The Toxicologist - Society of Toxicology

classifications were “non-severe”, these were considered to be GHS “Cat. 2”. The
tiered (bottom-up) approach combining the STE test, the EpiOcular assay / the
HET-CAM (a) assay and the BCOP assay allowed the evaluation of GHS eye irritation
rankings with a favorable predictivity (accuracy > 75%, under prediction rate
< 5%). From these results, this tiered approach might be a promising alternative
method for predicting eye irritation.
1002 TOWARDS ANIMAL-FREE TESTING FOR SKIN
SENSITIZATION: IN-HOUSE VALIDATION OF FOUR
METHODS: MUSST, H-CLAT, KERATINOSENS, AND
DPRA.
C. Bauch 1, 2 , T. Eltze 1 , E. Fabian 1 , S. N. Kolle 1 , C. Pachel 1, 3 , T. R. Hernandez 1 ,
B. Wiench 1, 4 , C. J. Wruck 2 , R. Landsiedel 1 and B. van Ravenzwaay 1 .
1 Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany,
2 Department of Anatomy and Cell Biology, RWTH Aachen University, Aachen,
Germany, 3 Technical University Kaiserslautern, Kaiserslautern, Germany and
4 Faculty of Chemistry, Pharmaceutics, and Geosciences, Johannes Gutenberg
University Mainz, Mainz, Germany. Sponsor: A. Van Cott.
Allergic contact dermatitis is induced by repeated skin contact with an allergen.
Assessment of the skin sensitizing potential of chemicals, agrochemicals, and cosmetic
ingredients is crucial to define their safe handling and use. Up to now, animal
tests have been used to identify skin sensitizing potential. Animal welfare as well as
the 7th Amendment to the Cosmetics Directive and REACH demands animal free
alternatives. The mechanisms of induction and elicitation of skin sensitization are
complex. To account for the multitude of events in the induction of skin sensitization
an in vitro test system will consist of a battery of various tests. Currently, we
perform in house validations of four in vitro assays addressing three different events
during induction of skin sensitization. 1) The peptide reactivity assay (DPRA, 1)
using synthetic peptides and HPLC analysis. 2) Two dendritic cell based assays on
the cell lines U 937 (MUSST) and THP-1 (h-CLAT) and flow cytometric detection
of the maturation markers CD54 and/or CD86 (2, 3). 3) ARE-dependent
gene activity in the reporter gene cell line KeratinoSens. We present the results of
these assays with more than 40 substances of known sensitizing potential including
the performance standards defined for the LLNA. The sensitivity, specificity and
accuracy of individual tests were obtained by comparison to human epidemiological
data as well as to data from the local lymph node assay.
1003 ASSESSMENT OF AIRWAY GENOTOXICITY
POTENTIAL USING THE EPIAIRWAY IN VITRO
HUMAN AIRWAY MODEL AND THE COMET ASSAY.
A. Armento, J. DeLuca, Y. Kaluzhny, H. Kandarova, M. Klausner and P. J.
Hayden. MatTek Corp., Ashland, MA.
Determination of genotoxicity potential is an important consideration for safety assessment
of chemicals that may be inhaled during exposure to consumer products,
occupational chemicals or environmental pollutants. Commonly used in vitro
genotoxocity assays produce a high rate of false positive results, limiting their practical
utility for predicting human genotoxicity hazard. For assessment of organ-specific
genotoxicity, 3D in vitro human tissue models which have in vivo-like barrier
function and metabolic capability are expected to have improved biological relevance
and predictive ability. Due to recently enacted legislation including REACH
and a ban on animal testing of cosmetics by the 7th Amendment to the Cosmetics
Directive, predictive organ specific genotoxicity tests are urgently needed. Previous
work has focused on development of skin specific genotoxicity tests including the
micronucleus assay and the comet assay. In the current poster, we describe development
of an airway specific genotoxocity assay using the EpiAirway model and the
Comet Assay. The EpiAirway model is produced from normal human airway epithelial
cells and reproduces the psudeostratified mucociliary phenotype of in vivo
proximal airways. The model has functional tight junctions, in vivo-like barrier
properties and in vivo-like xenobiotic metabolizing capabilities. EpiAirway tissues
were digested with (0.25%) trypsin to produce cell suspensions for use in Comet
Assay experiments. 100 comets were visually scored per tissue and each condition
was run in duplicate tissues. Visually scoring was conducted by assigning values for
% tail DNA on a 5 category scale (0 being undamaged DNA to 4 >80% DNA in
tail). Untreated control samples produced low background comet scores. Treatment
of the EpiAirway tissues with prototypical genotoxins such as methyl methane sulfonate
(MMS) prior to digestion produced statistically significant, dose-dependent
increases in % tail DNA. Thus, the EpiAirway Comet Assay appears to be a promising
approach to in vitro airway genotoxicity testing.
