Att2_Statement of Work.pdf

STATEMENT OF WORK (SOW)

Assessment of Immunotoxicity – In Vitro and In Vivo Assessment of Immune Function for the National Institute of Environmental

Health Sciences

December 20, 2023

1 General Information

1.1 General

This is a non‐personal service(s) contract under which the personnel rendering the service(s) are not subject, either by the contract’s terms or by the manner of its administration, to the supervision and control usually prevailing in relationships between the Government and its employees. The purpose of this contract is to provide all services and supplies necessary to conduct research studies to evaluate the potential for test agents selected by the Division of Translational Toxicology (DTT), National Institute of Environmental Health Sciences (NIEHS) to modulate immune function using established in vitro and in vivo models.

1.2 Description of Services

Independently, and not as an agent of the Government, the Contractor shall furnish all the necessary services, qualified personnel, procedures, materials, equipment, and facilities not otherwise provided by the Government as needed to perform the objectives set forth below. In general, studies shall be conducted according to the requirements described in a research outline (see below).

1.3 Background and Scope

DTT is an intramural division of NIEHS within the National Institutes of Health (NIH). DTT uses traditional and cutting‐edge approaches to better understand how factors in our environment may impact human health. Such approaches include the conduct of immunotoxicology studies on chemical, physical, and biological agents, as well as non‐chemical stressors, using in vitro and in vivo models of health and disease. Specific research activities include:

a) The development and establishment of methods with predictive value for potentially immunotoxic chemicals

b) Examination of selected agents employing a defined panel of immune function tests and observational measures

Immunotoxicity is used to describe the potential of an agent to alter the normal structure and/or function of the immune system as determined by established immunological techniques. In cases of immunomodulation, a chemical or physical agent may alter hematopoietic or immunological function in humans and animals, resulting in changes in the ability to recognize and eliminate threats such as self‐reactive or cancerous cells. When immune responses are suppressed, increased rates of infection and certain cancers may occur.

Conversely, when the immune system is stimulated and uncontrolled, chronic inflammation or autoimmune disease may manifest. In cases of hypersensitivity, the immune system responds to chemically modified ("non‐self") compounds (haptens) as part of a "specific" immune response. The most likely health consequences include respiratory tract allergies (e.g., asthma, rhinitis) or allergic contact dermatitis.

With the increasing awareness of the need for translational toxicology and to reduce the use of animals, the main objectives in DTT’s research efforts to examine altered immunity resulting from chemical exposures will be using in vitro methods to evaluate immune function. The focus of this requirement will be to provide DTT with a mechanism to evaluate the complete scope of chemical induced immunotoxicity by determining: 1) whether chemical or therapeutic agents can modulate the immune response using a battery of in vitro tests to measure immune suppression or stimulation in response to relevant antigens; 2) whether chemical agents have the potential to induce allergic responses using in chemico and in vitro Organisation for Economic Co‐operation and Development (OECD) guideline test methods for skin sensitization as part of an integrated panel of tests; 3) evaluate the potential of a test agent to modulate immune function using a comprehensive in vivo testing panel that evaluates the major components of the immune system in the developing rat and/or adult mice or rats; and 4) because immunology is a developing science, it is expected that new and improved methodology will be required to maintain "state of the art" evaluation. As an additional activity, in consultation with the Contracting Officer's Representative (COR), the Contractor shall identify research gaps and conduct methods development and validation.

1.4 Overview of Required Capabilities

The Contractor shall conduct research studies supported by the following Capability Areas described in more detail in Section 2. Required Capability Areas:

Evaluation of the Potential to Modulate Immune Function Using In Vitro Assays The Contractor shall have the capability to assess chemical modulation of immune function in primary human cells using in vitro screening methods and a panel of assays that provide functional and quantitative information on humoral, cell‐mediated, and innate immunity.

Evaluation of the Potential to Induce Hypersensitivity Using In Vitro Assays The Contractor shall have the capability to assess the potential for chemical agents to induce hypersensitivity using in chemico or in vitro models. To achieve this objective, research must be performed in accordance with OECD Guideline Test Methods as referenced in Section 7 of this Statement of Work (SOW) entitled “Applicable Publications.”

