Attachment III NSA Task 2 SOW.pdf

TASK ORDER 2

STATEMENT OF WORK

NIEHS DIVISION OF THE NATIONAL TOXICOLOGY PROGRAM

HUMAN NEURAL NEUROSPHERE ASSAY DATA ANALYSIS AND REPORTING

1.0. INTRODUCTION: Under this task order, the Contractor shall analyze data generated in Task 1 from the human neurosphere assay (NSA). All services, labor, equipment, and supplies provided under this task order shall be performed under accordance with the terms and conditions of the contract Statement of Work (SOW) and task order SOW.

2.0. TASKS: The contractor shall evaluate the neurodevelopmental effects of 115 environmental compounds using nine (9) neurodevelopmental endpoints in the NSA endpoints: 1) hNPC proliferation; 2) Viability (proliferating and differentiating hNPC); 3) Cytotoxicity (proliferating and differentiating hNPC); 4) Radial glia migration; 5) Neuronal differentiation; 6) Neuronal migration; 7) Neurite morphology; 8) Oligodendrocyte differentiation; and 9) Oligodendrocyte migration. In Task 2 the Contractor shall analyze and report the data generated from Task 1.

2.1. Analysis of Primary hNPC proliferation assay (NPC1): Data on cell viability, cytotoxicity and proliferation shall be analyzed to generate concentration response curves for each compound and endpoint with a BMC value as point of departure and corresponding confidence intervals.

2.2. Analysis of image-based endpoints (NPC2-5): The Contractor shall analyze image-based endpoints as described in section 2.2.1-2.2.4.

2.2.1. Nuclei identification: For nuclei identification the spot detector BioApplication of the Cellomics scan software (Version 6.6.0;Thermo Scientific) shall be used. The Spot Detector BioApplication shall analyze individual Hoechst-stained objects or spots (= cell nuclei) in the nuclei channel (386 nm) that fulfill pre-defined criteria on object intensity and object size that shall be established based on untreated NPC.

2.2.2. Annotation of neurons and oligodendrocytes: The identification of neurons (TUBB3 channel) and oligodendrocytes (O4 channel) shall be done automatically using a computational neural network (CNN) based on the Keras architecture implemented in Python 3.

2.2.3. Detection of migration area and cell type positioning: A density distribution mask shall be calculated automatically by Omnisphero using the Hoechst-stained nuclei, and the sphere core shall be determined. The density threshold shall be set, the outer boundaries shall be determined, the migration distances identified using the circular outer boundary for each migrated sphere shall be presented as one mean migration value/sphere. For assessing neuronal (NPC2b) and oligodendrocyte (NPC2c) migration, the distance of each annotated neuron and oligodendrocyte (see above) from the sphere core shall be determined and the mean migration distance calculated. NPC2b and NPC2c values shall be given as percent of NPC2a.

2.2.4. Morphological analysis: After the neurons are annotated by the CNN, the neurite endpoints are assessed by Omnisphero. First, the neurite channel image shall be pre-processed to a binary image to eliminate image- and staining-artifacts using the image processing algorithms in Omnisphero. The remaining neurite image shall then be projected onto the nuclei channel image and subsequently all nuclei that cannot be linked to at least one neurite are not annotated as neuron (see above) are removed.

In the next step, each neuron and its corresponding neurite(s) shall be isolated into “tiles” for determining the ratio between neurons and nuclei. A skeletonization algorithm shall be run on each isolated neuron tile, determining the individual length and area of each neurite. The final selection of neurite tiles for the calculation of the mean neurite area and mean neurite length is based on plausibility criteria including shape and the maximum length/area of a neurite, that were defined by manual inspection of artefacts in control experiments.

3.0. DATA RIGHTS: See IDIQ contract.

4.0. DELIVERABLES AND REPORTING REQUIREMENTS: See IDIQ contract.

4.1. 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:

4.1.1. A detailed description of all experimental procedures

4.1.2. A full set of all raw data

4.1.3. Guidance on the interpretation of the DNT in vitro NSA testing results.

4.1.4. Concentration response curves for each compound and endpoint with a BMC value as point of departure and corresponding confidence intervals.

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

4.1.6. Prioritization of specific DNT compounds using the ToxPi tool and the BMC of most sensitive endpoints.

4.2. Contract Management Reports: See IDIQ contract.

4.3. Periodic Status Reports: See IDIQ contract.

4.4. Draft Final Report: See IDIQ contract.

4.5. General Reporting Requirements: See IDIQ contract.

4.6. Electronic Deliverables: See IDIQ contract.

5.0. PLACE OF PERFORMANCE: See IDIQ contract.

6.0. PERIOD OF PERFORMANCE: 12 months.

7.0. Section 508 Requirements for Deliverables: See IDIQ contract.

8.0. CONTRACTOR FURNISHED MATERIALS: See IDIQ contract.