1. National Toxicology Program (NTP) Established in 1978 in DHHS Headquartered at NIEHS Not a “regulatory” agency Data used for regulatory actions Research on “nominations” Dept of Health and Human Services (DHHS) NIHCDC FDA National Toxicology Program NIEHSNIOSHNCTR

2. NTP Mandate Coordinate toxicology testing within the federal government Strengthen the science base in toxicology Develop and validate improved testing methods Provide information about potentially toxic chemicals to health regulatory agencies, research agencies, the scientific and medical communities, and the public

3. Standardization of testing Physical facility Personnel requirements Protocols and procedures Animal husbandry Chemical analysis Collection and microscopic analysis of tissues Minimum standards for study design All GLP

4. In-Depth Evaluation of Toxicology Prechronic toxicology: 3 months or less Chronic toxicology/carcinogenesis: 2-years Developmental and reproductive Immunotoxicology Behavioral and neurotoxicology

5. Documentation Complete record for each animal documenting all in-life data, necropsy record, and histopathology for 45 tissues Animal room record for entire study Feed and water analysis Chemical vehicle analyses Interim and final reports

6. Pathology Review Process Approximately 45 tissues/animal collected, fixed, sectioned, stained, analyzed by ACVP certified veterinary pathologist Initial diagnoses reviewed by QA pathologist Final review and resolution of all diagnoses by Pathology Working Group (PWG)

7. NTP Research Areas Herbal medicines Dietary supplements Radiofrequencies/cell phones Phototoxicology Endocrine disruptors Nanoscale materials DNA based therapeutics

8. ntp.niehs.nih.gov Results of all 540 completed 2-year studies and toxicology studies are searchable Historical control data base Nomination instructions

9. Assay for Insertional Mutagenesis 7d primary receipients 7mo secondary receipients 7mo additional leukemias low or high MOI Lin- or Sca1+ BM cells Serum-free culture ~ 50% incidence of leukemias

10. 12-Week Pilot Study Establish proficiency with procedures, methodology, and assays; Provide opportunity to make modifications prior to conducting definitive study; Demonstrate successful engraftment in a high percentage of recipients

11. SD SA dsRed2 U5U5 R SF U5R SF PRE* Gammaretro pRSF91dsRed2pre* ψ SD U5R Δ U5U5 R Δ SF dsRed2 PRE* Gammaretro SIN vector pSRS11.SFdsRed2 pre*  Key: SF: U3 enhancer from Spleen Focus-Forming Virus R, U5: elements within LTR Ψ: viral RNA packaging signal Δ: deletion of U3 from LTR SD and SA: splice donor and splice acceptor, respectively PRE*: Wood-chuck post-transcriptional regulatory element, without pX ORF

12. Animal Groups GroupVectorBattelleCCHMC 1LTR155 1-controlEmpty particle10 2SIN155 2-controlEmpty particle10 single Infectious titer Bone marrow donors: ♂C57B/6J (CD45.2) Irradiated recipients: ♀ C57B/6J (CD45.1; BoyJ) 60 animals total

13. 12 Week Pilot Study 6-Week interim evaluation: hematology transgene expression – flow cytometry donor chimerism – flow cytometry myeloid lineage differentiation – flow cytometry

14. 12 Week Pilot Study Necropsy Peripheral blood by cardiac puncture: hematology immunophenotyping transgene expression freeze cells DNA isolation Bone marrow isolation hematopathology immunophenotyping and transgene expression freeze cells for DNA isolation, qPCR; LM-PCR, Eco-env PCR; archive

15. 12 Week Pilot Study Necropsy Spleen and Thymus organ weight; section for histopathology single cell suspension for immunophenotyping and transgene expression DNA isolation cryopreserve remaining cells Lymph nodes section for histopathology imunophenotyping and DNA isolation if enlarged

16. 12 Week Pilot Study Necropsy Liver, kidney, brain, ovary, heart, small intestine: organ weight section for histopathology freeze tissue for DNA isolation All other organs will be collected and preserved in formalin for possible microscopic examination

17. Assay for Insertional Mutagenesis primary receipients 7mo secondary receipients 7mo additional leukemias low or high titer ~ 50% incidence of leukemias 50 primary recipients 2 secondary recipients per asymptomatic primary recipient

18. Definitive Study AnimalsVectorsInfectious Titers Total Phase-15022200 Phase-2*10022400 Control-15011 Control-25011 Total = 700 *Assumes all 50 phase-1 animals are asymptomatic after 7 months

19. Definitive Study Peripheral blood sample taken at 6 week intervals Leukemias – differential count, hematology Transgene analysis by flow cytometry or qPCR Necropsy – similar to pilot Histopathology on all tissues form all animals Unused frozen tissue will be archived in NTP frozen tissue archive and made available to scientific community