Evaluation of the Potential to Induce Immunotoxicity Using In Vivo Rodent Models The Contractor shall have the capability to conduct research studies in adult mice, adult rats, and/or developing rats, and to evaluate specific measures of immune function including:

a) Clinical Pathology

b) Humoral Mediated Immunity

c) Cell Mediated Immunity

d) Quantification of Leukocyte Subpopulations

e) Non‐Specific Immunity

Research studies shall be conducted using the assays described in Section 2.4. The exposure regimen for adult mice or rats shall normally (but not always) consist of daily dosing for 28 days by a route of exposure specified in a research outline. For studies including perinatal exposure, the exposure regimen shall normally (but not always) consist of daily dosing of time‐mated female rats beginning on day 6 of gestation (GD 6) and continuing through postnatal day (PND) 4. Offspring (F1) may continue to be dosed daily until PND 42 or other age as specified by the COR.

Development and Evaluation of Novel Tools and Methods to Assess Immunotoxicity The Contractor shall have the capability to assess novel methods that may be used to determine the potential for chemical agents to induce immune modulation or hypersensitivity with a focus on in vitro models. Assessment of new strategies to evaluate immune perturbations in the developing immune system should also be a priority. Studies to help establish cellular and/or molecular mechanisms of action for immune perturbations observed during the conduct of activities described in Sections 2.2, 2.3, and 2.4 may be appropriate. Development of novel screening strategies including the use of molecular techniques such as analysis of gene expression to assess xenobiotic‐induced immunologic alterations would be also desirable.

1.5 Policies and Procedures

For in vivo studies, the Contractor and applicable subcontractor facilities shall comply with the below regulations and specifications:

a. Test facilities shall be accredited by AAALAC International and shall have an approved Animal Welfare Assurance on file with the Office of Laboratory Animal Welfare (OLAW).

b. Test facilities shall comply with Public Health Service (PHS) Policy, the Animal Welfare Act and regulations, the current edition of the Guide for the Care and Use of Laboratory Animals, and all local, state, or federal regulations.

c. If animal species regulated by the U.S. Department of Agriculture (USDA) are involved, the Contractor shall be registered with the Secretary of Agriculture of the United States in accordance with 7 U.S.C 2136 and 9 Code of Federal Regulations (CFR) sections 2.25 through 2.28.

d. Animal experimental protocols, approved by the Contractor’s Institutional Animal Care and Use Committee (IACUC), shall be delivered electronically via PDF to the NIEHS COR prior to the commencement of a study. These Contractor‐approved protocols shall also be available to the NIEHS Animal Care and Use Committee (ACUC). The Contractor shall have an animal disease‐screening program that complies with the DTT Specifications, Chapter 6 (see below).

For in vitro studies:

a. The Contractor shall reference and utilize the Guidance Document on Good In Vitro

Method Practices (https://www.oecd.org/env/guidance‐document‐on‐good‐in‐vitro‐ method‐practices‐givimp‐9789264304796‐en.htm) for the conduct of in vitro work.

For all studies:

a. Studies shall be conducted in accordance with the Specifications for the Conduct of

Toxicity Studies by the Division of Translational Toxicology at NIEHS (https://doi.org/10.22427/NIEHS‐00), referred to hereafter as the DTT Specifications.

Specifically, Chapters 2, 3, 4, 6, 7, 8, 11, and 12 of the DTT Specifications are applicable to the activities outlined in this SOW. Any exceptions shall be reviewed and approved by the COR prior to study conduct.

b. When specified by the research outline, the study shall be performed in accordance with the Good Laboratory Practice (GLP) regulations established by the U.S. Food and Drug Administration (FDA) for Non‐clinical Laboratory Studies (21 CFR 58;

https://www.ecfr.gov/current/title‐21/chapter‐I/subchapter‐A/part‐58) or as most recently updated. The Government reserves the right to ensure compliance with FDA GLP regulations as specified in Part 58 “Good Laboratory Practices for Non‐Clinical Studies” (Federal Register, Friday, December 22, 1978, Part II, and any later interpretations published by the FDA) and to conduct Quality Assurance (QA) audits.

c. The Contractor shall have in place a Quality Management Plan. Information on quality management plans can be found at https://www.epa.gov/quality/epa‐qar‐2‐epa‐ requirements‐quality‐management‐plans.

