Inter-Laboratory Validation of an In Vitro Method to
Classify Skin Sensitizers
Amy J.
1
Clippinger ,
•
James M. McKim,
1PETA
2
Jr ,
Donald
2
Keller ,
Paul C.
2
Wilga ,
Hilda
3
Witters
and An R. Van
3
Rompay
International Science Consortium, Ltd., Norfolk, VA, USA; 2CeeTox Inc., Kalamazoo, MI, USA;
3Flemish Institute for Technological Research, Mol, Belgium
Abstract
Allergic contact dermatitis presents a concern for developers of personal care, chemical,
pharmaceutical, and medical device products. The development of non-animal methods to assess
skin sensitization is a priority due to the EU cosmetics testing ban, the 2018 REACH deadline, and
the goal of reducing animal use.
Induction of the Nrf2/Keap1/ARE
signaling pathway
Chemical
This study builds upon previous studies (McKim et al, 2010; McKim et al, 2012) showing that
CeeTox’s in vitro SenCeeTox® assay can correctly identify and categorize chemical sensitizers when
used in-house. The aim of this project was to further validate the SenCeeTox® assay by conducting
an inter-laboratory validation at the Flemish Institute for Technological Research (VITO).
In this study, MatTek’s three-dimensional human skin model, EpiDermTM (EPI-296, EpiDermTM 96-well
reconstituted human epidermis), was treated in triplicate with six concentrations of each test
article. Test articles were run in a blinded manner. The test articles evaluated were: metol,
isoeugenol, 2,3-butanedione, 2-mercaptobenzothiozol, eugenole, 1-chloro-2,4-dinitrobenzene,
glycerol, 2-hydroxyethylmethacrylate, 2-hydroxyethylacrylate, and lactic acid. Following 24 hr
exposure to the test articles, the following endpoints were measured: 1) cytotoxicity, 2) the ability
of each chemical to directly react with glutathione, and 3) expression of key genes. The gene
expression levels of seven target genes controlled by the Nrf2/Keap1/ARE signaling pathway were
examined: NADPH-quinone oxidoreductase 1, aldoketoreductase 1C2, interleukin 8, cytochrome
P450 1A1, aldehyde dehydrogenase 3A1, heme-oxygenase 1, and glutamate cysteine ligase catalytic
subunit C. The data were then analyzed in a blinded manner using a proprietary algorithm to predict
each chemical’s likelihood of causing a human sensitization reaction.
The results confirm the inter-laboratory reproducibility of SenCeeTox® which accurately predicted
the ability to elicit a sensitization reaction for all 10 blinded compounds tested at VITO.
Furthermore, it correctly predicted the sensitization potency category for 9 out of the 10
compounds, missing the 10th compound by only one potency category. In conclusion, SenCeeTox® can
predict the sensitization potency of chemicals ranging from non-sensitizers to strong-extreme
sensitizers. Because results from the SenCeeTox® assay have been so promising, further validation
of this assay will be undertaken by Cosmetics Europe, after which all results will be submitted to
EURL ECVAM.
AOP for Skin Sensitization
EGFR
PI3K
Akt
ERK
P38
MAPK
Ub
ligase
TNFα
CCL17
TARC
CCL5
RANTES
NFκB
Nrf2
CCL27
T-cell recruitment
Recruitment of
Langerhans cells
Nrf2
Elicitation
T-cell recruitment
Recruitment of
Langerhans cells
ARE
Allergic Contact Dermatitis
®
SenCeeTox
Proteosomal
Degradation
Keap1
NQO1
AKR1C2
IL8
CYP1A1
ALDH3A
HMOX1
GCLC
Method
SenCeeTox® measures:
1) Cytotoxicity
2) The ability of each chemical to directly react with glutathione. A chemical sensitizer will react
with glutathione based on its potency
3) Expression of 7 target genes controlled by the Nrf2/Keap1/ARE signaling pathway:
1) NADPH-quinone oxidoreductase 1
2) Aldoketoreductase 1C2
3) Interleukin 8
4) Cytochrome P450 1A1
5) Aldehyde dehydrogenase 3A1
6) Heme-oxygenase 1
7) Glutamate cysteine ligase catalytic subunit C
The data are then analyzed in a blinded manner using a proprietary algorithm.
