The Toxicologist - Society of Toxicology

airway model expressed higher levels of many XMEs. Surprisingly, many airway
XMEs were downregulated by chemical treatment – 30 genes were down regulated
and 11 were upregulated >3-fold. For the epidermal model, 9 XME genes were
downregulated and 18 were upregulated >3-Fold. Remarkably, CYP1A1 was induced
337-fold and 225-fold in EpiAirway and EpiDerm, respectively. To determine
induction of Phase I activity, a substrate cocktail was incubated with the
models and metabolites were determined by LC-MS/MS or fluorometry. Baseline
metabolism activity was very low. Significant induction of metabolic activity was
observed only for the CYP1A1/CYP1B1 substrate. The results indicate that numerous
changes in XME expression levels can be induced in epithelial tissues by nuclear
receptor pathways. However, with the exception of CYP1A1/1B1, Phase I XME
metabolic activity in human epidermal and airway epithelium appears to be low.
478 FURTHER DEVELOPMENT OF AN EPIDERM IN
VITRO SKIN IRRITATION TEST FOR THE GLOBALLY
HARMONIZED SYSTEM OF CLASSIFICATION AND
LABELING OF CHEMICALS.
A. Armento 1 , H. Kandarova 2, 1 , M. Klausner 1 and P. J. Hayden 1 . 1 MatTek
Corporation, Ashland, MA and 2 MatTek in vitro Life Sciences Laboratories,
Bratislava, Slovakia.
Determination of skin irritation potential is an international regulatory requirement
to ensure safe handling, packaging, labeling, use and transport of chemicals,
cosmetics and household products. Recent REACH legislation and a ban on animal
testing for cosmetics have heightened needs for validated in vitro Skin Irritation
Tests (SITs). A UN treaty endorsed by the US, EU, China, Japan, Australia and
others has outlined a Globally Harmonized System (GHS) of Classification and
Labeling of Chemicals. The GHS classifies skin irritancy of chemicals into three
categories: non-irritant (NI or no label), slight irritant (SI or class 3) or irritant (I or
class 2). The EpiDerm model has been validated for in vitro skin corrosion testing
endorsed by OECD as Test Guideline (TG) 431. An OECD TG for in vitro SIT
based on 3 ECVAM validation studies is under way. However, currently validated
SITs distinguish only 2 classifications – Class 2 and no label, thus they do not completely
satisfy needs of regulatory bodies requiring 3 classifications. Therefore, additional
efforts are underway to validate an EpiDerm SIT for GHS. Previous work
utilizing 15 reference chemicals established a preliminary EpiDerm-GHS-SIT prediction
model (The Toxicologist, 108(1):379 (2009)). Here we report new results
with an expanded set of 36 chemicals including 12 from each GHS category. Using
a tiered strategy with 2 MTT viability assay protocols, SI plus I chemicals were
identified with a sensitivity of 91.7% and specificity of 91.7%. Irritants were identified
with a sensitivity of 91.7% and specificity of 83.3%. Concordance for correct
classification of each category was 91.7%, 50% and 91.7% for NI, SI and I, respectively.
Overall concordance was 77.8%. One I and two SI were underpredicted as
NI. However, 2/3 underpredicted chemicals are NI in human patch tests. Four SI
were overpredicted as I. Interlaboratory transferability of the EpiDerm-GHS-SIT
protocols will be evaluated prior to formal validation studies.
479 PORCINE CORNEAL OCULAR REVERSIBILITY ASSAY
(PORCORA) PREDICTS EU R41 AND GHS CATEGORY 1.
M. Piehl, R. Soda, M. Carathers, G. L. DeGeorge and D. R. Cerven. MB
Research Laboratories, Spinnerstown, PA.
Currently, there is no alternative (non-in vivo) ocular irritation assay that can measure
corneal tissue damage and reversibility. With the support of two Colgate-
Palmolive Grants for Alternative Research, we have developed an alternative assay:
Porcine Corneal Opacity Reversibility Assay (PorCORA). PorCORA measures
corneal damage and recovery over extended time periods using porcine corneas excised
from by-product abattoir eyes. Test articles (liquid and solid) are dosed directly
onto the corneal surface, and tissue damage and recovery are assessed by
sodium fluorescein (NaFL) retention in the same corneas over time (up to 21 days).
We have confirmed NaFL retention results and corneal recovery in the PorCORA
system via several approaches. Both fluorescence and reflective confocal microscopy
confirm damage repair indicated by fluorescein retention in the cultured corneas.
In addition, we have shown histological evidence that also correlates well with
NaFL staining in the PorCORA assay. Here we report the results of a 32-reference
chemical validation including chemicals from the following classes: acetates, acids,
alcohols, alkalis, esters, hydrocarbons, inorganics, ketones, surfactants, and several
solid compounds. To determine if the PorCORA system can predict R41 or GHS
Category 1, we considered corneas that retained NaFL at 21 days post-dose to be
R41 and GHS Category 1. ECETOC historical rabbit eye data was used to classify
EU and GHS eye irritation for the 32 compounds tested. PorCORA predicted
11/11 compounds classified as R41 and 12/13 compounds classified as GHS
Category 1. Since PorCORA can predict these categories, then compounds that
cause damage that is reversible in the PorCORA system may be considered R36 or
Category 2. Thus PorCORA is a highly predictive method to distinguish between
ocular irritancy classifications R36 or R41 and Category 1 or 2 without the use of
live animals.