1.6 Reports and Deliverables

Information on specific reporting requirements is presented below. All final study reports shall be dated and signed by the individual(s) responsible. Electronic deliverables must be compliant with Section 508 regarding accessibility. In some cases, there may be accommodations for which the Government will provide information. Formats and additional requirements for monthly reports, study reports, and study files are described in more detail in the DTT Specifications.

All reports shall be sent to the COR electronically or at the address specified at the time of award. All reports shall be submitted in an electronic format that meets NIEHS/DTT hardware and software standards.

In addition to the reports mentioned herein, any incidence or unforeseeable occurrence not listed here, including physical modifications to the laboratory facilities and personnel changes which may impact study conduct, shall be immediately reported to the Government.

In addition to those documents required by the other terms of this contract, the Contractor shall prepare and submit the following reports to the COR electronically. An electronic copy of the Title Page shall also be sent to the Contracting Officer (CO) as a required deliverable in the manner stated below.

DELIVERABLE REFERENCE DUE/FREQUENCY SUBMIT TO

Quality Management Plan

Section 1.6.1.1 Submit prior to any work commencing and revise as needed or when major changes occur

COR, CO, and CS

Monthly Progress Report

Section 1.6.1.2 On the 20th day of each month COR, CO, and CS

Budget Summary Report

Section 1.6.1.3 Due once per month as an attachment with the monthly invoices

COR, CO, and CS

Test Article Study Report

Section 1.6.2.2

Submit draft report to the COR 180 days after completion of the in‐life portion of each study. Submit final report 60 days after receipt of COR’s requested revisions.

COR

Animal Use and Safety Protocols

Section 1.6.2.1

Submit draft protocols to the COR prior to submission to the Contractor’s IACUC or Safety Officer. Submit approved IACUC and related protocols no later than one week prior to commencement of the study.

COR

Laboratory Study Protocols

Section 1.6.2.1 Submit to the COR for review within 3‐5 weeks of receipt of the research outline

COR

Annual Report Section 1.6.1.4 Submit within 20 days following the close of each contract year

COR, CO, and CS

1.6.1 Periodic Reports

1.6.1.1 Quality Management Plan

A Quality Management Plan (QMP) shall be submitted before performance of work under the contract and updated versions submitted at regular intervals (e.g., biannually), or if the plan has been updated substantively, as described in the DTT Specifications.

1.6.1.2 Monthly Progress Reports

On the 20th day of each month, the COR shall receive a brief (1‐3 page) summary of the work currently in progress. This report shall include the status of all ongoing studies, reports, and manuscripts and shall be submitted to the COR and the CO via electronic mail. No QA Unit (QAU) audit of this progress report is required.

1.6.1.3 Budget Summary Reports

For each activity separately undertaken, and for the contract as a whole, the Contractor shall submit a monthly budget summary report. The report shall include amounts of each activity budget, total expenditures on the activity for the month, summaries of current contract year and cumulative expenditures, and projected expenses to complete each activity. Budget summary reports shall be submitted monthly to the COR as MS Excel spreadsheets (or in other spreadsheet software by agreement) and shall be sortable as required by COR.

Budget reports must be included as an attachment with submitted invoices. Monthly budget summary reports are due by the 20th day of each month.

1.6.1.4 Annual Reports

Within 20 days following the close of each contract year, the Contractor shall provide a report that summarizes the activities, progress and accomplishments for each activity conducted in the previous year. This report shall include the status of all ongoing studies, reports, and manuscripts and shall be submitted to the COR and the CO via electronic mail. No QAU audit of this progress report is required.

