The Toxicologist - Society of Toxicology

vapors or water insoluble chemicals. The EpiAirway model is an organotypic airliquid
interface culture produced from normal human airway epithelial cells.
EpiAirway more adequately reproduces in vivo airway epithelial morphology and is
more amenable to dosing since the apical surface is exposed to the atmosphere.
Here we describe an approach to validate the EpiAirway model for airway toxicology
applications. Two dosing protocols were developed and evaluated: 1) Direct application
of test chemicals in aqueous or corn oil vehicles, and 2) a vapor cup
method for dosing volatile chemicals. Dose response experiments were conducted
to determine EC50 concentrations for loss of culture viability, transepithelial electrical
resistance or release of inflammatory mediators. EC50 concentrations were
correlated with OSHA established Immediately Dangerous to Life and Health
(IDLH) concentrations. Direct application was found to perform well for chemicals
with IDLH values up to ~1,000 mg/m3. A prediction model based on EC50
(culture viability) after direct application was established for 15 chemicals. A correlation
coefficient of r2=0.79 was obtained for the entire chemical set. Alkyl amines
were found to be over-predicted by the direct application protocol. With alkyl
amines removed from the data set the correlation improved to r2=0.98. Direct application
of chemicals with IDLH values above ~1,000 mg/m3 was generally not
possible. A vapor cup dosing protocol was developed for these chemicals. To-date
vapor cup results for 11 volatile chemicals including a diverse range of chemical
classes produced a correlation coefficient of r2 = 0.83 with respect to in vivo IDLH
values. Thus, the prediction models appear to provide promise for in vitro determination
of airway toxicity. Future work will validate these models with larger chemical
sets.
2568 DEVELOPMENT OF THE REPLACEMENT OCULAR
BATTERY-ROBATT-TIERED TESTING STRATEGY OF
ALTERNATIVE TOXICOLOGY TESTS TO REPLACE THE
NEED FOR RABBIT EYE TESTS.
D. Cerven, G. DeGeorge and M. Piehl. MB Research Laboratories,
Spinnerstown, PA.
Using a series of non-animal assays in a tiered approach, the Replacement Ocular
Battery (ROBatt) accurately predicts the categories of acute ocular irritation corresponding
to OECD, EPA and GHS guidelines. At present, no single alternative
assay has been accepted by regulatory agencies to completely replace the use of live
animals. The Bovine Cornea Opacity/Permeability test (BCOP) has been accepted
by OECD as a screen for severe and corrosive materials. The Cytosensor
Microphysiometer has been accepted for sub-severe testing but is applicable only
for aqueous-based materials. The ROBatt approach uses a series of two to three
non-animal assays to categorize both aqueous and non-aqueous materials. An
NIH/FDA Grant has been awarded to MB for development the ROBatt decision
tree criteria. Initial screening will use the Chorioallantoic Membrane Vascular Assay
(CAMVA) to discriminate slight or non-irritants from moderate to severe irritants.
Slight or non-irritating materials will be categorized using the Porcine Cornea
Confocal Assay (PorFocal). The BCOP will be used for discriminating between
moderate and severe to corrosive materials and the Porcine Cornea Opacity
Reversibility Assay (PorCORA) will be used to categorize severe irritants and corrosives.
Fifty validation chemicals from the ECETOC database of ocular irritation
will be used initially. Having performed over 6,700 CAMVA, 5,700 BCOPs, and
nearly 100 PorCORA assays, the researchers are confident of the ability to completely
categorize any material to international standards.
2569 THE EYES HAVE IT: CALF VERSUS ADULT EYES IN
THE BOVINE CORNEAL OPACITY AND
PERMEABILITY (BCOP) ASSAY.
D. A. Donahue 2 , D. Cerven 1 , G. DeGeorge 1 and J. Avalos 2 . 1 MB Research
Laboratories, Spinnerstown, PA and 2 Kao Brands Company, Cincinnati, OH.
The Bovine Corneal Opacity and Permeability (BCOP) assay uses excised bovine
corneas obtained from cattle slaughtered for food use. Consequently, the age of the
cattle is generally not provided with the corneas. This introduces a variable at the
start of the assay, which can be reduced by using eyes from cattle at a defined age.
The OECD guidelines for the testing of Chemicals No. 437 recommends the use of
eyes from cattle 6 to 12 months of age but encourages investigators to report the estimated
age and/or weight of the animals providing the corneas. In a commercial
operation, capable of killing large numbers of cattle to provide suitable numbers of
eyes, it may not be possible to determine age and weight since animals are received
from a number of suppliers. Additionally, random-source animals typically have
not been raised in constant environment and are subject to numerous environmental
variables. This study looked at the effect of the cattle’s age on the BCOP assay.
