The Toxicologist - Society of Toxicology

analyzed quantitatively. A dose dependent induction of DNA breaks and pyrimidine
dimers was characterized using an adapted protocol of the single cell gel electrophoresis
(comet assay). P53 accumulation measured with an ELISA, 5 hours
post UV exposure, was shown to reach a plateau by exposure times over 15 minutes.
By then, the apoptosis rate related to Caspase-3 activity, measured 18 hours
post exposure, started to increase. Finally, quantitative RT-PCR on genes controlled
by p53 showed that (i) p21 and GADD45 were induced; (ii) whereas a clear induction
of MDM2 required relatively high UV doses. This study shows that reconstructed
skin is a useful tool as part of in vitro strategies for the assessment of photoprotection
and photogenotoxicity.
497 MST2000 - A NEW RECONSTRUCTED HUMAN
EPIDERMIS WITH INTEGRATED FUNCTIONAL
PRIMARY MELANOCYTES.
J. J. Hoffmann 1 , B. Becker 1 , A. Thiemann 2 , S. Weimans 2 , E. Heisler 2 and H.
W. Fuchs 1 . 1 R & D, CellSystems Biotechnologie Vertrieb GmbH, St. Katharinen,
Germany and 2 Laboratorium für Toxikologie und Ökologie, Evonik Stockhausen
GmbH, Krefeld, NRW, Germany. Sponsor: B. De Wever.
The development of new active compounds for skin bleaching and tanning are important
issues for the cosmetic and pharmaceutical industry. However active ingredients
have to be tested extensively for their efficacy and product safety. The aim of
the present study was to create a reconstructed epidermis containing keratinocytes
and melanocytes as a tool especially for efficacy studies. Based on the technology of
the Epidermal Skin Test - EST1000 (CellSystems® Biotechnologie Vertrieb
GmbH, Germany) several batches of skin models with different ratios of
melanocytes and keratinocytes were produced and characterised.
This characterisation included histological examinations to investigate the structural
properties of the epidermis and immunohistochemical staining of the
melanocytes by using anti-HMB45 antibodies.
An important feature for testing effects on skin pigmentation is the feasibility of
cultivating the models for at least ten days after substance application without significant
loss of viability. Therefore, the models were cultured for different periods at
the air liquid interphase followed by a standard MTT assay to determine their viability.
Finally the melanin content for each test approach was measured.
Increasing melanin contents were observed after direct UV-radiation which additionally
lead to a visible tanning of the epidermis. This effect was strongly affected by
several active ingredients that (i.a.) were known to directly inhibit tyrosinase activity.
We conclude from these results that the newly developed melanocyte containing
epidermal model is a useful tool for research and for characterisation of skin tanning
or bleaching substances and for cognate applications which require model systems
with melanocytes.
498 IN VITRO PREDICTION OF THE VALIDATED
SKINETHIC RHE 42 BIS SKIN IRRITATION TEST
METHOD FOR THE GLOBALLY HARMONIZED
SYSTEM (GHS) OF CLASSIFICATION AND LABELLING
OF CHEMICALS.
A. de Brugerolle de Fraissinette 1 , C. Tornier 1 , J. Cotovio 2 , J. Meunier 2 and N.
Alépée 2 . 1 SkinEthic Laboratories, Nice, France and 2 L’Oréal, Aulnay sous Bois,
France. Sponsor: G. Nohynek.
European legislations have heightened the need for scientifically validated in vitro
skin irritation test to replace the regulatory Draize eye irritation test. The SkinEthic
Reconstructed Human Epidermis (RHE) “42 bis” test method was recognized as a
stand alone test to assess the acute skin irritation by ECVAM by discriminating two
categories, irritant and non irritant substances, according to the European
Classification System (ESAC Statement, November 2008). An adaptation of the
Global Harmonization System (GHS) by the European Union (EU) has conducted
to a new EU-GHS Classification. According to the new rules for classification, the
cut off score to distinguish between no category to category 2 substances was
shifted to 2.3 from 2.0 (former EU system). Consequently, test substances with in
vivo scores between 2 and 2.3 are now considered as non-irritants. The aim of the
study was to determine in vitro prediction of the SkinEthic RHE test method. For
this purpose, the global performances of the test method were recalculated on a
large set of test substances representative of different physico-chemical properties
(including the catch-up validation set). The predictive capacity values (specificity,
sensitivity and accuracy) were at least comparable to the required predictive values
criteria defined by ECVAM as described in the Skin irritation background document.
Therefore, the SkinEthic RHE test method was considered as a test method
adopted in the draft in vitro skin iritation OECD Test Guideline.
499 MEETING REQUIREMENTS OF THE NEW OECD TG
404 FOR IN VITRO SKIN IRRITATION TESTING:
REPRODUCIBILITY OF THE EPIDERM SKIN
IRRITATION TEST (EPIDERM-SIT) FOLLOWING THE
ECVAM VALIDATION AND ACCEPTANCE AS A FULL
REPLACEMENT METHOD.
M. Klausner, H. Kandarova, P. J. Hayden, J. Kubilus and J. E. Sheasgreen.
MatTek Corp, Ashland, MA.
In autumn 2007, an international validation study was performed to evaluate the
reproducibility and confirm the predictive ability of the EpiDerm Skin Irritation
Test (SIT) method. In November 2008, ECVAM endorsed the EpiDerm SIT as a
full replacement method for the in vivo rabbit skin irritation test. As reflected draft
OECD guidelines for skin irritation testing (TG 404), US and EU regulators have
appropriately maintained that a test method must demonstrate reproducibility on
an ongoing basis so that regulators and commercial end users are assured that the
assay method is stable and continues to give valid test results over time. The purpose
of the present study was to investigate the reproducibility of the EpiDerm SIT
post-validation. Over a 4-month period in 2009, 12 independent lots of EpiDerm
tissue were exposed to 3 irritants (alpha terpineol, heptanal, and butyl methacrylate),
3 non-irritants (benzyl benzoate, benzyl salicylate, and isopropanol) and the
positive control (sodium dodecyl sulphate) and negative control (Dulbecco’s phosphate
buffered saline) using the SIT protocol. As per the SIT method, tissue viability
was determined using the robust and cost effective MTT assay following a single,
60-minute exposure and 42-hour post-exposure incubation. In all cases, the
SIT method correctly identified the irritants and non-irritants. Coefficients of variation
(CV) between the tests (n=12) for the tissue viability for all test articles were