1.6.2 Research Products

1.6.2.1 Animal Use, Laboratory Study, and Safety Protocols

Protocols shall be approved by the Contractor’s or Testing Facility’s IACUC. Health & Safety Summaries and Chemical Hygiene Plans for all in vivo and in vitro study protocols shall also be approved by the Contractor’s internal review committees. Draft protocols shall be provided to DTT for review and comment prior to evaluation by the appropriate internal review committee.

Approved protocols should be provided to DTT no later than one week prior to the commencement of the specific study. Non‐animal laboratory study protocols shall be submitted to the COR for review within 3‐5 weeks of receipt of the research outline. If study findings necessitate modifications to the laboratory study protocol, the Contractor shall submit an amendment to the study protocol to document the required changes for COR review and approval.

1.6.2.2 Draft and Final Test Article Study Reports

A draft study report shall be submitted to the COR one hundred and eighty (180) days after completion of the study. This report shall include a complete laboratory protocol, a summary of methods for the individual assays conducted in the testing of agents performed as part of all activities associated with that study, and a summary of all experimental findings. Individual animal data for each test conducted in the study shall be submitted as an Excel spreadsheet or other COR‐specified format and as a separate file, along with the draft study report for entry into the DTT database. It shall also include difficulties encountered and remedial action taken, including any deviations from the laboratory study protocol. The COR will review the draft and return comments to the Principal Investigator to be incorporated into the final report. Draft reports need not be Section 508 compliant. Within sixty (60) days of receipt of the COR’s requested revisions, the Contractor shall supply a full, unsigned copy of a revised report for final review by the COR. Upon approval by the COR, the Contractor shall provide a final report including covers and signed signature pages that is Section 508 compliant in PDF format and with all graphics attached. A separate MS Word file of the report and included tables that are Section 508 compliant shall also be submitted.

1.6.3 IT Security Plan and Other IT Deliverables

Refer to APPENDIX A ‐ HHS SECURITY AND PRIVACY LANGUAGE FOR INFORMATION AND IT PROCUREMENTS within this SOW.

1.6.4 HHS Section 508 and Accessibility Standards

Refer to contract requirements in Section H of the contract.

1.7 Post Award Conference/Periodic Progress Meetings

The Contractor shall attend any post award conference convened by the contracting activity or contract administration office in accordance with Federal Acquisition Regulation (FAR) Subpart

42.5. The CO, COR, and other Government personnel, as appropriate, may meet periodically with the Contractor to review the Contractor's performance. At these meetings the CO will apprise the Contractor of how the Government views the Contractor's performance and the Contractor shall apprise the Government of problems, if any, being experienced. Appropriate action shall be taken to resolve outstanding issues. These meetings shall be at no additional cost to the Government.

1.7.1 Progress Meetings

The Contractor shall attend, participate in, and furnish input to scheduled and unscheduled meetings, conferences, and briefings that relate to the functions and services herein as required by the Government to provide effective communication and impart necessary information. The Principal Investigator or designated representative shall attend meetings as requested by the Government. Meeting attendees shall at times include Contractor managerial, supervisory, and other personnel knowledgeable of the subject matter. Meetings may start or end outside of regular duty hours.

1.8 Contracting Officer’s Representative

As determined by the CO, a COR will be appointed and identified by letter of designation, a copy of which will be provided to the Contractor by the CO. The designation letter states the responsibilities and limitations of the COR.

1.9 Personnel

The following personnel are considered key personnel by the Government: Principal Investigator and Deputy Principal Investigator. The following personnel are considered critical personnel by the Government: Veterinarian, Study Directors, Quality Assurance Officer, and Health and Safety Officer.

The Contractor shall identify a Principal Investigator who shall be responsible for the performance of the work and provide the name of this person and that of a Deputy Principal Investigator, who shall act for the Contractor when the Principal Investigator is absent. The Principal Investigator and Deputy Principal Investigator shall have full authority on all contract matters relating to daily operation of this contract. Evaluation of key and critical personnel will be based on education, training, and experience with immunotoxicity studies to include but not be limited to:

1) Experience with in vitro assessment of immune function (innate, humoral‐ and cell‐ mediated immunity) using primary human cells or other relevant cell types.

2) Experience with in chemico skin sensitization and in vitro skin sensitization or other alternative methods used in immunotoxicity screening.

