ESAC Peer review of submission of external catch up validation study of In
vitro Reconstructed Human Epidermis (RHE) assay for Skin Irritation Testing
The above submission was sent to ECVAM in the Test Submission Template for
ECVAM/CORRELATE submissions recently developed by ECVAM and was reviewed byPeer Review Panel (PRP):
1. Independent scientific evaluation of the submitted Test Submission Template (TST)by three members of the ECVAM Scientific Advisory Committee (Dr M. Dambrova,
Dr D. Jirova and Dr C. Westmoreland) was performed.
The study was evaluated according to the principles outlined in the ECVAM document‘Performance Standards for Applying Human Skin Models to in-vitro skin irritation testing’(1 ). These performance standards are used to evaluate the reliability and accuracy of themethods which are based on reconstructed human epidermis and which measure or predict thesame biological or toxic effect as the fully validated and accepted reference method (seeESAC statement, 2007 and skin irritation validation study [SIVS] report) (2,4). In thiscontext, there was a request from ECVAM/CORRELATE for additional data, i.e. retesting ofdiethyl phthalate at test laboratory 3 (Oroxcell Laboratory) because of one invalid run and for
additional statistical evaluation. In addition, minor formal corrections/clarifications in thesubmision file were required.
1. Consensus views from scientific evaluation of submission
All procedures are clearly defined, explained and described in satisfactory details. The
description of participating units and management structure of the study gives impression of afully managed and coordinated study which afforded reliable data collection and statisticalanalysis. Following comments received from ECVAM in June 2008 regarding the original
TST submission, a new version of the TST was made available to the peer review panel
during the review process (TST V2.0) which integrated changes made to the original
document. These changes included retesting of diethyl phthalate at test laboratory 3 (Oroxcell
Laboratory) and formal corrections.
1.1

Data Collection

Are the data collection procedures and selection clearly defined ? YES
The presented data are obtained according to GLP compliant SOPs in test laboratories which
either are GLP certified or fully trained for procedures and the documentation practicerequired for this study. Data of the submission are sufficient to assess the study goal. The
study followed ECVAM SIVS Performance Standards and also reference data from ECVAMSIVS were employed.
The following comments were made regarding the conduct of the study. It was not felt by thepeer review panel that these comments significantly altered the overall summary (Section 3)of the panel:o Regarding the SOP for the IL 1α analysis, the Sections '5. Data Report andCalculations' including '5.1. Calculations' and '5.2. Assay acceptance criteria' of the IL
1α SOP were compiled as an Amendment only after the end of the study (26th March,
2008). They were missing in the original SOP for the IL 1α analysis.

o The real Project Plan, which should be elaborated before the start of the study, ismissing. The document called 'Protocol Amendement No:1 – Multicenter Study Plan'(MSP) was submitted to ECVAM on March 26, 2008, which means it was compiled
only in course of the study or even after the end of the study. It is written in a fast andnon-systematic form, missing good structure, pages are not numbered. It is signed by
the principal investigator, but not approved by study participants. The reason for theattachment of the sheet named 'Protocol Approval' (done by all laboratories) to the
MSP is not clear. However, the 'Multicenter Study Plan' lists study participants, there
is a reference to the company Vitroscreen responsible for coding/distribution of 20
reference substances and description of the way of distribution. Company Vitroscreen
provided the decoding to the contract statistician company before the statisticalanalyses and sent also decoding to the participating labs on Feb. 21, 2008 (specified in
the TST together with the type of statistical analyses applied). It means, that thestatistical analysis was not performed blinded.

1.2

Goal of the study

1.2.1 Was the goal of the study clearly understandable? YES
The goal of the study is understandable and clearly defined as evaluation of the predictive
capacity of the assay submitted to reliably discriminate skin irritants (I) from non-irritants(NI), as defined with EU risk phrases (R38; no label) according to the Dangerous Substances
Directive, 67/548/EEC. The study is submitted as an “External” Catch up validation study andemploys Reconstructed Human Epidermis (RHE) model provided by the biotech company
SkinEthicTM (Nice, France), which mimics the three-dimensional (3D) structure of the humanskin. The method aims to demonstrate that RHE model as an in vitro skin irritation assay issimilar/equivalent to the in vivo rabbit test for skin irritation.

