The Toxicologist - Society of Toxicology

espectively. And these solutions were mixed. In order to identify skin sensitizer, the
mixture of test chemical and GSH was incubated for over 12 hours, and then the
incubated mixture was analyzed by LC-MS. Where the GSH conjugate with test
chemical was detected, the test chemical was judged to be a skin sensitizer.
Additionally, in order to classify the potency of skin sensitizer, the mixture of test
chemical and GSH was incubated for 2 hours. Then, maleimide was added to the
incubated mixture to react with unconjugated GSH. Based on the determination of
peak area of GSH conjugate with maleimide by LC-MS, the GSH reactivity of each
test chemical was calculated. The GSH reactivity data was compared with GHS
classification obtained from animal studies. Compared the results of the Peptide
Binding Assay and in vivo study, it is shown that the Peptide Binding Assay gives a
high positive predictivity. In addition, it is indicated that most of chemicals which
had high GSH reactivity, are classified into Category 1 under the GHS
system.Therefore, it is concluded that the Peptide Binding Assay is a useful screening
method for evaluation of skin sensitization potency. This assay will be helpful to
reduce the number of animals and to shorten the experimental period.
975 A NEW RECONSTITUTED HUMAN CORNEAL
EPITHELIUM MODEL FOR THE ALTERNATIVE EYE
IRRITATION TEST.
K. Jung 1 , S. Lee 2 , Y. Ryu 2 , H. Jung 2 , W. Jang 1 , J. Han 3 , S. Seok 3 , J. Park 3 , Y.
Son 4 , J. Chung 3 , Y. Park 1 and K. Lim 1 . 1 , Amore Pacific. Co. R&D Center,
Gyeonggi-do, Republic of Korea, 2 Modern Cell&Tissue Technologies Inc., Seoul,
Republic of Korea, 3 Seoul National University, Seoul, Republic of Korea and 4 Kyung
Hee University, Gyeonggi-do, Republic of Korea.
Many efforts are being made to develop new alternative in vitro test methods for
the eye irritation test. Here we report a new reconstructed human corneal epithelial
model (MCTT HCE model) prepared from primary-cultured human limbal epithelial
cells as a new alternative in vitro eye irritation test method. In histological
and immunohistochemical observation, MCTT HCE model displayed a morphology
and biomarker expressions similar to intact human cornea. Moreover, the barrier
function was well preserved as measured by high transepithelial electrical resistance,
effective time-50 for Triton X-100, and corneal thickness. To employ the
model as a new alternative method for eye irritation test, protocol refinement was
performed and optimum assay condition was determined including treatment
time, treatment volume, post-incubation time and rinsing method. Using the refined
protocol, 25 reference chemicals with known eye irritation potentials were
tested. With the viability cut-off value at 50%, chemicals were classified to irritant
or non-irritant. When compared with GHS classification, the MCTT HCE model
showed the accuracy of 88%, sensitivity of 100% and specificity of 77%. These results
suggest that the MCTT HCE model might be useful as a new alternative eye
irritation test method. #Acknowledgement: This research was supported by a grant
(09162KFDA557 and 10182KFDA508) from Korean Food & Drug
Administration in 2009-2010.
976 AN ACUTE 1 HOUR EXPOSURE TO 2, 3, 7, 8-
TETRACHLORODIBENZO-P-DIOXIN (2, 3, 7, 8-TCDD)
INHIBITED VITELLOGENIN (ZFVTG 1-3) INDUCTION
BY 17α-ETHYNYLESTRADIOL IN ZEBRAFISH (DANIO
RERIO).
S. M. Bugel 1 , L. A. White 2 and K. R. Cooper 2 . 1 Department of Environmental
Sciences, Rutgers University, New Brunswick, NJ and 2 Department of Biochemistry
and Microbiology, Rutgers University, New Brunswick, NJ.
Zebrafish (Danio rerio) were used to test the hypothesis that exposure to 2,3,7,8-
TCDD down-regulates the induction of 3 vitellogenin genes (zfVTG 1-3) by 17αethynylestradiol
(EE2). Vitellogenin is a hepatic protein required by developing
oocytes for growth and maturation and has been demonstrated by our lab to be
down-regulated and correlated with decreased egg production in killifish (Fundulus
heteroclitus) inhabiting Newark Bay (NJ, USA). 2,3,7,8-TCDD was chosen because
of its abundance in Newark Bay and for the ubiquity of AhR agonists in aquatic
systems reporting reproductive impacts on oviparous organisms. Preliminary experiments
established dose-response relationships for significant inductions of zfVTG1
and zfCYP1A mRNA by EE2 and 2,3,7,8-TCDD, respectively (p ≤ 0.05). To investigate
the effect of 2,3,7,8-TCDD on the EE2-dependent induction of zfVTG
mRNA expression, zebrafish embryos were either treated with EE2 (1000 pptr)
from 6 hpf to 4 days post fertilization or were pre-treated with 2,3,7,8-TCDD (25
and 400 pptr) for 1 hour at 3 hpf and then exposed to EE2. When treated with EE2
alone, zfVTG 1, 2 and 3 were significantly induced 42, 22 and 14-fold, respectively,
over control. Exposure to 400 pptr TCDD for 1 hour prior to EE2 treatment
208 SOT 2011 ANNUAL MEETING
significantly inhibited induction of zfVTG 1, 2 and 3, by 97, 98 and 92%, respectively.