214 SOT 2011 ANNUAL MEETING
1004 APPLICATION OF PRECISION-CUT LIVER SLICES FOR
THE INVESTIGATION OF XENOBIOTIC-INDUCED
CELL PROLIFERATION.
E. Fabian 1 , F. Schuck 2, 1 , C. Jaeckh 1 , S. Groetters 1 , B. van Ravenzwaay 1 and R.
Landsiedel 1 . 1 Expermental Toxicology and Ecology, BASF SE, Ludwigshafen,
Germany and 2 Johannes Gutenberg-University, Mainz, Germany.
Precision-cut liver slices (PCLiS) are a commonly used in vitro system for the assessment
of metabolism and transport of xenobiotica in the liver. Within the current
study, PCLiS were evaluated for their applicability to detect xenobiotic induced
cell proliferation, since cell proliferation is a non-genotoxic contributor to hepatocarcinogenesis.
Therefore, a rotating culture system was established and characterized
by viability parameters such as membrane integrity (LDH, ALT), energy status
(ATP) and mitochondrial activity (MTT). In this system FCS-supplemented
Williams’ Medium E sustained liver slices for four to six days. Intracellular LDH activities
of slices showed activities of more than 200 U/L/mg wet weight after three
days of culture and declined to 5% of this activity after six days. ATP assessment
showed similar results. Slices contained levels of about 100 pmol ATP/mg wet
weight after three days of cultivation and declined to 3% of this level after six days.
Data from the MTT assay confirmed these findings. The mitochondrial reduction
declined to levels of about 40% of the control after six days of cultivation. Based on
the established culture conditions, the system was tested for its capability to induce
hepatocyte proliferation after treatment with Wy-14,643, a known PPARα agonist
in rodents. The proliferation in cultured liver slices was assessed as an integral over
five days by BrdU co-incubation and immuno-staining of histological sections of
PCLiS by counting stained cells. Incubations of 1 to 100 μM Wy-14,643 resulted
in a concentration-dependent increase of proliferating cells up to a factor of 1.5fold
compared to the control. This study demonstrated a proof of principle for the
detection of xenobiotic-induced induction of cell proliferation in PCLiS.
1005 EVALUATION OF XENOBOTIC METABOLISM IN
HUMAN RECONSTRUCTED SKIN MODELS FOR
GENOTOXICITY TESTING.
C. Jaeckh 1 , V. Blatz 1 , E. Fabian 1 , K. Reisinger 2 , B. van Ravenzwaay 1 , S. Trappe 3
and R. Landsiedel 1 . 1 BASF SE, Experimental Toxicology and Ecology, Ludwigshafen,
Germany, 2 Henkel AG and Co., KGaA, Duesseldorf, Germany and 3 Bundesinstitut
for Risikobewertung, Berlin, Germany.
Skin models are used to assess the irritation potential of chemicals. Chemicals and
their metabolites are also reported to induce DNA-damage. Information on chemical
biotransformation within the skin is therefore demanded for genotoxicity testing.
Metabolic capacities of native rat, human and, porcine skin have been reviewed
by Oesch et al. 2007. Less is known about the metabolic capacity of human reconstructed
skin models, a potential alternative in vitro test system to replace rodent in
vivo testing.
The objective of this study was to investigate enzymatic activities of three reconstructed
skin models compared to native human skin with the perspective to select
a regulatory accepted human skin model for genotoxicity testing. Enzyme activities
were investigated by incubation of model substrates within S9 or microsomal fractions
of the epidermal skin model EpiDerm(MatTek), the full-thickness skin
model EpiDermFT(MatTek) and the Phenion® full-thickness models
Phenion®FT (Henkel). Selected enzymes included CYP-enzymes, Flavin- dependent
monooxygenases, UDP-glucuronyltransferase as well as N-acetyltransferases.
Contradictive to reported CYP-enzyme transcription, CYP-enzyme activities remained
under the limit of quantification. FMO, UDP-GT and NAT–catalyzed reactions
were quantified in all reconstructed skin models, however with differing
catalytic activity. In summary, EpiDerm, EpiDermFT and Phenion®FT
show certain metabolic capacities. Catalytic activities differ between the skin models
and in between skin layers. The results obtained recommend the use of fullthickness
rather than epidermis skin models. Finally, we demonstrate that biotransforming
capacity of reconstructed skin models allows the detection of genotoxic
potential of promutagenic substances using the COMET Assay. We acknowledge
funding from the German Federal Institute of Research and Education (BMBF;
0315226D).
1006 CUTANEOUS METABOLISM.
J. Eilstein, G. Léreaux, J. Meunier, J. Leclaire and D. Duché. Life Sciences
Research-Safety Research Department, L’Oréal Advance Research, Aulnay-sous-Bois,
France. Sponsor: H. Toutain.
According to the literature, the metabolism of other organs than the liver such the
skin seems much less studied. Indeed, skin represents the major protective barrier of
the body to the environment and chemicals exposure but is not really yet consid-