1.2.2 Is the scientific rationale given? YES
The scientific rationale of the assay is based on previously published data, which indicate that
SkinEthicTM RHE model is promising tool for the in vitro skin irritation and skin corrosion
testing (publications by Kandárová et al., 2006). The SkinEthicTM RHE model also presents a
differentiation pattern comparable to EpiSkinTM and EpiDermTM models and to normal humanepidermis. Even though the submitted assay protocol has been developed recently, the RHE
tissues are used routinely by several contract laboratories and companies, and the resulting
data have been already used for assessing the skin irritancy potential of the test substances. By
using SkinEthicTM RHE model, the irritant substances are identified by their ability todecrease cell viability below the threshold of 50% level using the MTT assay. In addition, theinflammatory responses are evaluated by the quantification of the IL-1• assay.
The rationale is given and documented. The principle of the assay is based on the ability ofirritant chemicals to penetrate the stratum corneum by diffusion and their subsequent
cytotoxicity to living epidermal cells. Moreover, if cytotoxic effect is absent or weak,
inflammatory mediators released might be considered in a tiered approach to increase thesensitivity of the test (similarly to the tiered approach scientifically validated for the
EpiSkinTM test assay, ESAC, May 2007). Irritant substances were identified by their ability todecrease cell viability using the MTT below the defined 50% threshold level. The
inflammatory responses were evaluated by the quantification of interleukin 1α using ELISA.
1.2.3 Is the regulatory rationale given? YES
The regulatory rationale is clearly defined, based on an urgent need for a full replacement forin vivo rabbit skin test according to Method B.4 of Annex V to Directive 67/548/ECC or
OECD TG 404. The test is developed as a stand alone method for the assessment of the skinirritancy potential of the test substances and could be used for hazard identification andlabelling of new and existing chemicals (including cosmetic raw materials andpharmaceuticals) according to the EU classification system (R38 or no label).
1.3.

Test definition (Module 1)

1.3.1 Are the test and its purpose well defined? YES
The test and its purpose are clearly given . SkinEthicTM RHE assay is being proposed as a full
replacement method of the in vivo rabbit skin irritation test for the assessment of dermal
irritation according to the performance standards for applying human skin models to in vitro
skin irritation testing, using the RHE model which is manufactured according to definedquality assurance procedures and commercialized by SkinEthicTM (Nice, France).
SkinEthicTM RHE is a highly differentiated and stratified epidermis model comprising themain basal, supra basal, spinous and granular layers and a functional stratum corneum. The
SkinEthicTM RHE model presents a histological morphology comparable to the in vivo humantissue and is used for skin irritation testing that involves topical application of test materials tothe surface of the epidermis, and the subsequent assessment of their effects on cell viability.
The submitted SkinEthicTM RHE protocol employs a single exposure time of 42 minutes forboth liquids and solids (powders) and a 42-hour post-exposure incubation before assessmentof tissue viability. The twenty reference test substances were selected for test to give anadequate distribution of irritancy scores derived from in vivo rabbit skin irritation tests.The
main endpoint used in the study is cell viability (measured as MTT reduction). Even thoughIL-1• was also measured, it did not turn out to improve the predictive ability of the assay. The
provided SOP of the test method fully describes all procedures concerning tissue
characteristics and handling, as well as application of test compounds and data processing.
The mean relative tissue cell viability above 50 % was taken as a predictive measure for a nonirritancy potential of the test substance.
The prediction model for the SkinEthicTM RHE assay is overtaken from the proposed
Performance Standards for applying human skin models to in vitro skin irritation testing, in
the ECVAM Skin Irritation Validation Study (4 ) and adequately serves the proposed purpose.
1.3.2 Are the proposed standardised protocol and prediction model adequate?
YES
The detailed, GLP compliant SOP for MTT and IL 1α are provided. Training with 20
substances before the study is reported in the TST, but only partly documented in the Training
Report of Coty and Oroxcell, no raw data are provided. The training report of Oroxcell is notadequately filled in. Training of L´Oréal laboratory is not documented at all.

