The Toxicologist - Society of Toxicology

Cleveland, while others were found more universally (Group 2). Asthma exacerbations
were lower in remediated homes compared to non-remediated homes. The
study objective was to compare the allergy induction potential of these groups of
molds to house dust mite (HDM) in our mouse allergy/asthma model. Female
BALB/c mice received 1 or 4 exposures by intratracheal aspiration of mold or
HDM extract (0-80 μg). Serum and bronchoalveolar lavage fluid (BALF) were collected
2 days after the final exposure. The allergic impact of the various mold extract
exposures was different for each regardless of its categorization. Multiple exposures
were required to induce increased BALF eosinophil counts and serum total
IgE. To achieve similar results to those induced by HDM in the antigen-specific
IgE assay, Group 1 molds required 1.5X more Scopulariopsis brevicaulis (SBE) or
6.7X more Trichoderma viride (TVE). Group 2 molds required: 0.2X as much
Cladosporium cladosporiodes (CCE) or 2.3X more Epicoccum nigrum (ENE) to
produce similar results Both CCE and ENE induced BALF eosinophil counts and
responses to methacholine (Mch) challenge similar to or higher than HDM.
Neither Penicillium crustosum (PCG, Group 1) nor Wallemia sebi (WSE, Group
1) induced robust cellular or IgE responses but PCG did induce elevated responses
to Mch challenge at the higher doses. The data suggest that the categorization of
certain molds as Group 1 or 2 does not indicate their capacity to induce allergic responses
in a mouse model. However, the current study does not allow us to draw
conclusions regarding the capacity of Group 1 and 2 molds to non-specifically exacerbate
asthmatic responses. (This abstract does not reflect U.S. EPA policies.)
299 THE INFLUENCE OF GENETIC BACKGROUND IN A
MOUSE MODEL OF CHEMICAL-INDUCED
OCCUPATIONAL ASTHMA.
K. Luyts, V. De Vooght, B. Nemery, P. H. Hoet and J. A. Vanoirbeek. Research
Unit for Lung Toxicology, K.U. Leuven, Leuven, Belgium.
The development of occupational asthma is the result of interactions between environmental
factors and individual susceptibility. In this study we assessed the influence
of the genetic background - as present in different mouse strains - in a model
of chemical-induced occupational asthma, using a typical respiratory sensitizer,
toluene diisocyanate (TDI). On days 1 and 8, male mice of different strains
(BALB/c, BP/2, A/J, C57Bl/6, DBA/2, CBA and AKR) were dermally treated with
TDI (0.3%) or vehicle (acetone olive oil, AOO, 2:3) on each ear (20μl). On day
15, they received an oropharyngeal challenge of TDI (0.01%) or AOO (1:4).
Airway reactivity to methacholine was measured (pulmonary resistance) 22 h later.
Total and differential cell counts, IL2, IL5, IL6, IL13, TNFα and KC were assessed
in bronchoalveolar lavage (BAL) fluid. Total serum IgE and IgG2a levels were measured.
Lymphocyte subpopulations (CD4, CD8, CD25 and CD19) in auricular
lymph nodes and release of IL2, IL4, IL10, IL13 and IFNγ by lymphocytes were assessed.
Airway hyperreactivity was observed in the TDI-treated BALB/c, BP/2, A/J
and AKR mice. Airway inflammation was most pronounced in TDI-treated
BALB/c mice compared to their own control. No significant differences in BAL cytokines
were found. Numbers of helper T cells (CD4), activated T cells
(CD4CD25), cytotoxic T cells (CD8) and B cells (CD19) were increased in the auricular
lymph nodes of TDI-treated BALB/c, BP/2, A/J and CBA mice. In TDItreated
BALB/c, BP/2, A/J, C57Bl/6 and CBA mice elevated concentrations of IL4,
IL10, IL13 and IFNγ were detected in the supernatant of the auricular lymphocytes
along with an increase in total serum IgE. These experiments indicate that the genetic
background has a large impact on different aspects of an asthmatic phenotype.
In this model of chemical-induced occupational asthma, the human phenotype is
best reflected by the BALB/c mice. Acknowledgments: The Interuniversity
Attraction Pole Program (P6/35) and the Research Foundation - Flanders (FWO
G.0547.08)
300 COMPARISON OF LLNA RESPONSES BETWEEN CBA
AND BALB/C MOUSE STRAINS.
T. Burns 1 , J. Strickland 1 , E. Salicru 1 , D. Allen 1 and W. Stokes 2 . 1 ILS,
Inc./NICEATM, NIEHS, Research Triangle Park, NC and 2 NICEATM, NIEHS,
Research Triangle Park, NC.
