratinocytesgrown under defined conditions. The resulting 3D tissue is a non-keratinized multi-layered andstratified (but non-cornified) epithelium intended to model properties of human corneal epithelium.
The Cosmetics Europe Report
The Cosmetics Europe Report is not a Validation Study Report (VSR): the study evaluated theperformance of a bio-analytical endpoint measurement system (which is not itself a test method).
The Scientific Committee on Consumer Safety (SCCS) of the European Commission Directorate
General for Health and Consumers previously advised that “...for coloured substances, a different
endpoint, not involving Optical Density (OD) quantification, should be envisaged. Analytical methods
such as HPLC/UPLC might be more appropriate to detect formazan in the in vitro assay...” (McNameeet al. 2009). An alternative endpoint detection measurement for RhT/MTT reduction product test
methods that overcomes this current limitation would extend the applicability domain of these RhT
tests.
The Cosmetics Europe study proposes the use of suitably qualified HPLC/UPLC endpoint
measurement in this context. The ESAC believes The Cosmetics Europe Report describes a bioanalytical system that has the potential to overcome a current limitation on the use of these andother RhT test methods.

ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 5 of 15

2.2

Design and conduct of the study

2.2.1

Definition of the study objectives

EIVS
The ESAC believes these are clearly defined.
The Cosmetics Europe Report
The ESAC considers the study objectives to have been sufficiently clearly defined.

2.2.2

Study design for the prospective part

EIVS
The ESAC concludes that, subject to specific qualifications set out elsewhere in this Opinion, thestudy design was generally appropriate and sufficiently robust with respect to determining therelevance and reliability of these test methods for the purposes defined in the VSR.
SkinEthic™ HCE
The protocol for the SkinEthic™ HCE test method described the following testing strategy:(1) The first step was an "Eye irritation Peptide Reactivity Assay" (EPRA).
(2) For EPRA reactive chemicals the SkinEthic™ HCE Short-time Exposure protocol (HCE SE).
(3) For EPRA non-reactive chemicals the SkinEthic™ HCE Long-time Exposure protocol (HCE LE).
However, for the purposes of the EIVS study all chemicals were tested using both the HCE SE and HCE
LE protocols.
104 coded chemicals were blind-tested using both the SE and LE protocols in three runs with three
replicate tissues per run, in three laboratories. A test strategy combining, in a sequential manner, theEye Irritation Peptide Reactivity Assay (EPRA) with both the SE and LE variants of the SkinEthic™ HCEtest method was also assessed.
EpiOcular™ EIT
In the ring trial 104 coded chemicals were blind-tested (three runs with two replicate tissues per run,in three laboratories) using EpiOcular™ EIT SOP (V 6.0), using separate protocols for liquid and solid
chemicals, and two prediction models (50% and 60% cell viability).
With the original EpiOcular™ EIT protocol (SOP V6.0) all of the VMG acceptance criteria were met for
liquid chemicals: however, some of the VMG predictive capacity acceptance criteria were not met forsolid chemicals.
The VMG considered that test method performance might be improved if a. Test optimisation wasundertaken by the test developer within EIVS.

ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 6 of 15

To improve the performance of the test methods with solid chemicals, test optimisation wasundertaken within EIVS to produce a balanced increase in sensitivity offset by some decrease in
specificity using 11 of the most challenging EIVS solid chemicals. An amended optimised for solid
chemicals protocol, V 8.0, was used for a post-optimisation validation study of the EpiOcular™ EIT
optimised in one laboratory with 60 solid chemicals. The post-optimisation findings are incorporated
and analysed in the VSR, and form the basis of the VMG conclusions on the performance of the
EpiOcular™ EIT.
The Cosmetics Europe Task Force Eye Irritation Report
The ESAC believes the Cosmetics Europe study was well-designed, and is satisfied that a sufficientnumber of RhT MTT-reduction product test methods, chemicals, and laboratories were used.
In the first phase of the study, three laboratories qualified HPLC/UPLC systems using appropriatequalification criteria in accordance with the May 2001 US Food and Drug Administration (FDA)
guidance for industry “Bioanalytical Method Validation”.
Next, three laboratories tested up to 26 chemicals using one of three RhT test methods measuring
three different toxicological endpoints (EpiOcular™ EIT as per the EIVS protocol v6.0 for eye irritation,
SkinEthic™ RhE as per OECD TG 431 for skin corrosion, and the EpiSkin™ as per OECD TG 439 for skinirritation). The three laboratories with the suitably qualified HPLC/UPLC systems for tested thesamples and applied the Prediction Models using both endpoint measurements.