The prediction model is fully adequate. The reconstructed human epidermal model (RHE)
exhibits the structure of human epidermis. Human-derived epidermal keratinocytes have been
cultured to form a differentiated model of human epidermis including basal, spinous andgranular layers, and a multi-layered stratum corneum. RHE tissues contain 4 to 7 viable layers(approximate thickness of viable epidermis: 40-100 µm) which is typical for skin models andthe thickness of the stratum corneum is approximately 20-60 µm. The SkinEthic RHE model
contain 13.2 +/- 2.1% lipids. The stratum corneum (SC) and its lipid composition is sufficient
to resist the rapid penetration of cytotoxic marker chemicals, e.g. SDS or Triton X-100. This
property is estimated by the exposure time required to reduce cell viability by 50% (ET-50)
upon application of 1% Triton X-100.
1.4

Data quality

The presented data are obtained according to GLP compliant SOPs in test laboratories which
either are GLP certified or fully trained for procedures and the documentation practicerequired for this study. Data of the submission are sufficient to assess the study goal. The
study followed ECVAM SIVS Performance Standards and also reference data from ECVAMSIVS were employed.
Evaluated data are of required quality, well described and discussed. Raw data from training
experiments are not provided. Raw MTT and IL 1α data for 20 coded experimental
substances are provided for all experimental runs of all laboratories, as well as for thetransferability experiments (MTT endpoint) performed in week 48 with uncoded 20
substances.
1.4.1 Are they sufficient to assess the study goal? YES
The data presented in the TST for the SkinEthic RHE skin irritation test appear to be of very
high quality. They are sufficient to assess the study goal.
The work was conducted to ‘GLP-like’ standards. The three of the participating institutions(L’Oreal, Coty Laboratories, Oroxcell) have either official GLP compliance as determined by
AFSSAPS or have defined in-house quality systems. Q. It is unclear why the GLP complianceof the work at Oroxcell did not include evaluation of test substances and the report.
1.4.2 Quality of the reference data
Reference data from ECVAM SIVS were employed. Reference data, i.e. the classification
data using the skin irritation effects in the rabbit, were used, as the rabbit represents theregulatory accepted species. Although the intention is to predict the human health hazard,
neither in vivo nor in vitro skin irritation tests are currently calibrated to match human
response.
1.5

Test materials

1.5.1 Is the number of evaluated substances sufficient ?
The 20 reference chemicals (10 skin irritants [R38 label], 10 non irritants [no label])recommended by ECVAM as providing a representative distribution of the 58 chemicals usedin the ECVAM international skin irritation validation study (Performance standards
document: Skin irritation validation study, 2007) were used. This list allows comparison of
results with those obtained originally with EpiSkinTM in the Skin Irritation Validation study

(SIVS). The list includes two chemicals which were ‘false positives’ in the EpiSkinTM model(1-bromo-4-chlorobutane and 4-methyl-thio-benzaldehyde) and three ‘false negatives’ (hexyl
salicylate, terpinyl acetate and dipropyl disulphide).
The independency in coding and distribution of substances were ensured in line with the study
plan.
1.5.2 Are they representative of proposed applicability domain? YES
The selection and number of evaluated substances comply with the requirements of the
ECVAM SIVS Performance Standards.
Applicability domain of the SkinEthicTM RHE assay is the same as for both models(EpiSkinTM, EpiDermTM) that were the subject of validation under the ECVAM SIVS.
Consequently, the information regarding the method applicability on volatiles, emulsions,mixtures, hydrolyzing and polymerizing chemicals, acids and bases is considered as
insufficient. SkinEthic should not be considered validated for such classes of chemicals. For
chemicals that directly reduce MTT, correction techniques were developed.
1.6
Within-laboratory variability (Module 2) – assessment of reproducibilityof the data in the same laboratory
The within-laboratory variability was properly assessed. The within-laboratory variability
was assessed for each laboratory by means of the assessment of the frequency of non
qualified experimental runs, by one-way ANOVA statistics, by analysis of the withinlaboratory standard deviation, by calculation of Bravais-Pearson correlation of the mean cell
viability for the three pairs of runs and by evaluation of the proportion of identically classified
test substances.
Regarding the MTT endpoint, only one laboratory (Oroxcell) for one test substance showed
between-runs SD > 18 as unacceptable. Only in this laboratory was 1 out of 20 tested
substances not consistently classified. For L´Oréal and COTY all 20 substances wereconsistently classified. Comparison of variability via the cumulative distribution of the SD of
all runs in the three laboratories revealed the highest between-runs variability at Oroxcell andthe lowest at COTY. However, the overall results for MTT endpoint for twenty reference test
substances demonstrated a low variability for all laboratories. Additionally submitted data,that were generated in July 2008 on the ECVAM request for substance No.2 (diethyl
phthalate), proved compliance with results from previous 2 valid runs. Data for 3 valid runs
were recalculated. In this context, the PRP expressed the opinion, that for future validation
studies a clear guidance should be given from ECVAM/CORRELATE on how many invalid
runs can be repeated and not included in calculations.
Regarding the IL-1α, a substantial within-laboratory variability was observed within assay
and between runs for experimental chemicals and for PC and NC. Between six to nine out of
20 test substances had a p-value below the level of 1% indicating significant differences
between the independent runs. Overall, the results on the within-runs variability using thetwenty reference test substances seemed to demonstrate variability for all laboratories using
the RHE assay.

