The Toxicologist - Society of Toxicology

0-15, 0-24, 0-30 or 0-48 hours post fertilization (hpf) or 15-120, 24-120, 30-120
and 48-120hpf. Embryos treated until 15hpf or treated after 30hpf did not exhibit
a significant increase in lesions, while embryos treated between 15-30hpf had a significant
increase in MTBE specific vascular lesions (p≤0.05). We hypothesized that
MTBE disrupts the regulation of genes in the vascular endothelial growth factor
(VEGF) pathway. The effect of 5mM MTBE on VEGF associated genes was analyzed
by qPCR at 24h, within the critical period. MTBE significantly (p≤0.05) decreased
expression of VEGF-a and -c by 40 and 70% respectively, by had no effect
on VEGF receptor-2 or HIF1-α. Expression of Wnt3a, VE-Cadherin5 and βactin
were significantly decreased, while MMP-2 and -9 were not. To further understand
the effect of MTBE on zebrafish development at time course analysis of global gene
expression patterns was conducted using the Zebrafish Affymetrix GeneChip.
Embryos were exposed to 6.25μM, 0.625mM or 5mM from 0-15, 0-24 or 0-
30hpf. Preliminary evaluation of the data show the maximal number of genes with
a greater than 2 fold-change occurred at 24hpf for all treatments. The least number
of gene changes occurred at 30hpf. Taken together, these data suggest that vascular
lesions observed with MTBE exposure develop as a result of disruption of genes expressed
in the first 24h of development despite the fact that lesions are first apparent
at 48hpf. These studies demonstrate critical periods in zebrafish vascular development
and identify pathways sensitive to cardiovascular toxicants. Training Grant
#ES07148 Center Grant #ES05022
2549 EFFECT OF TRIAZINES ON TESTOSTERONE
BIOSYNTHESIS IN BLTK1 MURINE LEYDIG TUMOR
CELLS.
A. L. Forgacs 1 , Q. Ding 1 , I. T. Huhtaniemi 2 , N. A. Rahman 2 and T. R.
Zacharewski 1 . 1 Michigan State University, East Lansing, MI and 2 University of
Turku, Turku, Finland.
Atrazine (ATR), Simazine (SIM), and Propazine (PPZ) are equipotent triazine herbicides
that have been implicated in the etiology of testicular dysgenesis by altering
steroidogenesis. To further investigate their effects on testosterone biosynthesis,
ATR, SIM, and PPZ were examined in BLTK1 cells, a novel murine Leydig cell line
(BLT-1 cells, clone K1) that possess an intact steroidogenic pathway producing low
basal levels of testosterone (T), which can be induced by recombinant human
chorionic gonadotropin (rhCG). ATR, SIM and PPZ (1, 3, 10, 30, 100, 300 and
600 μM) elicited dose-dependent increases in progesterone (P) and T levels up to
4-fold relative to DMSO at 24 hrs as determined by ELISA. Temporal analysis (300
μM at 1, 2, 4, 8, 12, 24 or 48 h) elicited comparable P and T profiles by all three
triazines (ATR > PRO >> SIM) that were similar to the P and T profiles induced by
rhCG. ATR elicited time- and dose-dependent induction of Star and Hsd17b3
mRNA levels while Hsd3b1, Cyp17a1 and Srd5a1 mRNA expression was repressed.
PPZ elicited similar, albeit weaker, gene expression effects while SIM had
minimal effects on steroidogenic gene expression. These results indicate that triazines
alter P and T levels by regulating gene expression profiles of specific steroidogenic
enzymes.
2550 IMPROVEMENTS AND LIMITATIONS OF THE BOVINE
CORNEAL OPACITY AND PERMEABILITY TEST (BCOP,
OECD TEST GUIDELINE 437) IN ROUTINE TESTING
FOR SEVERE OCULAR IRRITANTS.
H. Raabe 3 , A. Schrage 1 , R. Curren 3 , K. Norman 3 , S. N. Kolle 2 , M. Rey-
Moreno 2 , B. van Ravenzwaay 2 and R. Landsiedel 2 . 1 Product Safety, BASF SE,
Ludwigshafen, Germany, 2 Experimental Toxicology and Ecology, BASF SE,
Ludwigshafen, Germany and 3 Institute for in vitro Sciences, Inc., Gaithersburg, MD.
Data on eye irritation are generally needed for hazard identification of chemicals.
Since the bovine corneal opacity and permeability (BCOP) test has been accepted
by many regulatory agencies for the identification of corrosive and severe ocular irritants
since September 2009 (OECD Test Guideline 437), we evaluated this alternative
method using a broad variety of chemicals and formulations. Ten reference
standards recommended in the TG437 and 21 substances (including historical false
negatives [FN] and false positives [FP]) with published BCOP and in vivo data
were tested in two labs (BASF and IIVS). Our results matched the published in
vitro data very well, the concordance was 77% (24/31) with FN and FP rates of
20% (2/10) and 24% (5/21). However, one of the solid test substances, Dibenzoyl-
L-tartaric acid, had a high variability in both labs resulting in false negative (FN)
classifications in about half of the BCOP assays performed (n=8). Additionally as
suggested by the guideline, we used histopathological evaluation of cross sections of
treated corneas to identify false FN. This endpoint corrected the classification of
some FN, but increased the number of false positives at the same time. Thus it did
not increase of the overall predictivity. In parallel, we compared the opacitometer
546 SOT 2011 ANNUAL MEETING
which was originally used in the development of the BCOP in the 1980s to a new
opacitometer model with certified components, electronic data processing and calibration
standards (glass filters). The data obtained with the new instrument correlated
almost perfectly with the traditionally machine. Regardless of the utilized
equipment, we recommend the use of calibration filters for stability, reproducibility
and comparability of BCOP results from different laboratories.
