The Toxicologist - Society of Toxicology

645 EVALUATION OF THE IN VITRO EPISKIN AND
SKINETHIC RHE SKIN IRRITATION TEST METHODS
FOR HAZARD IDENTIFICATION OF CHEMICALS.
D. Lelièvre 1 , N. Alépée 1 , J. Cotovio 1 , M. H. Grandidier 1 , A. De Brugerolle de
Fraissinette 2 , C. Tornier 2 and J. R. Meunier 1 . 1 L’Oréal, Aulnay sous Bois, France
and 2 SkinEthic Laboratories, Lyon, France. Sponsor: H. Toutain.
On July22nd, 2010, the in vitro skin irritation OECD Test Guideline TG439 and
the supportive Background Document were adopted. As reflected in the OECD
TG439, the EpiSkin, designated as the Validated Reference Method and the
SkinEthic RHE method can be considered to be applied to a wide range of physicochemical
substances and mixtures. The evaluation of both the reproducibility and
the reliability of these methods was performed to confirm their ability to predict the
classification of chemicals in the former and the recent implemented Global
Harmonization System (GHS) classifications applied in Europe. The present study
purpose was to investigate the performances of both test methods with an expanded
set of 62 chemicals in both classifications. Chemicals with in vivo scores between 2
to 2.3 are now considered as non-irritants. Consequently to the new Classification
Labelling Packaging (CLP) system, the number of No category substances were increased
from 37 to 45 whereas the irritant category number decreased to 17. When
comparing the obtained results in the old versus the new European classification
systems, the sensitivities of both, the in vitro Validated Reference EpiSkin method
and the SkinEthic RHE test method were improved to 88.2 and 94.1%, respectively.
Accuracies of both test methods were 83.9 and 77.4 respectively. Overall performances
were in accordance with criteria defined in the Annex 2 of OECD
TG439. These results confirm that these two test methods can be considered as reliable
in vitro screening tools to assess the skin irritation potential of chemicals for
the REACh registration and the European Cosmetic Directive legislation.
646 ADAPTATION OF THE VALIDATED SKINETHIC RHE
SKIN CORROSION TEST METHOD TO 0.5 CM2 TISSUE
SAMPLE.
C. Tornier 1 , M. Roquet 1 , N. Alépée 2 , J. R. Meunier 2 and A. De Brugerolle de
Fraissinette 1 . 1 SkinEthic Laboratories, Lyon, France and 2 L’Oréal, Aulnay sous Bois,
France. Sponsor: H. Toutain.
The Globally Harmonised System for the classification and labelling of chemical
substances and mixtures (GHS) defined skin corrosion as the production of irreversible
tissue damage in the skin. In vitro human reconstructed epidermal models
have been used to develop protocols able to discriminate corrosives from non corrosives.
The SkinEthic RHE test method, using 0.63 cm2 inserts, was validated by
ESAC (ECVAM advisory board) in 2006. Due to manufacturing constraints, the
SkinEthic RHE model is now produced on 0.5 cm2 instead of 0.63 cm2. The present
study first aim to demonstrate that the RHE skin corrosion protocol could be
adapted from 0.63 cm2 to 0.5 cm2 RHE tissues samples. For such purposes, the
protocol size adaptation was performed using the 25 chemicals including the 12
OECD TG431 reference test substances. To test the robustness and relevance of the
test method, particular attention was given to choose chemicals correctly classified
(non corrosive, corrosive) but also misclassified chemicals (false positives / negatives).
In addition, the objective was to show that the quality of RHE tissues was
not only maintained but also improved. The quality control, performed on 136
(0.63 cm2) and 262 (0.5 cm2) RHE batches, showed a mean of 0.991 and 1.169,
respectively. This similarity over the 9 years demonstrates the high quality production
of the tissues using both viability and morphology parameters. Results obtained
with the 0.5 cm2 skin corrosion test method showed that all corrosives (12)
were correctly classified, and 11 out of 13 chemicals were identified as non corrosives.
The 2 misclassified chemicals were also identified as false positives in the reference
validated EpiSkin test method. The overall accuracy over the 25 chemicals
was 92%. The specificity and the sensitivity of the 12 OECD TG431 reference
chemicals were 100%. In conclusion, the quality of 0.5 cm2 SkinEthic RHE tissues
was thoroughly maintained over 9 years and the performance of the skin corrosion
test method fully meet the OECD and ECVAM requirements.
647 EXPRESSION AND INDUCTION OF XENOBIOTIC
METABOLISM GENES IN THE STRATATEST® HUMAN
SKIN MODEL.
C. Rasmussen, K. Gratz, N. Simon, M. VanderZanden, C. Johnston and L.
Allen-Hoffmann. Stratatech Corporation, Madison, WI.