Chemical selection:
EIVS
The ESAC believes the method by which the test chemicals were selected, and the number andnature of the test chemicals, were appropriate.
The Cosmetics Europe Report
The ESAC believes that the 26 chemicals selected were adequate for this study, and represent thealmost the full dynamic range of the relevant toxicological endpoints.

2.2.3

Study design for the retrospective partNot applicable.

2.2.4

Statistical analysis used in both part of the study

The ESAC believes that whist the data management systems for both studies were robust, elementsof the statistical analysis were not best practice.
EIVS
Almost complete datasets are available for both RhT test methods, but ESAC has some concerns
about some of the statistical methods applied - see 2.3.3 below.

ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 7 of 15

The Cosmetics Europe Report
The ESAC believes that all of the reference and new data relied upon are of sufficient quality.
However, the statistical method by which the formazan measurements by OD-photometry and
HPLC/UPLC were correlated in the study report is not ideally suited to situations where the true valueof the variable of interest in unknown, and in producing this Opinion the ESAC has taken account ofan alternative approach developed for use in these situations (Bland and Altman, 1986).

2.3

Study results and conclusions

2.3.1

Standardised use

EIVS
The ESAC considers that the SOPs used in the EIVS validation study are sufficiently detailed andcomplete, with SOP V 8.0 being considered the definitive protocol for EpiOcular™ EIT.
The Cosmetics Europe Report
The ESAC considers the qualification process and parameters described for suitably qualified
HPLC/UPLC to be appropriate and sufficiently clear.

2.3.2

Within- and between laboratory reproducibility

Within-Laboratory Reproducibility (WLR)
EIVSBoth RhT test methods proved to be highly reproducible within laboratories, satisfying theacceptance WLR criteria established by the VMG.

EpiOcular™ EIT
The possible short-comings of the VMG WLR calculations are described elsewhere in this Opinion.
The ESAC WG, having re-analysed the data set on the basis of the number of chemicals tested
estimated the 95% Confidence Interval (95%-CI) for the WLR using the 60% cut off to be 89.0% to94.4%, satisfying the VMG acceptance criteria (WLR ≥ 85%).
The WLR of the EpiOcular™ EIT optimised for solid chemicals protocol (SOP V8.0) at the onelaboratory that conducted the supplementary testing satisfied the VMG acceptance criteria.
The Cosmetics Europe Task Force Eye Irritation Report
During the Phase 1 (qualification) reproducibility was addressed with both intra- and inter-day
evaluations of the formazan calibration reproducibility calculated by regression analysis. Whilst the
ESAC accepts that variations on this method of assessing agreement between two test methods arecommonly used, the use of correlation for this purpose can be misleading and make it difficult to
ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 8 of 15

recognise differences, and the significance of the differences, between the findings with different
measurement systems when the true value of the variable of interest is unknown. The ESAC takes
account of an alternative approach (Bland and Altman, 1986), developed for use in these situations,which also confirms the limits of agreement are good.
Within laboratory reproducibility (WLR) of formazan measurement by HPLC/UPLC was evaluated in
one laboratory: the results obtained were 100% concordant.