1.7 Transferability (Module 3) - how easy is it to transfer the tests to a second
laboratory?
Transferability of the SkinEthicTM RHE assay was assessed during the training that included
3 test substances (2 liquids and 1 solid) according to template of method transfer (1 day or 3days training) that was established within SkinEthicTM. Both Oroxcell and L’Oréal
laboratories were naïve, having never used the SkinEthic’s protocol. Furthermore, Coty
Laboratory routinely used the RHE model to assess skin irritancy potential, but was notfamiliar with SOP for the protocol of 42 min exposure time and 42-hour post-exposure
incubation. The training exercise gave evidence that SkinEthicTM RHE assay can be
efficiently transferred. Thus, the within-laboratory variability was acceptable (SD smaller
than 18 for all tested substances, and positive and negative controls met the acceptancecriteria in MTT assay). However, the IL-1• assay showed a highest variability. This allowed
identifying MTT test cell viability parameter as a relevant endpoint, whereas the IL-1• might
be a questionable endpoint for classification of substances.
1.8
Between-laboratory variability (Module 4) - assessment of reproducibilityof the data in different laboratories
Using the proportion of identically classified tests substances as a measure of between
laboratory reproducibility, the results showed that all of the 20 test substances were identified
identically (R38 or no-label) in the three laboratories.
Between laboratory variability was also assessed using the ANOVA/t-test p values and the
ANOVA sum of squares of the MTT results. 19 out of the 20 tested sustances produced
results that were reproducible between laboratories. Between laboratory standard deviations
for the MTT results for all 20 test chemicals were <18%. The data reproducibility wasproperly assessed.
Concerning the MTT endpoint, and using the 1-way ANOVA evaluation, 19 out of 20 tested
chemicals were reproducible between the three laboratories. Analysis of SD betwen runs
showed that none of the 20 substances exhibited a SD larger than 18. Results for all
substances were highly reproducible between the three labs. All test substances wereidentically identified in the three labs in three valid runs.
Regarding the values obtained for IL-1• a lack of standardisation of this secondary endpoint
was seen, irregular low values for irritants and high values for non-irritants can be seen in
Table 43 (pg.78 of TST).
.

1.9

Predictive capacity (Module 5)

1.9.1 Has the predictive capacity of the methods been properly assessed ?
YES – but further statistical anaylsis is required
Regarding the MTT endpoint, three out of twenty test substances were consistantly
misclassified in all laboratories in all runs. Two non-irritants (1-bromo-4-chlorobutane and 4methyl-thio-benzaldehyde) were classified as irritants and one irritant was classified as nonirritant (hexyl salicylate). In addition, the Oroxcell laboratory misclassified one more

