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HEALTH
AN VAN ROMPAY1, KAREN HOLLANDERS1, ELS ADRIAENS2, NATHALIE ALEPEE3,
VIRGINIE LEBLANC3, HELENA KANDAROVA4, AGNIESZKA DRZEWIECKA5, PRZEMYSLAW
FOCHTMAN5, KATARZYNA GRUSZKA5, ROBERT GUEST6, GARETH MAGLENNON6, JANE
SCHOFIELD6, JAMIN A. WILLOUGHBY7 and SANDRA VERSTRAELEN1
1VITO nv, Applied Bio & molecular Systems, Mol, Belgium; 2Adriaens Consulting BVBA,
Aalter, Belgium; 3L’Oréal Research & Innovation, Aulnay-sous-Bois, France; 4MatTek In
Vitro Life Science Laboratories, Bratislava, Slovakia; 5Institute of Industrial Organic
Chemistry Branch Pszczyna, Pszczyna, Poland; 6Envigo, Cambridgeshire, United
Kingdom; 7Cyprotex US, LLC, Kalamazoo, MI 49008, USA
CON4EI: Funded by CEFIC LRI – AIMT6 – VITO, Eye irritation testing in vitro in practice –
Database and testing strategy. Cosmetic Europe contributed in the chemical selection.
Introduction
Assessment of ocular irritation is an international regulatory requirement
in the safety evaluation of products. Although a number of in vitro assays
exist, none are capable of fully categorizing chemicals as stand-alone
assays. In CON4EI (2015-2016) the reliability of 8 in vitro test methods
and computational models for 80 reference chemicals was assessed and
an integrated testing strategy for eye irritation was established. One of
the 8 assays was the SkinEthicTM HCE EIT (Reconstructed Human Corneal
Epithelium Eye Irritation Test).
Results will be published in ATLA.
Methods
Assessment of the acute eye irritation potential is part of the international
regulatory requirements for testing of chemicals. The objective of the
CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project is
to develop tiered testing strategies for eye irritation assessment for all
drivers of classification. For this, a set of 80 reference chemicals (38
liquids and 42 solids) was tested with eight different alternative methods.
Here, the results obtained with SkinEthicTM HCE EIT (Reconstructed
Human Corneal Epithelium Eye Irritation Test) model are shown. The
primary aim of this study was an evaluation of the performance of the test
method to discriminate chemicals not requiring classification for serious
eye damage/eye irritancy (No Category) from chemicals requiring
classification and labelling (Category 1 and 2). In addition, the predictive
capacity in terms of in vivo driver of classification was investigated. In a
second step, it was investigated if SkinEthicTM HCE EIT can be used as part
of a tiered-testing strategy for eye irritation assessment. The chemicals
were tested twice, the first run was performed by VITO and the second
run was performed by L’Oréal.
For the SkinEthicTM HCE EIT, 100% concordance in predictions (I versus NI)
between the two participating laboratories was obtained. The accuracy of
the SkinEthicTM HCE was 97.5% with 100% sensitivity and 96.9%
specificity. The results of this study seem promising with regard to the
inclusion of this test method in an integrated testing strategy for eye
irritation assessment.
This research is funded by CEFIC-LRI. We acknowledge Cosmetics Europe
for their contribution in chemical selection.
Keywords: CON4EI, SkinEthicTM HCE EIT, eye irritation/corrosion, ocular
irritation assay, in vitro novel irritation testing; testing strategy
Abstract
SkinEthicTM HCE EIT discriminates between No Cat and Cat 1/Cat21,2. The
80 blind-coded chemicals (15 No Cat, 27 Cat 2 and 38 Cat1) were tested by
the laboratories VITO and L’Oréal.
1Alépée et al., Toxicology in Vitro 2016; 31,43-53; 2Alépée et al., Toxicology
in Vitro 2016; 34,55-70.
Results
Concordance in prediction: in terms of the classification I versus NI, a 100% agreement in prediction between
VITO and L’Oréal was obtained.
Reproducibility of the viability: The high
reproducibility between the runs between VITO
and L’Oréal was reflected in the correlation plots.
These plots illustrates the two-by-two relation
between the experiments for liquids and solids
separately. Dots close to the line of equality (solid
line), indicate similar levels of viability for the
independent experiments. The dotted lines
correspond with the cut-off that distinguishes I
from NI.
Predictive capacity: The predictive capacity was calculated for each laboratory and for the cumulative results of
the two laboratories. A sensitivity of 96.9% and specificity of 100% with an accuracy of 97.5% was obtained
overall and in both laboratories separately.
Overall, four false negatives were
reported, the following in vivo Cat 2
chemicals (both solids) were consistently
predicted NI by the two laboratories:
(2R,3R)-3-((R)-1-(Tert-
butyldimethylsiloxy)ethyl)-4-oxoazetidin-
2-yl acetate (in vivo Cat 2A with CO
mean ≥ 1 as driver) and m-
Dinitrobenzene (in vivo Cat 2B with Conj
mean ≥ 2 as driver).
Boxplot showing the distribution of the viability in function of GHS Driver of classification.
The dotted lines correspond with the cut-off for liquids (60%) and solids (50%) that distinguishes I from NI.
Distribution of the liquid chemicals (chem.) according to the drivers of
classification (UN GHS Cat 1 and Cat 2) and according to the subgroups
(UN GHS No Cat) as defined by Barroso and co-workers (2016).
Distribution of the solid chemicals (chem.) according to the drivers of
classification (UN GHS Cat 1 and Cat 2) and according to the subgroups (UN
GHS No Cat) as defined by Barroso and co-workers (2016).
Summary
• 100 % concordance in prediction between VITO and L’Oreal was obtained.
• The accuracy was 97.5% with 96.9% sensitivity and 100% specificity.
• Yes, the results seem promising with regard to inclusion of SkinEthicTM HCE EIT in an
integrated testing strategy for eye irritation assessment
Cefic LRI-AIMT6-VITO CON4EI project: defines
applicability domains, strength and limitations of these
8 in vitro test systems:
1) BCOP (Bovine Corneal Opacity and Permeability
2) BCOP-LLBO (BCOP-Laser Light Based Opacitometer),
3) ICE (Isolated Chicken Eye),
4) EpiOcular-EIT (EpiOcular Eye Irritation Test)
5) EpiOcular-ET50
6) SkinEthicTM HCE EIT (Human Corneal Epithelial),
7) STE (Short Term Exposure)
8) SMI (Slug Mucosal Irritation) to define eye irritation
potential.
It identifies tiered-testing strategies for eye irritation
assessment.
Classification
(Prediction)
In vitro EITL
(Liquids)
In vitro EITS
(Solids)
Not classified
(No category)
Mean tissue viability >
60%
Mean tissue viability >
50%
Classified
(Category 1 /
Category 2)
Mean tissue viability ≤
60%
Mean tissue viability ≤
50%
SkinEthic™ HCE
EITL - Liquid:
Exposure: 30 min
Post Soak: 30 min
Rinse
EITS - Solid:
Exposure: 4 hours
Post incubation: 18 hrs
Rinse
Post Soak: 30 min
Receipt :
At least overnight
incubation at
37C, 5% CO2,
≥ 95% humidity
Rinsing with PBS
without Ca2+ Mg2+
Topical application
37°C, 5% CO2,
≥ 95 % humidity
with control and test
substances
Quantification(OD570
reading )
MTT Incubation for
3 hrs at 37C
Formazan extraction (4 hrs
at RT or ON at 4C)