Between-Laboratory Reproducibility (BLR)
EIVS
The ESAC notes that all of the BLR values reported in the VSR are significantly above the acceptancecriteria set by the VMG (BLR ≥ 80), but that there was no BLR study conducted using the EpiOcular™
EIT optimised protocol for solids (SOP V 8.0).

The Cosmetics Europe Task Force Eye Irritation Report
There was 100% concordance between OD-photometry and HPLC/UPLC formazan endpoint
measurements for non-interfering chemicals.

2.3.3

Predictive Capacity

EIVS
The ESAC believes that the VMG’s determination of confidence limits for the predictive capacity
based on the number of test runs, rather than the number of chemicals tested, produces confidence
limits which may be inappropriately narrow, and has taken account revised calculations based on thenumber of chemicals tested.
Furthermore, the ESAC considers that accuracy figures should reflect the way decisions will be madein practice. Specifically, as chemical classification will be based upon one qualifying test run, thestudy results should have been evaluated to produce an accuracy range determined by the best- andworst-cases based on data from single runs rather than aggregating data from different test runs
with the same chemical. Additional analysis was undertaken along these lines.

SkinEthic™ HCEThe SkinEthic™ HCE, SE and LE protocols, failed to meet the 'definitely acceptable' criteria forsensitivity and overall accuracy defined by the VMG (specificity ≥ 60%; sensitivity ≥ 90%, overall
accuracy ≥ 75%).

ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 9 of 15

The SkinEthic™ HCE sensitivity, as judged by the VMG criteria (specificity < 50%; sensitivity < 80%,
overall accuracy < 65%), was 'definitely unacceptable' 2.
EpiOcular™ EIT
ESAC believes that with SOP V 8.0 and a 60% cut off all of the VMG acceptance criteria can bedeemed to have been met.
When used for liquid chemicals, the original EpiOcular™ EIT SOP V6.0 protocol the VSR reported
results satisfying all of the VMG predictive capacity acceptance criteria. A 60% cut-off resulted in abetter sensitivity. For solid chemicals the results with protocol version (V6.0) did not satisfy all of the
VMG acceptance criteria: six chemicals were under-predicted, one of which (misclassified by two of
the laboratories) is classified in vivo as Category 1.
When the solid chemicals were tested in one laboratory by the protocol optimised for solid chemicals(SOP V 8.0) and a 60% cut-off all of the VMT definitely acceptable values were achieved.
The ESAC notes that with respect to accuracy using the best-case/worse-case calculation model even
the worst-case values satisfy VMG criteria for sensitivity and accuracy, and that the worst-case
specificity is very close to the VMG acceptance criterion. As the ESAC considers that the true
predictive capacity falls somewhere between the best- and worst-case calculations based on all test
runs, the VMG predictive capacity criteria are deemed to have been met with the 60% cut-off value.
The Cosmetics Europe Task Force Eye Irritation Report
Where comparisons can be made between HPLC/UPLC data and in vivo classification data, theHPLC/UPLC data did not under-estimate the in vivo classification. In case of EpiOcular™ EIT data, fortest substances that do not exhibit colour interference or direct MTT reduction, the tissue viability
values obtained are almost identical whether using absorbance (OD-photometry) or HPLC/UPLC, with
the OD-photometry values being consistently, marginally above the values obtained with the
HPLC/UPLC test method.
2.3.4

Applicability and possible limitations

EIVS
The EpiOcular™ EIT and SkinEthic™ HCE test methods were not developed, designed, or evaluated todifferentiate between UN GHS / EU CLP Category 1 (serious eye damage) and Category 2 (eyeirritation) classifications. There is limited information available on the performance of the test
methods with chemical mixtures.

2

The VMG defined a three tier system for judging the acceptability of performance of the assays by using a three categorysystem – “definitely acceptable” and “definitely unacceptable”, with the values between these figures being borderline
acceptable. The same approach had been chosen by EURL ECVAM for validating the skin corrosion assays (Fentem et al.,1998).

ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 10 of 15

EpiOcular™ EIT
The ESAC believes that the available evidence on the relevance and reliability of the EpiOcular™ EIT
supports its consideration for use within an integrated, tiered approach using strategic combinationsof alternative test methods to replace the Draize eye test, either as the initial step in a Bottom-Up
Approach or a second or subsequent step in a Top-Down Approach, to discriminate chemicals(substances and chemical mixtures) not requiring classification for serious eye damage/eye irritancy(No Category) from chemicals requiring classification and labelling (Category 1 and Category 2)
according to the UN GHS Classification and Labelling of Chemicals (UN GHS) and as implemented by
the EU CLP regulation (EU CLP).
With respect to the chemicals that were misclassified when the EpiOcular™ EIT findings were judged
against the Draize eye test reference data, the ESAC has identified no specific additional limitations
or exclusions.

SkinEthic™ HCE
The ESAC believes that the reported predictive capacity of the SkinEthic™ HCE test method does notjustify it being considered for regulatory use.
The Cosmetics Europe Task Force Eye Irritation Report
The ESAC believes that the evidence presented suggests that for chemicals compatible with the three
RhT methods used within this study comparable classifications are obtained using both ODphotometry and HPLC/UPLC endpoint detection systems; that with these RhT test methods
HPLC/UPLC endpoint detection measurement can be used in the case of strongly coloured chemicals
which are incompatible with formazan measurement by OD-photometry; and that there are plausible
and reasoned arguments for why this endpoint measurement system might be applicable to other
RhT test methods relying on measurement of the same endpoint.
2.3.5

Identified gaps between study design and study conclusions

EIVS
On the basis of the EIVS data the VMG concluded that the use of 2 tissue replicates in similar or
modified RhT/MTT-based test method aiming at identifying chemicals not requiring classification forserious eye damage/eye irritation is statistically and scientifically justified. However, the ESAC does
not accept that this can be generalised to all RhT test methods on the basis of the available evidence.
The Cosmetics Europe Task Force Eye Irritation Report
Nil of note.

ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 11 of 15

2.4

Potential regulatory use of the test method

EIVS
EpiOcular™ EIT
The ESAC believes that the available evidence on the relevance and reliability of the EpiOcular™ Eye
Irritation Test (EIT) supports its consideration for use within an integrated, tiered approach using
strategic combinations of alternative test methods to replace the Draize eye test, either as the initial
step in a Bottom-Up Approach or a second or subsequent step in a Top-Down Approach, todiscriminate chemicals (substances and chemical mixtures) not requiring classification for serious eye
damage/eye irritancy (No Category) from chemicals requiring classification and labelling (Category 1
and Category 2) according to the UN GHS Classification and Labelling of Chemicals (UN GHS) and as
implemented by the EU CLP regulation (EU CLP).
SkinEthic™ HCE
The ESAC believes that the predictive capacity of the SkinEthic™ HCE test method reported in the
EIVS VSR does not currently justify it being considered for regulatory use.

The Cosmetics Europe Task Force Eye Irritation Report
The ESAC judges that the Cosmetics Europe Report provides evidence, and sets out plausible andreasoned arguments, for in the case of regulatory testing this endpoint measurement system being
considered for RhT test methods relying on the quantification of formazan.

2.5

Recommendations

EIVS
SkinEthic™ HCEESAC agrees with the VMG and the test method developer's decision to not advance the SkinEthic ™
HCE test method in this validation on the basis of insufficient predictive capacity (in particularspecificity) for considering the test method, at present, for a bottom up test strategy within a tiered
approach for inclusion into an Integrated Testing Strategy (ITS) / Integrated Approach to Testing andAssessment (IATA). ESAC understands that the test method developer is currently optimising the test
method protocol before re-entering the validation process. ESAC supports the decision for
optimisation, including considering developing and evaluating different protocols for liquid chemicals
and solid chemicals.
EpiOcular™ EIT
1. The ESAC recommends that the EpiOcular™ EIT test method SOP V 8.0 using the 60% cut off beconsidered for use within an integrated, tiered approach using strategic combinations of
ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 12 of 15

