Attachment I NCC Blanket SOW FINAL.pdf

STATEMENT OF WORK

NIEHS DIVISION OF THE NATIONAL TOXICOLOGY PROGRAM

HUMAN NEURAL CREST CELL MIGRATION ASSAY

1.0. BACKGROUND: The National Institute of Environmental Health Sciences (NIEHS), located in the Research Triangle Park, North Carolina, is one of the 27 Institutes and Centers composing the National Institutes of Health. The NIEHS is home to the Division of the National Toxicology Program (DNTP), a Health & Human Services (HHS) interagency program dedicated to testing and evaluating substances in our environment whose mission is to evaluate agents of public health concern by developing and applying tools of modern toxicology and molecular biology. The program maintains an objective, science-based approach in dealing with critical issues in toxicology and is committed to using the best science available to prioritize, design, conduct, and interpret its studies. To that end, NTP is continually evolving to remain at the cutting edge of scientific research and to develop, validate and apply new technologies

The DNTP is exploring effects of toxicants during neurodevelopmental stages that may be uniquely sensitive to environmental insults. This potential window of sensitivity is due in part to the enormous changes in key neurodevelopmental processes of proliferation, migration, apoptosis, and neural network maturation. As a consequence, the DNTP has a requirement to investigate the sensitivity to environmental chemicals in a battery of assays that cover key neurodevelopmental events during embryonic and neonatal development. The human neural crest cell (NCC) migration assay is one component of the battery used by the DNTP. One of the key processes in neurodevelopment is the differentiation and migration of NCCs. During development, NCCs migrate to target sites where they differentiate and become sensory neurons, glia, melanocytes, adrenal medulla and cranial cartilage/bones. If migration and differentiation of NCCs occurs incorrectly, malformations may occur.

DNTP requires a high-content, specific and reproducible NCC assay using NCCs differentiated from human induced-pluripotent stem cells. The NCC assay will allow for batch screening of environmental compounds with unknown DNT potential that might be used to prioritize compounds for targeted in vitro and in vivo DNT testing.

2.0. SCOPE: This Statement of Work (SOW) will be used to execute testing effects of compounds in the NCC assay testing services under the Indefinite Delivery/Indefinite Quantity (IDIQ) contract. During the contract the Government will identify tasks required to complete this testing effort. Task orders with specific details will be placed as needed by the DNTP. Specific performance dates and quantities will be specified on each individual task order.

3.0. OBJECTIVES: The purpose of this effort is to explore effects of environmental compounds with unknown developmental neurotoxicity potential in the NCC. Specifically, the Contractor shall test a set of 115 environmental compounds in the NCC.

4.0. TASKS: The contractor shall evaluate the neurodevelopmental effects of environmental compounds using two (2) neurodevelopmental endpoints in the NCC: 1) NCC migration; 2) Viability of migrating NCCs. Evaluation of the effects of environmental compounds in the NCC shall be conducted as two tasks. In Task 1 the Contractor shall run the NCC assay and collect data. In Task 2 the Contractor shall analyze and report the data generated from Task 1.

4.1. Chemicals: All test compounds and dimethylsulfoxide (DMSO) will be supplied by the NIEHS/DNTP blinded in a one-time shipment. All other assay controls shall be supplied by the contractor. The Contractor shall be responsible for supplying all other reagents, equipment and facilities needed to conduct the specific assays

4.2. Task1 (NCC migration assay data collection): Neural crest cells shall be seeded into 96-well plates in N2-S medium containing FGF2 and EGF. Cells shall be seeded around stoppers to create a circular cell-free area. Stoppers shall be removed after 24 hours to allow cells to start migration on day of migration 0’ (DoM0’). On DoM1’ cells shall be exposed for 24 hours to test compounds; eight concentrations shall be assayed as well as a DMSO vehicle control. Test concentrations shall be prepared in a 1:3 dilution series from a starting concentration of 100 µm. Thereafter, cells shall be stained with calcein-AM and H-33342 and high-content imaging shall be performed. Total number of viable (calcein-AM and H-33342 double labeled) NCC that have migrated into the region of interest (ROI) shall be determined using high-content imaging.

4.3. Task 2 (NCC migration assay data analysis and reporting): NCC that have migrated into the region of interest (ROI) shall be determined using both an automated imaging algorithm and the RingAssay software tool (http://invitrotox.uni-konstanz.de/) to generate concentration-response curves from at three separate experiments. Migration results shall be normalized to untreated controls combining the migration and viability data into a prediction model to classify test compounds as either inactive or specific inhibitors of NCC migration, or non-specific inhibitors of NCC migration using both an automated imaging algorithm and software tool to generate concentration-response curves from at least three separate experiments. Migration results shall be normalized to untreated controls combining the migration and viability data into a prediction model to classify test compounds as either inactive or specific inhibitors of NCC migration, or non-specific inhibitors of NCC migration. If cytotoxicity is noted in greater than three (>3) concentrations, then the dilution series shall be repeated at a lower range. Experiments shall be performed at least three times. Test compounds identified as specific inhibitors of NCC migration shall be repeated in a second test of concentration response experiments Test concentrations shall be prepared in a 1:3 dilution series from a starting concentration of 100 µm.