substance (allyl phenoxy-acetate) in one run with no impact on the median approachclassification. Taking all individual classifications (n=93) into account or considering only
three valid runs (n=90) a similar specificity of 80% and sensitivity of 90 % was achieved
using the MTT endpoint.
Regarding the IL-1• endpoint, the predictive capacity was evaluated analysing the real IL-1•
values, the IL-1• values corrected by NC and fold increase of the IL-1• values above 5. In
general, none of the MTT non-irritant test substances exhibited a release of IL-1• above 60
pg/mL (resp. 50 pg/ml) and none of the MTT non-irritant test substances showed foldincrease above 5 of released IL-1•. However, high between-run variability in IL-1• values
and SD for non-irritants can be seen in Tabs.68-72 for the Oroxcell laboratory. In addition,
one of the MTT classified non-irritant substances exhibited a fold increase above 5 in all 3
runs (Tab.74 of TST) in the Oroxcell laboratory. The irritant classified substances tended
irregularly to induce higher IL-1• amount. No additional correctly classified irritants aspositives were identified using the IL-1• endpoint. The specificity of 80% and sensitivity of
90% was recognized independently of the added IL-1• parameter compared to those obtainedusing the MTT parameter only. When considering the mean IL-1• fold increase release, twoof the nine MTT identified non-irritants were missclassified as irritants in one laboratory in
one run. For the combined prediction based on MTT and the IL-1• fold increase, theclassification reached a sensitivity of 80% and specificity of 76.7%. On contrary to
performance of the EpiSkinTM test in the SIVS (3), the predictive ability of the SkinEthicTM
RHE assay was not improved by introduction of the IL-1 a endpoint.
1.9.2 Is the assay relevant for its stated purpose? Yes.
No problem was recognized using the two SOP protocols (MTT and IL-1• detailed
procedures). Overall results of the within-laboratory variability show SD smaller than 18 for
all tested substances using the MTT parameter whereas it was obvious that the IL-1• showed
the highest variability. Concordance of results for positive and negative controls in alllaboratories was shown, which met the acceptance criteria. The concordance in the reliability
and predictivity using the MTT parameter was proved as well. The MTT cell viability
parameter was identified as a relevant endpoint, whereas the IL-1• might be a questionableendpoint for classification of a given test substance according to the EU classification (R38,
no label). The MTT results of the SkinEthicTM assay are recognized as equivalent to the
EpiSkinTM test. The sensitivity of the SkinEthicTM assay is equal or higher than 70%, and thespecificity is equal or higher than 80%, shown for the EpiSkinTM test.
10. Applicability domain (Module 6) - Is the proposed applicability domain welldefined ? YES
The applicability domain comprises chemicals as liquids and solids, not gases, vapours or
aerosols. Quickly hydrolysing or polymerising chemicals or chemicals with other type ofdegradation were not included in the ECVAM SIVS. Consequently, for this type of chemicals
also the validity of the SkinEthicTM method cannot be considered as assessed. Highly volatile
substances and chemicals that react with the plastic material of the cell culture inserts may
provide higher levels of variability and may give inconsistent results.
1.11 Performance standards (Module 7) - Have appropriate performancestandards been defined for the test ? YES

The ECVAM SIVS Performance Standards Document was exactly followed in this study. However,

the appearance of heptanal on the list of standards is surprising due to the fact that during the
ECVAM SIVS it was tested in one laboratory only and only during the test optimization
study on EpiSkinTM. In addition, a possibly wrong calculation of the dominating rabbit skinirritation median value for heptanal was identified (3.35 instead of 4).
Regarding the number of acceptable invalid runs and the possibility to repeat the invalid run,the PRP expressed the opinion, that for future validation studies a clear guidance should begiven from ECVAM/CORRELATE on how many and how many times the invalid runs canbe repeated and not included in calculations. Such specification should be included in“modified” ECVAM SIVS Performance Standards Document.

It is suggested, that existing human data should not be ignored, as they provide valuable
toxicological information. A considerable number of chemicals classified R38 by the rabbit
test do not cause irritation in human skin in vivo. Reconstructed human skin models consist ofcells of human origin. They were developed to predict human skin hazard. That is why theyclassify some of rabbit irritants as human non-irritants (e.g. Hexyl salicylate is one of the R38
reference chemicals of the PS document which was repeatedly shown to be non-irritating tohumans).
Although volatile and rapidly changing/decomposing chemicals should be excluded from thelist of standards, some of the standard substances exhibit these characteristics (e.g. 1bromohexane, 4-methyl-thio-benzaldehyde, a-terpineol).
1.12 Readiness for regulatory purposes
The method is ready for regulatory purposes using as the main endpoint the cell viability(MTT reduction), with a threshold of 50% viability. Interleukin 1 alpha (IL-1 a) was alsomeasured to determine if this additional endpoint would improve the predictive ability of theassay. For SkinEthicTM RHE model, a sensitivity of 90% and specificity of 80% was
identified for the MTT only. It was not improved by IL-1 a endpoint. The overall accuracy
was 85%. It can be suggested as a stand alone in vitro toxicity test to replace the Draize rabbit
skin irritation test for classification of skin irritants and non-irritants.
2.