Exposure to 25 pptr 2,3,7,8-TCDD down-regulated induction of zfVTG 1,
2 and 3 by 85, 75, 58%, respectively. These results demonstrate that acute exposure
to 2,3,7,8-TCDD can inhibit the induction of vitellogenin mRNA expression by
EE2 and to our knowledge are the first to report a direct effect on vitellogenin gene
regulation in an in vivo aquatic model. These findings offer insight for reproductive
impacts observed in oviparous species inhabiting aquatic systems heavily contaminated
by AhR agonists.
977 TOXICOLOGICAL INVESTIGATION OF VOLATILE
ORGANIC COMPOUNDS ON NON-MAMALIAN
MODEL SPECIES, DROSOPHILA MELANOGASTER
AND CAENORHABDITIS ELEGANS FOR INDOOR AIR
POLLUTANTS BIOMONITORING.
Y. Chung 1 , H. Eom 1 , K. Song 2 , S. Kim 3 , M. Rhee 4 , T. Chon 5 and J. Choi 1 .
1 University of Seoul, Seoul, Republic of Korea, 2 Korea Conformity Laboratories, Seoul,
Republic of Korea, 3 Seoul National University, Seoul, Republic of Korea, 4 Chungnam
National University, Seoul, Republic of Korea and 5 Pusan National University, Pusan,
Republic of Korea. Sponsor: D. Ryu.
Indoor air quality has been recognized as a significant health and environment
issue. Some air pollutants are reported to occur more frequently and at a higher
concentration in indoor air than in outdoor air, including volatile organic compounds
(VOCs). In this context, the indoor environment can be of crucial importance
because modem society spends most of their time indoors, and exposure to
VOCs may result in a spectrum of illnesses ranging from mild to very severe effects.
Most of researches on indoor air pollution, however, have been based on the measurement
of VOC in the environment and human samples. In this study, we investigated
toxicity of VOCs on non-mammalian species. There has been a strong need
to reduce, refine or replace mammalian species in toxicological testing with alternative
testing methods and non-mammalian models. Fully sequenced genomes of
Drosophila melanogaster and Caenorhabditis elegans have showed an unexpectedly
high level of conservation with the vertebrate genome, which makes them attractive
animal models for biological studies, including toxicology. In this study, applicability
of D. melanogaster and C. elegans functional genomics tools to indoor air pollutant
monitoring was tested; the responses of D. melanogaster and C. elegans to
VOCs exposure were screened across various mutant strains and sensitive mutant
strains were selected to be developed as biomonitoring tools for indoor air pollutants.
Detailed results will be presented in the conferences.
Acknowledgments : This work was supported by the “ The Eco-technopia 21 project”
of Korean Ministry of Environment through and by the Basic Science Research
Program through the National Research Foundation of Korea(NRF) funded by the
Ministry of Education, Science and Technology (2010-0016195)
978 DETERMINING SKIN SENSITIZATION POTENTIAL OF
MEDICAL DEVICE MATERIALS USING A NEW IN
VITRO METHOD.
D. Keller 1 , B. Wallace 1 , H. Wagner 1 , J. Gorski 2 and J. M. McKim 1 . 1 CeeTox,
Inc., Kalamazoo, MI and 2 North American Scientific Associates, Inc. . (NAMSA),
Northwood, OH.
The ISO 10993 series of standards require that medical devices be evaluated for
sensitization potential. This is accomplished using a method that subjects the device
to polar and non-polar extraction vehicles and testing the extracts in either the
mouse local lymph node assay (LLNA) or the guinea pig maximization test
(GPMT). Regulatory issues in the EU, consumer pressure, and testing efficiency
make in vitro tests desirable. Cells cultured in aqueous media may not provide sufficient
exposure for non-polar extracts. The aim of this study was to determine if a
new in vitro method for detecting chemical sensitization in HaCaT cells could be
used in human reconstituted skin models cultured at an air-liquid interface. In
order to determine whether the skin models would respond in a manner similar to
HaCaT cells, twelve chemicals used in medical devices were selected and evaluated.
The chemicals were administered at concentrations ranging from 0.1 to 2500 μM
in 0.5% DMSO and incubated for 24 hr at 37 o Cwith5%CO 2 . Direct reactivity
was determined by GSH binding. The expression of eleven genes under the control
of Nrf2, (e.g. NADPH-dehydrogenase quinone 1 and glutamate-cysteine ligase)
was measured by qRT-PCR. The data obtained were analyzed for GSH binding,
cell viability, magnitude of gene expression, and potency. These data were processed
by a proprietary algorithm and a toxicity index determined. The test compounds,
which included toluene diisocyanate and nickel sulfate, were accurately identified