Microsoft PowerPoint - 2006 JSAAE kojima.pptHajime Kojima, Ph.D., JaCVAM, NIHS
Current validation and peer review in Japan
Planning on Validation Comet assay (in vivo or in vitro)Mutagenicity
Planning on Validation Lumi-cell, CER-estrogen reporter assay
Endocrine disrupter
Peer Review in progressCulture modelCorrocivity
Pre-validation in progressh-CLAT
Validation in progressLLNA-BrdU
Peer Review by ICCAM
Materials Cultured skin model Kit Name Components Maker EpiDermTM 24well/kit (EPI-200 +culture medium :KURABO
Vitrolife-SkinTM 23well/kit Gunze +culture medium
EpiDermTM
Vitrolife-SkinTM
Test protocol used was the blind trial of phase III in ECVAM validation*. The pre-test and main trial obtained cut-off percentage cell viability values (viability after 3 minute or 1 hour exposure). *Ref.:Liebsch, M., et al., ATLA 28, 371-401, 2000
Methods
NC4-Amino-1,2,4-triazole7 CPhosphoric acid8
NCSodium perborate12 CChromium trioxide13
*C: corrosive, NC: non-corrosive Ref.:Fentem, J. H., et al., Toxicol. In Vitro, 483-524, 1998, Liebsch, M., et al., ATLA 28, 371-401, 2000
CPotassium hydroxideKOH(10%)1 C/NC*ChemicalNo.
Pergormance of EpiDermTM and Vitrolife-Skin TM
188%0 0/12
EpiDermTM
Summary
Reliability of these two models was similar to the results obtained on ECVAM validation. Though peer review of these models is not yet completed , the ad hoc.committee of toxicology at MHLW in Japan has approved the utilization of these models to evaluate the corrosivity of a chemical.
Battery System for Prediction of Phototoxicity: the Yeast Growth
Inhibition Phototoxicity Assay and the Red Blood Cell Photohemolysis
Assay
Probably phototoxicant
Non phototoxicant
Yeast Growth Inhibition Phototoxicity Assay
Preparation of 2.5 % (v/v) RBC suspension
Preparation of test chemical solutions by ten-, four- or
five-fold dilution
Dispensation of 990 µL of RBC suspension or completely hemolyzed
solution into 24-well plate
Application 10 µL of test chemical solutions, solvents or PBS into 24-well plates
15.0 J/cm2 irradiation with a solar simulator
Unrradiation
+UV group -UV group
Dispensation of 100 µL of the irradiated and unirradiated RBCs suspension and complete hemolyzed solution into 96-well plates
Measurement of the absorbances at both 540 nm and 525 nm or an adjacent wavelength with a microplate reader
Classifications: Photohemolysis 5 : 5 Photohemolysis 10 :± 10 Photohemolysis :
Red Blood Cell Photohemolysis Assay
Summary Battery system for prediction of phototoxicity:
the yeast growth inhibition phototoxicity assay and the red blood cell photohemolysis assay was concluded to be a good method to predict the phototoxic potential of chemicals, though some improvements to the assay method are still required.
Validation
Day123 Chemical application Day123 Chemical application
5Hr later Extract Lymph node, measure of
H-TdR uptakecell growth
H-TdR uptakecell growth
LLNALocal lymph node assay OECD guideline 429 (2002)
Deduction Refinement Low cost& short term
GPMTBuehler
LLNA-DA
At 8 day, extract lymph node, Measure lymph node weight and ATP contents/mice(n=4)
At 8 day, extract lymph node, Measure lymph node weight and ATP contents/mice(n=4)
mouse CBA/JN 812w
At 1237daysapply of chemicals Before Expose 1 hr, apply with 1%SLSsolution At 1237daysapply of chemicals Before Expose 1 hr, apply with 1%SLSsolution
crush and suspension Diluted X100 in PBS
Materials
Date:April-July/2006 Participated lab.:10, 2-3 tests/Lab. Chemical used: Total 12, 4-6substances/Lab. 3 chemicals were examined by all 10 experimental laboratories
while 9 chemicals were each tested by 3 different laboratories. Chemicals with the fixed 3 doses were distributed to each laboratory coded to disguise their type. The value of 3 was set as the cut-off point of the stimulation indices (SI), which summarize the ATP amount.
Director: Dr. T. Ohmori (Kyoto Univ.) Organizer: Validation Committee in JSAAE Support: JaCVAM (Selection, coding & supply of chemical and
materials )
Results and Discussion
The results for the 3 chemicals examined by all laboratories and 5 of the other 9 chemicals were consistent and have small variances in the SI. There were 4 chemicals which produced inconsistent results between 3 laboratories. 2 chemicals showed the clearly dose response relationships. On the other hand, for the other 2 chemicals it seemed that the type of solvent in these chemicals caused the large variations. Sensitivity, specificity and concordance of the LLNA-DA compared to the GPMT/BT were 87.5% (7/8), 100% (3/3) and 90.9% (10/11), respectively. We conclude that, considering the published data of the LLNA, the results from this study are acceptable as a catch-up validation study, at least within the range of examined chemicals.