alternative test methods to replace the Draize eye test, either as the initial step in a Bottom-Up
Approach or a second or subsequent step in a Top-Down Approach, to discriminate chemicals(substances and chemical mixtures) not requiring classification for serious eye damage/eye
irritancy (No Category) from chemicals requiring classification and labelling (Category 1 andCategory 2) according to the UN GHS Classification and Labelling of Chemicals (UN GHS) and as
implemented by the EU CLP regulation (EU CLP).
2. Based on the data generated during validation, the ESAC recommends that EpiOcular™ EIT beconsidered applicable to a wide range of liquid and solid chemicals. However, confidence in theability of the EpiOcular™ EIT test method to detect solid irritants (using SOP version 8.0) would
be increased if more chemicals were tested under blind conditions by the other two laboratories.
3. The ESAC recommends that the SOP should be amended to better define the protocol to be used
for test chemicals with an unclear physical state, specifically that all viscous, waxy and gel-likechemicals are placed in a water bath for 15 minutes at 37°C before deciding if they should betested with the liquids or the solids protocol; and that the test chemical should be applied
directly from the water bath and should not be brought to room temperature before testing.

General4. The ESAC recommends that a range of labelled chemical mixtures be identified for use as test
substances within future eye irritation validation studies.
The Cosmetics Europe Task Force Eye Irritation Report
5. The ESAC recommends that the use of suitably qualified HPLC/UPLC systems for thedetection and quantification of formazan be proposed for inclusion in relevant regulatory
guidelines to extend the applicability domain of RhT test methods to include coloured
chemicals interfering with formazan measurement by OD-photometry. However, theHPLC/UPLC provided marginally but systematically lower values for formazan levels than OD
photometry. This may impact on some of the prediction models which currently rely on
formazan quantification by OD-photometry

ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 13 of 15

3.

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Annex 1

ESAC and ESAC Working Group charged with thescientific review

ECVAM Scientific Advisory Committee (ESAC)
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Prof. Jürgen BORLAK
Dr. Neil CARMICHAEL
Dr. Edward CARNEY
Dr. Harvey CLEWELL
Prof. Lucio G. COSTA
Dr. Kristina KEJLOVÁ
Prof. David John KIRKLAND
Prof. Annette KOPP-SCHNEIDER
Dr. Renate KRÄTKE
Prof. Claus-Michael LEHR
Dr. José Maria NAVAS
Prof. Aldert PIERSMA
Dr. Jonathan RICHMOND
Dr. Erwin L ROGGEN
Dr. Dorothea SESARDIC

ESAC Working Group*:

Dr. Jon RICHMOND (ESAC Member, WG Chair)Prof. Annette KOPP-SCHNEIDER (ESAC Member)Dr. Renate KRÄTKE (ESAC Member)Dr. Kristina KEJLOVA (ESAC Member)Dr. Tohru MARUNOUCHI (Fujita-Health University; ICATM nomination by JaCVAM)Dr. Jill MERRILL (FDA; ICATM nomination by NICEATM/ICCVAM)*) One member of the ESAC WG (JM) had been the ICCVAM liaison to the EIVS VMG during the validation study. She had
not been a member of the core Validation Management Group (VMG). Although all decisions during the validation study
were taken by the core VMG, JM advised and commented on some elements of the study design. The ESAC WG does notbelieve that this liaison role constitutes a significant conflict of interest with respect to the work of the ESAC WG.

EURL ECVAMDr. Claudius GRIESINGER (ESAC Coordinator)
Dr. Michael SCHÄFFER

ESAC OPINION on the EURL ECVAM Eye Irritation Validation Study (EIVS)Page 15 of 15