Further statistical analysis requested from ECVAM

Within ECVAM document ‘Performance Standards for Applying Human Skin Models to invitro skin irritation testing’ there is a requirement that 'the sensitivity of a 'me too' test mustbe higher than 70%, and the specificity must be equal to or higher than 80%'. These figures
are based on the analysis of the 20 reference chemicals in the SIVS with EpiSkinTM using
median test results from the dataset.
It is therefore important when quoting sensitivity and specificity results for a new test that thesame statistical methodology is used as was used when defining these requirements within the
Performance Standards for Applying Human Skin Models to in-vitro skin irritation testing.
The peer review panel therefore asked ECVAM to recalculate sensitivity, specificity andaccuracy figures for the SkinEthicTM RHE test using exactly the same methodology (i.e. using
median test results) to that used for the same 20 chemicals in the SIVS using the EpiSkinTM

test. These results are presented in Appendix 1 (EpiSkinTM) and 2 (SkinEthicTM RHE test) andsummarised below
EpiSkinTM (SIVS)SkinEthicTM RHE test

70%
80%
75%

90%
80%
85%

Sensitivity
Specificity
Accuracy3.

Overall Summary

Given the consistently positive comments within the initial scientific peer review of thesubmission of the follow-up validation study of the SkinEthicTM RHE assay t together withthe additional statistical analysis of sensitivity, specificity and accuracy, the combined veiwsof the peer review panel can be summarised as:
The performance of the SkinEthicTM RHE assay met the criteria outlined to be considered tohave sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (nonskin irritating) test substances compared to the validated and accepted method. Limitations
associated with the previously validated and accepted in-vitro reference method 1 (EpiSkinTM) for skin irritation e.g. applicability domain (1, 4) also apply to the SkinEthicTM RHE assay.
Dr M. Dambrova,
Dr D. Jirova
Dr C. Westmoreland
Date : November 20, 2008
References
1. ECVAM (2007) Performance Standards for Applying Human Skin Models to In Vitro
Skin Irritation Testing. Online: http://ecvam.jrc.ec.europa.eu
2. ECVAM (2007) Statement of the ECVAM Scientific Advisory Committee (ESAC) on
the Validity of In Vitro Tests for Skin Irritation. Online: http://ecvam.jrc.ec.europa.eu
3. ECVAM (2007) Skin Irritation Validation Study Phase II: Analysis of the primary
endpoint MTT and the secondary endpoint IL-1•. Online:
http://ecvam.jrc.ec.europa.eu.
4. Spielmann, H., Hoffmann, S., Liebsch, M., Botham, P., Fentem, J., Eskes, C., Roguet,
R., Cotovió, J., Cole, T., Worth, A., Heylings, J., Jones, P., Robles, C., Kandárová, H.,
Gamer, A., Remmele, M., Curren, R., Raabe, H., Cockshott, A., Gerner, I. and Zuang,
V. (2007) The ECVAM International Validation Study on In Vitro Tests for Acute Skin
Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin
Integrity Function Test. ATLA 35, 559-601.

Appendix 1: Statistical analysis of sensitivity, specificity and accuracy of EpiSkinTM test using 20 reference chemicals
tested in the full prospective exposure study
Individual Laboratory Predictions for the 20 Reference Chemicals ONLY. Used for calculating the predictive values (sensitivity, specificity, accuracy)
MedianIn Vitro
Prediction

1

1

I

0

0

0

NI

0

0

0

0

NI

no label

0

0

0

0

NI

Allyl phenoxy-acetate

no label

0

0

0

0

NI

36

Isopropanolno label

0

0

0

0

NI

8

4-methyl-thio-benzaldehyde

no label

0

1

1

1

I

39

Methyl stearate

no label

0

0

0

0

NI

10

Allyl heptanoate

no label

0

0

0

0

NI

33

Heptyl butyrate

no label

0

0

0

0

NI

34

Hexyl salicylate

R38

0

0

0

0

NI

FN

55

Terpinyl acetate

R38

0

1

0

0

NI

FN

58

Tri-isobutyl phosphate

R38

1

1

1

1

I

4

1-decanol

R38

1

1

1

1

I

20

Cyclamen aldehyde

R38

1

1

1

1

I

3

1-bromohexane

R38

1

1

1

1

I

15

a-terpineol

R38

1

1

1

1

I

23

di-n-propyl disulphide

R38

0

1

0

0

NI

18

Butyl methacrylate

R38

1

1

1

1

I

Heptanal

R38

1

.

.

1

I

Nr.