LLNABrdU
Validation Plan of LLNA-BrdU
of chemical and materials )
Comparison with results obtained from alternative methods in GPMT
Class in GPMT*Chemicals DEREK TOPKAT Pept ide- b inding assay
h-CLAT LLNA-Br U
LLNA-DA LLNA
Cinnamic aldehyde Posit iv e Posit ive Posit ive Posit ive Posit ive Posit iv e Posit ive
2,4-Dinitrochlorobenzene Posit iv e Posit ive Posit ive Posit ive Posit ive Posit iv e Posit ive
α-Hexy lc innamic aldehyde Posit iv e Posit ive Posit ive Posit ive Posit ive Posit iv e Posit ive
Formaldehyde Posit iv e Posit ive Posit ive Posit ive Posit ive Posit iv e Posit ive
p-Phenylened iamine Posit iv e Posit ive Posit ive Posit ive Posit ive Posit iv e Posit ive
Lact ic a cid Negat iv e Negat iv e Negat ive Nega tive Nega t iv e Nega tive
Resorc inol Posit iv e Posit ive Negat ive Posit ive Posit iv e Nega tive
Sodium laury l sulfate Negat iv e Posit ive Negat ive Nega tive Nega ti v e Posit ive
Posit ive (5
Chemical name LLNA-DA LLNA GPMT/ BA HMT HPTA
DNCB + + +
pPDA + + + + +
Current Validation Status of Reporter Gene Assays
in Japanese METI and MHLW
Comparison with ER /subtypes Transient - transfection method for human ER and ER-
Human ER/ HeLa & AUGp Reporter vector
Available Reporter Gene Assay in Japan -Stable-
· Under Development
· Showed 10 fold-induction at 10 nM of DHT · Completed data acquisition of 770 compounds · Currently pre-validated and validated under multi-
lab. validation in Japan
· Showed ca. 10 fold-induction at 1 nM of E2 · Completed data acquisition of 1457 compounds · Showed favorable relationship as the result of
comparison to Uterotrophic assay using 48 chemicals · Pre-validated using 55 chemicals listed by ICCVAM · Validated under multi-lab. validation in Japan (reported at 2nd VMG-NA)
StatusHost CellReceptor
CHO-K1human AR
human TR
HeLahuman ER
· Showed ca. 20 fold-induction at 1 nM of E2
· Showed ca. 60 fold-induction at 10 nM of DHT · Completing data acquisition of 250 compounds · Currently pre-validated and validated under
multi-lab. validation in Japan
· Showed ca. 10 fold-induction at 1 nM of E2 · Completed data acquisition of 250 compounds
· Showed ca. 10 fold-induction at 1 nM of E2 · Completed data acquisition of 300 compounds · Pre-Validated · Validated under multi-Lab. validation in Japan
StatusHost CellReceptor
HeLarat ER
CV-1human AR
HeLahuman ER
HeLahuman ER
Robot arm
Promoter
reporter-gene
HeLa cells
Collaboratory of MHWMETI HTSP of Human Estrogen Reporter Mediated Reporter Gene Assay
LUMI-CELLTM Estrogen Receptor Assay Xenobiotic Detection Systems, Inc
BG1Luc4E2 cell line (human ovarian carcinoma)
JaCVAM
ICCVAM
Management Team Protocol Chemicals Lab. International guideline
Proposal of OECD etc.
Xenobiotic Detection Systems, Inc
HeLa cell assay
Funding Sponsors National Institute of Environmental Health Sciences (NIEHS)
The European Centre for the Validation of Alternative Methods (ECVAM) The Japanese Center for the Validation of Alternative Methods (JaCVAM)
Study Management Team NICEATM
Consultation ICCVAM Endocrine Disruptor Working Group (EDWG)
ECVAM Endocrine Disruptor Task Force (EDTF) OECD Validation Management Group for Non-Animal Testing (VMG- NA)
LUMI-CELL® ER –TA Agonist and Antagonist Assays
hER HeLa-9903 Agonist Assay
Draft validation of Estrogen Receptor Assay
International validation study of In vitro/in vivo Comet assay
Genotoxicity test
DNA damage
Point mutation
Unscheduled DNA synthesis Comet assay
Unscheduled DNA synthesis Comet assay
Utilization of Transgenic animal Utilization of Transgenic animal
Ames assay Mouse lymphoma assay
Ames assay Mouse lymphoma assay
5
Chromosome aberration assay using mammalian cells
Study Management Team M.Hayashi (NIHS)
L.Schectmann (FDA) R.Tice (NICEATM) T.Hurtung (ECVAM) Y.Uno (Mitsubishi) H.Kojima (JaCVAM)
Consultation B. Burlinson (Huntington)
Y. Sasaki (Hatinohe) T. Ohmori (Kyoto Univ.):Statician N. Nakajima OECD Validation Management Group (VMG)
Prevalidation of Comet Assays
K.Morita (NIHS) T. Asano(Nittodenko) M.Nakajima (Biosafety Res. Ctr.) Y. Yamakage(Hatano, Inst.FDSC)
Biosafety Res. Ctr.
Safety Ctr.
Draft validation of Comet assay
Establishment of Management Team Researcher of USA, EU, Japan and Asian Secretary: JaCVAM
Kick-off meeting Management teamLead Lab.3-5Lab. Protocol of pre-validation
Pre-validation Lead Lab. 23 Samples(codedprotocol
Validation Lead lab. + 10 Lab. 20 samples
Proposal of test guideline OECD
*Telephone meeting
-- Schedule (unauthorized draft)Schedule (unauthorized draft) --
Pre-Validation Study* (2006-2007 To pick up practical (technical) issues of Comet assay In limited laboratories (lead laboratories) Using limited positive/negative compounds (2 or 3)
Definitive Validation Study (2007-2008) To make basic data for preparing the OECD guideline In the expanded number of laboratories** (if possible) Using 10-20 compounds (under a blind condition)
* Studies will be done in accordance with recommendations of 3rd IWGT (Environ. Mol. Mutagen., 2000) and 4th International Comet Assay Workshop (Mutagenesis, 2003)