Human in vitro skin models have shown broad utility for toxicological testing due
to the similarity to the in vivo state and the ability to evaluate compounds as dosed
in actual use or exposure. Importantly like human skin, which is metabolically
competent, numerous studies have confirmed the expression and induction of
phase I and phase II metabolic enzymes in keratinocytes and skin models. In the
present study, the metabolic competency of the StrataTest® human skin model was
investigated by evaluating the expression and induction of genes involved in xenobiotic
metabolism. The StrataTest® full-thickness human skin model, containing
both epidermal and dermal components, faithfully recapitulates many of the biological
characteristics of human skin. The model is generated using NIKS® keratinocytes,
a clinically-tested and consistent source of non-tumorigenic, pathogenfree
human keratinocyte progenitors. To confirm that NIKS® keratinocytes
provide appropriate metabolic capacity, gene expression profiles of phase I and
phase II enzymes from NIKS® and primary keratinocytes were compared. Gene
expression profiling was performed. Differences in expression of two-fold or greater
between NIKS® and primary keratinocytes were considered significant.
Concordance for expression of 51 phase I and phase II metabolic enzymes in
NIKS® and primary human keratinocytes was 98%. RT-PCR and qPCR were employed
to evaluate baseline gene expression and induction after exposure of the skin
model to xenobiotic agents. Cytochrome p450 1A1 (CYP1A1) and 1B1 (CYP1B1)
were weakly expressed constitutively, while N-acetyltransferase 1 displayed more robust
constitutive expression. Upon exposure to 3-methylcholanthrene, CYP1A1
and CYP1B1 expression was strongly induced. Induction of CYP1A1 was also confirmed
in skin tissues exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin. These results
show that similar to other in vitro skin models, StrataTest® skin tissues express
genes critical to xenobiotic metabolism, further demonstrating the utility of this
model for toxicological testing applications.
648 INTRAMUSCULAR INJECTION SITES: IS THE
QUALITY OF THE DIAGNOSIS IMPROVED WHEN
MORE THAN ONE TISSUE SECTION IS EXAMINED?
C. Thuilliez, M. Perron-Lepage, C. Clément, J. Briffaux and C. Botteron.
Ricerca Biosciences SAS, Lyon, France.
Microscopic evaluation of intramuscular injection sites at our Laboratory involves
the examination of three tissue pieces from each site, in order to maximize the
chance of examining the area where the injected material was deposited. We retrospectively
studied the extent to which the quality of the diagnosis is maintained if
only one section per injection site is examined.
Two rabbit and two rat studies with intramuscular injection of the vehicle or test
item were reviewed by one pathologist. In rabbit studies (160 animals and 800 injection
sites) intramuscular injections were given in the dorsolumbar, quadriceps
femoris or gluteus medius muscles and each site received one injection. In rat studies
(120 animals and 320 injection sites) injections were given in the quadriceps
femoris or gluteus medius muscles and each site was injected 4 or 7 times. For both
species, the injection was performed in the centre of this area, which was sampled at
necropsy. Three pieces were trimmed from the sample, each piece separated by 0.3
cm in rats and 0.5 cm in rabbits. Piece 1 was in the middle, piece 2 lateral to it and
piece 3 medial to it. Sections from each piece were re-examined and each of the
original diagnoses, which summarized findings in all 3 sections together, was assigned
to the section(s) where it was observed. The studies in each species were
pooled but control and treated animals were evaluated separately.
Section 1 had a statistically significantly higher number of relevant findings when
compared to sections 2 and 3 in treated rats and rabbits. In treated rats more than
85% of the original diagnostic features were found on this section. In rabbits, this
percentage reached more than 75%. In control rats and rabbits, section 1 had a
higher or equal number of findings than the other sections.
This study reveals that examination of more than one tissue piece does not greatly
improve the quality of the diagnosis. One middle piece is considered to be sufficient
to provide an accurate representation of lesions at intramuscular injection sites.
649 ESTABLISHMENT OF T-CELL INDEPENDENT
ANTIBODY RESPONSE IN RATS AS AN
IMMUNOTOXICITY EVALUATION METHOD.
S. Ito, H. Hattori, R. Kawai, S. Komatsu, K. Muramatsu, M. Yagi, T.
Matsuyama, T. Furukawa and A. Sanbuissho. Medicinal Safety Research
Laboratories, Daiichi Sankyo Co., LTD., Fukuroi, Shizuoka, Japan. Sponsor: M.
Teranishi.
T-cell independent antibody response (TIAR), as well as T-cell dependent antibody
response (TDAR), plays an important role in the biological defense. In this study,
we investigated the optimal conditions for evaluating TIAR in rats and performed a
validation study of TIAR using well-known immunosuppressive agents, cyclophosphamide
(CPA) and cyclosporine A (CsA).
To investigate the optimal conditions for TIAR, female Crl:CD (SD) rats were immunized
with trinitrophenylated Ficoll (TNP-Ficoll) as a T-cell independent antigen.
Based on the results, we selected the following conditions: the dose of TNP-
Ficoll: 30 μg/rat; number of immunization: once; and timing of blood collection to
measure anti-TNP antibodies by ELISA: 7 days after immunization.
SOT 2011 ANNUAL MEETING 139