550 SOT 2011 ANNUAL MEETING
The calf eyes used in this study were obtained from a well-managed barn-raised
herd, which had weekly veterinary monitoring and controlled feed and medication.
In this study we compared the In Vitro Scores (IVS) from random source cattle
corneas with those of corneas from a well-managed calf herd of 4 to 5 months of age.
Thirty over the counter cleaners, hair dyes, hair sprays, deodorants and moisturizers,
which had IVS scores obtained from random-age animals were used in the evaluation.
The IVS scores from the random aged animals ranged from -0.62 to 111.58.
The IVS scores from age-defined calf eye corneas ranged from -1.11 to 22.86.
2570 APPLICATION OF AVAILABLE ALTERNATIVE
METHODS TO PREDICT SKIN SENSITIZATION OF
PRESERVATIVES.
S. Martinozzi Teissier, C. Piroird, S. Ringeissen, J. Eilstein, D. Duché, J.
Ovigne and J. Meunier. L’Oréal, Aulnay sous Bois, France. Sponsor: H. Toutain.
Contact sensitizers are reactive molecules that modify skin proteins to form antigen
recognized by specific T cells. In addition to the haptenation mechanism, contact
sensitizers induce several phenotypic and functional changes of dendritic cells that
allow them to migrate to the lymph node, present antigen and prime efficiently
hapten-specific T cells. Risk assessment of chemicals for skin sensitization so far relies
on animal assays. Concerned by ethical issues and regulatory changes in the EU,
the cosmetic industry is focused on finding nonanimal approaches to assess the sensitizing
potential of chemicals. Due to the complexity of the sensitization process it
is now commonly agreed that hazard identification and risk assessment could only
be addressed by combining a battery of assays. Such a combination should face the
physicochemical diversity of cosmetic ingredients as well as the need to quantify the
skin sensitization potency of chemicals to inform future risk assessment decisions.
Among the in vitro methods so far available for pre-validation studies, we developed
the Myeloid U937 Skin Sensitization Test (MUSST) based on the induction
of CD86 on U937 cells. More recently, we enlarged our set of assays by incorporating
the well-described direct peptide reactivity assay (DPRA). In this study we will
present how in silico tools, DPRA, MUSST could be jointly used for the evaluation
of a particular type of ingredients: we show the results obtained on a benchmark of
25 well known preservatives. This pragmatic exercise, allowed us to get an insight
into the feasibility, but also the actual limitations of using nonanimal methods for
the screening and/or evaluation of new chemicals for skin sensitization potential.
This case study shows that in silico and in vitro tools can already be used, to determine
profiles that correspond to « risk categories » based on benchmark chemicals
in a defined ingredient class or chemical class family.
2571 IL-18 SECRETION AS A MARKER FOR
IDENTIFICATION OF CONTACT SENSITIZERS IN THE
EPIDERM IN VITRO HUMAN SKIN MODEL.
W. Deng, J. Oldach, A. Armento, S. Ayehunie, H. Kandarova, S. Letasiova,
M. Klausner and P. J. Hayden. MatTek Corporation, Ashland, MA.
Assessment of the allergic potential of chemicals has traditionally been conducted in
animal models such as the local lymph node assay (LLNA). However, recent legislation
has prohibited the use of animals for conducting such tests on cosmetics or cosmetic
ingredients. Thus, animal alternative tests for contact sensitization are urgently
needed. Interluekin-18 (IL-18) secretion has recently been identified as a
useful endpoint for determination of contact sensitization potential in keratinocyte
monolayer cultures (Naik, SM, et al, J. Invest. Dermatol. 113:766–772, 1999;
Corsini, E., et al., Toxicology In Vitro 23: 789-796, 2009). The irritation potential
of a chemical may also influence the potency of sensitizing potential. Because epidermal
irritation and sensitization are often dependent on chemical penetration and
metabolism in the skin, 3D organotypic skin models that possess in vivo-like barrier
properties and metabolizing capabilities may provide significant benefits over
monolayer culture models for determination of contact sensitization potential. The
goal of the current work was to evaluate IL-18 as an endpoint for sensitization potential
after topical application of chemicals to the EpiDerm in vitro human skin
model. Eleven contact sensitizers and 4 non-sensitizer chemicals were tested based
on the chemical set specified by Corsini et al, 2009. A protocol was developed using
aqueous or ethanol based vehicles for topical application. Preliminary range-finding
experiments were conducted to determine chemical toxicity profiles using the MTT
viability assay. Additional doses were then chosen for definitive toxicity and IL-18
secretion experiments. EpiDerm tissue viability and IL-18 secretion by ELISA were
evaluated 18-24 hours following topical application of test chemicals. 10/11 contact
sensitizers were correctly identified by the assay (90.9% sensitivity) with no
false positive results (100% specificity). Thus, the EpiDerm IL-18 assay appears to
be a promising tool for in vitro determination of contact sensitization potential.