While CBA is currently the recommended strain for the LLNA, the assay was originally
developed using BALB/c mice. Since the introduction of the LLNA, several
groups have published LLNA studies using BALB/c mice, including the National
Toxicology Program, the National Institute for Occupational Safety and Health,
and the Dow Chemical Corporation. BALB/c mice are used in countries where
CBA mice are difficult to obtain. This has resulted in reference databases for the
LLNA that include studies conducted with both CBA and BALB/c mice. However,
there is little published literature that directly compares the performance of the
LLNA in studies done on the same substances in the two mouse strains. The study
reported here is a retrospective evaluation of the performance of the LLNA when
using CBA mice with those using BALB/c mice. NICEATM evaluated 108 independent
studies representing 15 substances in four vehicles in which 86 studies
used CBA mice and 22 used BALB/c mice. Thirteen of these substances had guinea
pig reference data and 12 had human reference data. LLNA outcomes using
BALB/c are in agreement with LLNA outcomes obtained with CBA for 87%
(13/15) of the test substances. LLNA outcomes with CBA agree with guinea pig
outcomes for 92% (12/13) of the test substances and with human outcomes for
92% (11/12) of the test substances. LLNA outcomes with BALB/c agree with
guinea pig outcomes for 77% (10/13) of the test substances and with human outcomes
for 75% (9/12) of the test substances. A correlation analysis of log transformed
EC3 values calculated using LLNA data from each of the two strains indicates
that the results from the two strains are correlated (r = 0.75). Overall, these
data indicate that LLNA outcomes do not differ appreciably when either CBA or
BALB/c mice are used as test animals. ILS staff was supported by NIEHS contract
N01-ES-35504.
301 PREDICTION OF SKIN SENSITIZATION POTENTIAL
OF CHEMICALS BY HUMAN CELL LINE ACTIVATION
TEST (H-CLAT) AND AN ATTEMPT OF
CLASSIFICATION OF SKIN SENSITIZATION POTENCY.
Y. Nukada 1 , T. Ashikaga 2 ,T.Abo 1 , S. Sono 2 , H. Sakaguchi 1 , H. Itagaki 2 and
N. Nishiyama 1 . 1 Kao Corporation, Tochigi, Japan and 2 Shiseido Co., Ltd.,
Kanagawa, Japan.
We have developed the h-CLAT, an in vitro skin sensitization test using THP-1
cells (human monocytic leukemia cell line). This test is based on the augmentation
of CD86 and CD54 expression in THP-1 cells following exposure to chemicals. In
this study, we evaluated about 106 chemicals with different potentials of skin sensitization
in the h-CLAT and compared our results with LLNA or human test data.
Moreover, we tried to clarify the applicability domain of this method based on
physico-chemical property. The accuracies of the h-CLAT vs. LLNA or human test
data were about 84 % or 80%, respectively. Among several types of physico-chemical
properties of each chemical, molecular weight, boiling point, melting point and
Log Kow are not related to the applicability domain of the h-CLAT. On the other
hand, many of false-negatives did not dissolve to the medium at the highest applying
dose. Therefore, it seems that in the current protocol of h-CLAT, poor solubility
of samples to the medium is the most important limitation. We also evaluated
the utility of h-CLAT to classify the skin sensitization potential by using various
calculated values. From the data of 66 chemicals, which was both positive in h-
CLAT and LLNA, several values were calculated as follow: 1) CV75, 2) maximum
RFI of each chemical, 3) EC150, and 4) EC200. Correlational analyses between
LLNA EC3 and the four values were performed. A statistically significant correlation
was observed between CV75, EC150, and EC200 values with LLNA EC3.
The EC150 value showed the better correlation compared to other values. From
EC150 and EC200, Minimum Induction Threshold (MIT) was determined as a
minimum value, smallest of either EC150 or EC200. MIT also show the good correlation
with EC3. From these data, the h-CLAT values might be a one of the useful
tool to predict the allergic potency of chemicals after improving the detailed
conditions.
302 ESTABLISHMENT OF AN EX VIVO METHOD TO
DETECT ALLERGEN SPECIFIC IGE MEDIATED
HISTAMINE RELEASE IN BASOPHILS FROM
CYNOMOLGUS MONKEY WHOLE BLOOD.
P. Skov 2 , A. Lucock 1 , S. Kirk 1 and D. Everett 1 . 1 Covance Laboratories Ltd.,
Harrogate, North Yorkshire, United Kingdom and 2 RefLab ApS, Copenhagen,
Denmark.
Many biotechnology derived pharmaceuticals intended for human use have the potential
to be immunogenic, especially in animals. During pre-clinical safety assessment
it is important to determine if an antibody response has been made against
the test substance and to determine if such a response has the potential to be detrimental.
One possible outcome is that an immune response to a biological may generate
IgE antibodies which can lead to a Type 1 hypersensitivity and the possibility
of anaphylactic shock. When an antibody response against a test substance is established
it is important to characterize this response and establish possible adverse
consequences. In this study we have established a method which can measure IgE
mediated release in basophils from cynomolgus monkey whole blood.
White blood cells (WBC) including basophils were isolated from whole blood in
EDTA, by dextran sedimentation, from 4 primates suspected of having a type I hypersensitivity
reaction to a test substance designed for the treatment of clotting disorders.
Harvested white blood cells were pre-incubated with IL-3, then aliquoted
onto microtitre plates and incubated at 37°C for 1 hour. The experiment was designed
such that WBC’s from each animal were incubated with 4 different batches
SOT 2010 ANNUAL MEETING 65