480 ESR EVIDENCES OF NO2 DERIVED PEROXYNITRITE
AS TRIGGERING NEWER DIESEL ENGINE EMISSION
LUNG OXIDANT INJURY- OXIDATION CATALYSIS
RESPONSIBILITY.
J. Morin 1 , V. Hasson 1 , D. Preterre 2 , V. Keravec 2 and F. Dionnet 2 . 1 Inserm,
Rouen, France and 2 Certam, Saint Etienne du Rouvray, France. Sponsor: R. Forster.
Diesel particulate matter has frequently been claimed as the main trigger but some
studies suggest that gas phase pollutant might be responsible of oxidative stress induced
health effects. NO2 could be one of the major potential triggers. A continuous
flow exposure device to continuously sampled and diluted engine exhausts
using a biphasic air/liquid culture of rat lung tissue is used. Physical properties of
the aerosol and pollutant bioavailability remain unaltered in the delivered aerosol.
Study of the impact of treating Diesel exhaust by oxidation catalysis and diesel particulate
filter on lung tissue oxidative stress and reactive oxygen species production
measured by electron spin resonance was conducted. ESR measurement using CPH
as a spin probe was also measured in exposed culture media to a continuous flow of
aerosol in the absence of lung tissue and proved to be very useful for assessing the
oxidant potential of the aerosol. A highly significant correlation between NO2 concentrations
in the aerosol and the ESR signal was suggestive of a potential major
role of NO2 compared to Diesel soot for triggering Aerosol filtration for removing
soot did not alter the ESR signal from the aerosol.Diesel exhaust induced oxidant
stress has been identified. To verify the potential role of NO2, synthetic NO2 was
delivered from a cylinder and a series of antioxydants was tested for their ability to
prevent the formation of ROS assayed by ESR. It is demonstrated that MEG (a
peroxinitrite scavenger), prevents the occurrence of ROS production upon NO2
exposure. Glutathione, ascorbate and sodium dithionite exhibited similar activities
as MEG. Diesel exhaust treated by oxidation catalysis induced major oxidative
stress and lung tissue glutathione depletion, compared to untreated emissions.
Evidences that NO2 might trigger these effects through the formation of peroxinitrite
which is known to be detrimental to biological tissues especially to lung and
heart are diszcussed.
481 THE INFLUENCE OF PHORTRESS ON CYTOKINE
LEVELS AND TISSUE VIABILITY IN PRECISION-CUT
RAT LUNG TISSUE.
M. J. Furniss 1 , R. E. Parchment 1 , J. E. Tomaszewski 2 and H. P. Behrsing 1 .
1
Predictive Toxicology Section, Laboratory of Human Toxicology and Pharmacology,
SAIC-Frederick/NCI-Frederick, Frederick, MD and 2 Division of Cancer Treatment &
Diagnosis, National Cancer Institute, Bethesda, MD.
The effects of Phortress, an anti-proliferative chemotherapeutic compound currently
in clinical trials, were further examined using the precision-cut lung slice
(PCLS) model. Initial results using PCLS demonstrated increased cytokine levels
for rat and human tissues. Here a more thorough evaluation of Phortress’s concentration
response is made by examining pro-inflammatory cytokine (IL-1β, TNF-α,
CINC, and IFNγ) levels and histologically assessing tissue viability in rat PCLS.
PCLS were prepared from adult male Fisher 344 rats and incubated for 6 days in
serum-free M199 medium. Treatment with 10, 25, 50, and 100 μM Phortress (72
hr) was started after a two-day pre-incubation and was followed by a 24-hr drugfree
period before tissue harvest. Cytokine levels were measured in the lung slice
lysates at 24, 48, 72, and 96 hr post-dosing. A concentration-dependent cytokine
response was seen following treatment of lung slices with Phortress. At 10 μM
Phortress a cytokine response above the control levels was not seen. At 25 μM a
transient spike was observed during the treatment period, but this subsided once
Phortess was removed and replaced with medium. At 50 and 100 μM Phortress, a
greater and sustained cytokine response was observed that did not diminish following
Phortress removal. The concentration-dependent cytokine response correlated
well with the loss of lung parenchymal cells (pneumocytes and bronchioalveolar) viability,
as determined histologically. This study demonstrates that PCLS can be
used as an in vitro tool for examining the histological consequence and pro-inflammatory
cytokine response of investigational drugs. Comparative work with human
tissue will further evaluate PCLS as a model to make cross-species assessments for
the toxicological evaluation of new investigational drugs.
Funded by NCI Contract No. HHSN261200800001E.
104 SOT 2010 ANNUAL MEETING