Results
Adverse outcome pathway (AOP) for skin sensitization initiated by covalent binding to proteins. The
boxes outlined in red represent events measured by SenCeeTox. Adapted from OECD
ENV/JM/MONO(2012)10/PART1. 04-May-2012.
®
SenCeeTox
Advantages
•Accuracy, sensitivity and specificity as good as or better than in vivo or other in vitro sensitization
assays
•Provides potency categorization
•Can be used with soluble compounds, insoluble compounds or finished products because SenCeeTox®
is used in combination with MatTek’s EpiDermTM, a 3-dimensional human skin model
Test Article #
(Blinded)
Test Article Name
(Unblinded)
In Vitro Toxicity
Index (IVTI)
LLNA Potency
Category
5
5.5
3
5
3
6.5
1
4
5.5
1
6
SenCeeTox
Predicted Potency
Category
Moderate
Moderate-Strong
Weak
Moderate
Weak
Strong-Extreme
Non-sensitizer
Moderate
Moderate-Strong
Non-sensitizer
Strong
CTX01
CTX02
CTX03
CTX04
CTX05
CTX06
CTX07
CTX08
CTX09
CTX10
p-benzoquinone
(positive control)
Benzoic acid
(negative control)
Metol
Isoeugenol
2,3-butanedione
2-mercaptobenzothiozol
Eugenole
1-chloro-2,4-dinitrobenzene
Glycerol
2-hydroxyethylmethacrylate
2-hydroxyethylacrylate
Lactic acid
p-benzoquinone
Benzoic acid
1
Non-sensitizer
Non-sensitizer
Strong
Moderate
Weak
Moderate
Weak
Strong
Non-sensitizer
Moderate
Moderate
Non-sensitizer
Strong
•Mechanistically based - measures key events involved in sensitization including direct protein binding
and up-regulation of genes regulated by the Nrf2/Keap1/ARE signaling pathway
•Complies with current European Union, Indian and Israeli cosmetic testing regulations
•Reduces time and cost versus animal testing
•Provides animal welfare advantage over the animal tests. The guinea pigs or mice who are commonly
used in skin sensitization testing are injected with or have a substance applied to their skin and often
experience redness, itchiness, scaling, and inflammation. The guinea pig tests [Magnusson Kligman
Guinea Pig Maximization Test (GPMT) and the Buehler Test] cannot determine potency of the test
substance, rely on subjective endpoints, and are especially painful because they cover both the initial
exposure to the substance (“induction”) and the subsequent elicitation phase.
Conclusions and Future Direction
SenCeeTox® accurately predicted the ability to elicit a sensitization reaction for all 10 blinded
compounds tested at VITO.
SenCeeTox® correctly predicted the sensitization potency category for 9 out of the 10 compounds
tested, missing the 10th compound by only one potency category.
Further validation of this assay will be undertaken by Cosmetics Europe’s Skin Sensitization Task
Force, after which all results will be submitted to EURL ECVAM.
References
MatTek's EpiDerm™ System consists of normal, human-derived epidermal keratinocytes (NHEK) which
have been cultured to form a multilayered, highly differentiated model of the human epidermis.
Images from MatTek.com and Curren, R.D., Mun, G.C., Gibson, D.P., and Aardema, M.J. Development of
a method for assessing micronucleus induction in a 3D human skin model (EpiDerm™). Mutation
Research, 2006. 607(2): p. 192–204.
1. McKim, J.M., Jr., D.J. Keller, 3rd, and J.R. Gorski. A new in vitro method for identifying chemical
sensitizers combining peptide binding with ARE/EpRE-mediated gene expression in human
skin cells. Cutan Ocul Toxicol, 2010. 29(3): p. 171-92.
2. McKim, J.M., Jr., D.J. Keller, 3rd, and J.R. Gorski. An in vitro method for detecting chemical
sensitization using human reconstructed skin models and its applicability to cosmetic,
pharmaceutical, and medical device safety testing. Cutan Ocul Toxicol, 2012. 31(4): p.
292-305.