XXX

ChemicalEU Class

L’OréalUnilever2

1-bromo-4-chlorobutane

no label1

1

22Diethyl phthalate

no label

024

di-propylene glycol

no label41

Naphthalene acetic acid11

SanofiFP

FP

FN

1) CALCULATION ON THE BASIS OF ALL LABORATORY PREDICTIONSSENSITIVITY
Sensitivity = TP / ( TP+FN)
Sensitivity

75

SPECIFICITY
Specificity = TN / (TN + FP)
Specificity

83.3

ACCURACY
Sum (TP + TN) / (TP+TN+FN+FP)
Accuracy

79.3

30 individual predictions for NEGATIVES
but only 28 for positives = total of 58 predictions
absolut
percent
TP
21
75
25
FN
7
Sum
28
TN
FP
Sum

25
5
30

83.3
16.7

1a) CALCULATION ON THE BASIS OF ALL LABORATORY PREDICTIONS
Including 3 concordant laboratory predictions for Heptanal (same weighting as for the other chemicals)
30 individual predictions per class = total of 60 predictions
SENSITIVITY
absolut
percent
Sensitivity = TP / ( TP+FN)
TP
23
76.7
76.7
23.3
Sensitivity
FN
7
Sum
30
SPECIFICITY
Specificity = TN / (TN + FP)
TN
25
83.3
83.3
16.7
Specificity
FP
5
Sum
30
ACCURACY
Sum (TP + TN) / (TP+TN+FN+FP)
80.0
Accuracy

2) CALCULATION ON THE BASIS OF THE FINAL DECISION MAKING (MEDIAN)SENSITIVITY
Sensitivity = TP / ( TP+FN)
Sensitivity

70

SPECIFICITY
Specificity = TN / (TN + FP)
Specificity

80

ACCURACY
Sum (TP + TN) / (TP+TN+FN+FP)
Accuracy75-> led to PS
Value
for metoo's-> led to PS
Value
for metoo's

10 individual predictions per classabsolut
percent
TP
7
70
30
FN
3
Sum
10

TN
FP
Sum

8
2
10

80
20

Appendix 2: Statistical analysis of sensitivity, specificity and accuracy of the SkinEthicTM RHE Test
Individual Laboratory Predictions for the 20 Reference Chemicals used for calculating the predictive values (sensitivity, specificity,
accuracy)No

Chemical

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

1-bromo-4-chlorobutane
diethyl phthalate
di-propylene glycol
naphthalene acetic acidallyl phe0xy-acetate
isopropa0l
4-methyl-thio-benzaldehyde
methyl stearate
allyl hepta0ate
heptyl butyrate
hexyl salicylate
terpinyl acetate
tri-isobutyl phosphate
1-deca0l
cyclamen aldehyde
1-bromohexane
a-terpineol
di-n-propyl disulphidebutyl methacrylate
heptanal*

In
vivo
score§
0
0
0
0
0.3
0.3
1
1
1.7
1.7
2
2
2
2.3
2.3
2.7
2.7
3
3
4

EU

EU

label

GHS
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1

Lab1

Lab 2

Lab 3

Over
all

0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1

1
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
1
1
1

1
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
1
1
1

1
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
1
1
1

1
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
1
1
1

FP

FP

FN

1) CALCULATION ON THE BASIS OF ALL LABORATORY PREDICTIONS
40 individual predictions per classabsolut
percent
TP
27
90
10 ok (• 30%)
FN
3
Sum
30

SENSITIVITY
Sensitivity = TP / ( TP+FN)
Sensitivity90

ok (• 70%)

SPECIFICITY
Specificity = TN / (TN + FP)
Specificity80

ok (• 80%)85

no values provided in the
PS

TN
FP
Sum

24
6
30

80
20

ok (• 20%)

ACCURACY
Sum (TP + TN) / (TP+TN+FN+FP)
Accuracy2) CALCULATION ON THE BASIS OF THE FINAL DECISION MAKING (MEDIAN).
10 individual predictions per classabsolut
percent
TP
990
10 ok (• 30%)
FN
1
Sum
10

SENSITIVITY
Sensitivity = TP / ( TP+FN)
Sensitivity90

ok (• 70%)

SPECIFICITY
Specificity = TN / (TN + FP)
Specificity80

ok (• 80%)85

no values provided in the
PS

TN
FP
Sum

ACCURACY
Sum (TP + TN) / (TP+TN+FN+FP)
Accuracy

8
2
10

80
20

ok (• 20%)