5.0. DATA RIGHTS: The Government has unlimited rights to all documents/material produced under this contract to the extent permitted by the data rights clauses. The parties mutually acknowledge their understanding that this is the government’s intent. These documents and materials may not be used or sold by the contractor without written permission from the contracting officer. All materials supplied to the Government will be the sole property of the government and may not be used for any other purpose. This right does not abrogate any other government rights.

6.0. DELIVERABLES AND REPORTING REQUIREMENTS: A deliverable is the product of work done on this contract; the finished result of a step of work; or any unique and verifiable product, result, or capability to perform a service that must be produced to complete a process, phase, or project under this contract.

Required Reports (below) for all contract activities shall comply with requirements in respect to submission milestones, report formats and content and reporting timeframe. All final deliverables received from the Contractor shall be essentially error free. All submitted electronic copies of final deliverables shall be Section 508-compliant. Additional details regarding 508-compliance of electronic documents are found at http://www.hhs.gov/web/508/index.html.

6.1. Required Reports: A draft and final report for this effort shall provide a summary of the methods used and the results obtained in sufficient detail and quality to meet the requirements for peer reviewed literature. The draft report shall be delivered in both hard and electronic formats (MS Word) for review and comment by the Contracting Officers Representative (COR); the final report shall be delivered in hard and electronic (PDF) formats. All final electronic documents shall be section 508 compliant. All raw data shall be attached as an appendix to the report and submitted electronically to the COR as an Excel spreadsheet. The final report shall include but not be limited to:

6.1.1. A detailed description of all experimental procedures

6.1.2. A full set of all raw data, summarized/normalized data

6.1.3. Guidance on the interpretation of the DNT in vitro NCC testing results.

6.1.4. Concentration response curves for each compound

6.1.5. Identification of compounds as "specific DNT compounds," unspecific DNT compounds," or "negative compounds" using an NCC-specific classification model

6.2. Contract Management Reports: These shall consist of Standard Operating Procedures (SOP) related to conduct of the differentiation of NCCs and the NCC assay.

http://invitrotox.uni-konstanz.de/ http://www.hhs.gov/web/508/index.html

6.3. Periodic Status Reports: These shall detail the status of active assignments and any problems encountered and remedial actions taken to resolve those problems should be provided quarterly to the COR. Deviations from the SOW and/or protocols shall be noted.

6.4. Draft Final Report: This shall detail all significant outcomes resulting from the requirements as described within the SOW and contract, problems encountered and remedial actions taken to resolve the problem, as well as any deviation from the SOW and/or SOPs shall be submitted to the COR for review and comment within 15 working days of completion of the last of the studies; the Final Report responding to any comments shall be submitted to the COR and the CO within 15 working days of receipt of COR comments.

6.5. General Reporting Requirements: Final electronic deliverables shall be Section 508 compliant according to the checklists at http://www.hhs.gov/web/508/index.html under Helpful Resources.

The Contractor shall ensure that the reporting timeframes for each milestone are met. In general, specific formatting requirements will be provided by the COR. If changes are required by the COR, revised electronic version of the document shall be submitted. The COR will notify the PI when original or revised reports are approved.

Unless otherwise instructed, all reports submitted under this contract shall reference the contract number, the title and types of report, and the represented period on the first page of the report.

6.6. Electronic Deliverables: Unless otherwise directed, electronic deliverables shall be submitted to the COR, Alternate COR, and contract officer (CO). Electronic copies of the Contract Management Report and Monthly Status Reports shall be submitted to the COR and Alternate COR. The COR will designate a forum for these reports. Electronic copies of Monthly Status Report, Invoices/Financial Reports and other reports as directed shall be emailed to the CO. The CO will provide email addresses to the Contractor.

7.0. PLACE OF PERFORMANCE: The opportunities for sample degradation and compromising the quality of data are greater with the continued movement of samples to various sites for analysis, therefore; analysis shall take place at the contractor’s facility.

8.0. Section 508 Requirements for Deliverables: All Final Reports and all documents shall be prepared, formatted, and checked for conformance with Section 508 standards, prior to delivery to NIEHS or posting to the websites, as appropriate. Draft reports need not be Section 508 compliant.

The Department of Health and Human Services has written procedures and checklists based on the US Access Board Standards (http://www.access-board.gov/about.htm) for preparing documents to meet all requirements of Section 508 standards, using either Microsoft Word or Adobe Professional, version 8.0 or higher. The Contractor shall refer to the checklists and standards found on HHS’s Section 508 website (http://www.hhs.gov/web/508/checklists/index.html) for further guidance. Submission of electronic documents which satisfy the appropriate HHS Checklists and Standards will be considered acceptable.

Should an accessibility problem be found in the documents, the Government will return them for remediation, with a timeframe to be negotiated at that time.

The following Section 508 accessibility standards apply to the content or material identified in this SOW:

36 CFR Part 1194.22 “Web-based Intranet and Internet Information and Applications” 36 CFR part 1194.41 “Information, Documentation, and Support” Section 508 compliance for PDF files: The NIH Section 508 Coordinator approved a Section 508 accommodation for certain DNTP reports. As the accommodation must be renewed annually, the COR will inform the Contractor about any changes. Currently, the accommodation applies to the posting of public comments.

9.0. CONTRACTOR FURNISHED MATERIALS: The Contractor is required to supply all equipment, property and services needed to fulfill the SOW.

http://www.hhs.gov/web/508/index.html