2551 ECVAM PREVALIDATION STUDY ON SKIN
SENSITISATION ALTERNATIVES: UPDATE ON
PROGRESS.
A. Angers-Loustau 1 , D. Basketter 2 , P. Aeby 3 , S. Aiba 4 , N. Alépée 8 , T. Ashikaga 5 ,
T. Cole 1 , A. Compagnoni 1 , G. Gerberick 6 , S. Hoffmann 7 , J. Ovigne 8 , J.
Richmond 9 , H. Sakaguchi 10 and S. Casati 1 . 1 European Commission (Joint Research
Centre), Ispra, Italy, 2 DABMEB Consultancy Ltd., Sharnbrook, Bedfordshire, United
Kingdom, 3 Colipa, Skin Tolerance Task Force, Brussels, Belgium, 4 Tohoku University
Graduate School of Medicine, Sendai, Japan, 5 Shiseido Quality Assessment Center,
Yokohama, Japan, 6 Procter & Gamble, Cincinnati, OH, 7 Seh Consulting & Services,
Koln, Germany, 8 L’Oréal Research, Aulnay-sous-Bois, France, 9 Home Office,
Dundee, United Kingdom and 10 Kao Corporation Safety Science Research
Laboratories, Tochigi, Japan.
Recent progress with in vitro assays in skin sensitization toxicology has resulted in
the development of test methods which could make a valuable contribution to the
replacement of the existing animal tests. ECVAM is currently evaluating three approaches:
the Direct Peptide Reactivity Assay (DPRA), the Myeloid U937 Skin
Sensitization Test (MUSST) and the human Cell Line Activation Test (h-CLAT) in
a prevalidation study conducted in liaison with JaCVAM and ICCVAM. The main
purpose of the study is to assess the robustness and reliability (within- and betweenlaboratory
reproducibility) of the test methods. To this end, they are challenged
with a set of 24 coded chemicals in three laboratories each. Results generated in this
study will allow exploring the possible contribution of these tests to the prediction
of skin sensitisers and their potency. An overview of the challenges and conclusions
of the Transferability phase will be presented, as well as additional preliminary results
from the testing phase with coded chemicals. In the case of the DPRA, the experimental
part will be finalized and the results presented.
Disclaimer: The views expressed in this poster are purely those of the authors, and
should not be regarded as an official position of the European Commission.
2552 LEYDIG CELLS RESPONSES TO STIMULATION WITH
LUTEINIZING HORMONE IN CULTURES FROM RATS
OF DIFFERENT AGE.
P. Balbuena, J. L. Campbell, R. Clewell and H. J. Clewell. The Hamner Institutes
of Health Sciences, Research Triangle Park, NC.
Leydig cells, which synthesize and secrete testosterone, are essential in the development
and maintenance of male phenotype and reproductive capacity. Most of the
information available in the literature on Leydig cells originated from evaluation of
whole animals, intact testes, or tumor-derived cell lines. However, alterations in cellular
function associated with immortalization limits the use of cell lines for studying
normal function or chemical disruption of the Leydig cell itself. A viable system
based on primary Leydig cell cultures would present a great advantage in elucidating
mechanisms of action for compounds affecting Leydig cells steroidogenesis.
Primary cultures of Leydig cells were established from testes of 1, 3, and 5 month
old Sprague Dawley rats. Passages of these cultures were performed until cells
reached senescence. Cells were then tested for production of testosterone in response
to Luteinizing hormone (LH) at 2, 4, 8, 16, and 24 hr. Since adult Leydig
cells require LH stimulation to produce testosterone, LH induction and measurements
of hormone production allowed us to assess variability due to time of exposure,
passage of cells, and age of the primary cells in culture.
LH induced testosterone concentrations increased over time, with maximum levels
8-fold that of the control at 16 hr. for all ages. LH-induction of testosterone in the
1 and 3 months old rat cells reached 5-7 ng/ml, but in the 5 months old rat cells the
levels were 0.05 ng/ml. Cells from 3 month old rats were viable up to 5 passages.,
although testosterone production decreased at passage 3. Passages of cells from 5
month old rats were not viable. This system shows considerable potential for the
study of age dependent differences in normal Leydig cell function without the additional
complications inherent in an in vivo system. Furthermore, the ability to
maintain testosterone production in passaged cells presents an opportunity for
long-term study of molecular mechanisms of compounds associated with Leydig
cell abnormal function.