3) Experience with rodents, health assessments of rodents, and toxicology studies, including demonstrated capability in the conduct of studies in laboratory rodents to evaluate modulation of immune function following exposure to a test compound.

4) Number and variety of compounds tested using in vitro and in vivo screening methods as well as years of experience testing these compounds.

5) Demonstrated experience in the interpretation of immunotoxicity data and preparation of technical reports.

6) Experience of personnel working together as a team.

The Contractor shall identify appropriate technical and support personnel to conduct all aspects of the studies, including, but not limited to, staff to provide animal care/husbandry; administer test articles to rodents (including pregnant dams, neonates/pups, juvenile, and adult animals as required) via required route(s) of exposure (e.g., dosed feed or water, oral gavage, intravenous or intraperitoneal injection); collect in‐life data (e.g., body weights, clinical observations, food/water consumption); and technical staff proficient in sterile and other laboratory techniques for in vitro studies.

The Principal Investigator, Deputy Principal Investigator, and study directors shall be employees of the Contractor (i.e., not consultants or subcontractors). Technical and support staff may be subcontractors (upon approval by the COR and CO), however, performance of all work must occur at the prime contractor’s facility. The daily interaction and constant coordination of efforts needed to conduct multiple tests with the same test article throughout the studies make it critical that they be physically together.

1.10 Data Rights, Patents, and Copyrights

The Government has unlimited rights to all documents and materials produced under this contract. All documents and materials, to include the source codes of software, produced under this contract shall be Government owned and are the property of the Government with all rights and privileges of ownership and copyright belonging exclusively to the Government.

These documents and materials may not be used or sold by the Contractor without written permission from the CO. All materials supplied to the Government shall 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.

1.10.1 Source Documentation

All original source documentation is the property of NIEHS. Data summaries and other relevant information generated during the performance of this contract shall be submitted to the COR in the form of status reports and agent specific reports as specified in the Deliverables section above. In addition, annual reports shall be submitted for all activities as specified in the Deliverables section above. To the extent possible, the Government will retain unrestricted access and rights to all tissues, data, and technical information generated under the contract.

The Contractor shall arrange for delivery of all raw data to the National Toxicology Program (NTP) Archives upon completion of each individual study. Archival study data materials shall be submitted to the NTP Archives as specified in the DTT Specifications.

1.11 Non‐Disclosure Requirements

Performance under this contract may require the Contractor to access data and information proprietary to a Government agency, another Government Contractor, or of such nature that its dissemination or use other than as specified in this work statement would be adverse to the interests of the Government or others. The Contractor and Contractor’s employees (including subcontractors), shall not divulge, or release data or information developed, or obtained under performance of this contract, except to authorized Government personnel or upon written approval of the CO. The Contractor shall not use, disclose, or reproduce proprietary data, which bears a restrictive legend, other than as specified in this contract. All documentation showing individual names or other personal information shall be controlled and protected under the provisions of the Privacy Act of 1974, Public Law 93‐579, 5 United States Code (U.S.C.) Section 552a.

1.12 Replication, Dissemination, or use of the Results

It is anticipated that the results of the research conducted under the contract shall be published in the open, peer‐reviewed scientific literature and/or in a NTP or NIEHS technical report series.

Specific details concerning authorship and technical issues surrounding the publication of the research shall be discussed and agreed upon by the Principal Investigator and the DTT COR on a case‐by‐case basis.

Because premature release of information developed under the contract could be harmful to the public, the Contractor and the Government agree that it is important to consider issues related to the timing of the release of research results when submitting information for publication. With regard to public disclosures of the research to be carried out pursuant to this contract, the Contractor agrees to submit all proposed articles to the COR at least thirty (30) days in advance of submission for publication. Within this thirty‐day period the COR shall inform the Contractor if there are serious concerns raised by the timing of said publication.

Upon receipt of a notification that the Government objects to the publication of said information the Contractor agrees to withhold submission of said publication while the author and the COR discuss the issues of concern. The Contractor agrees to give good faith consideration to all such issues and further agrees that it will not knowingly submit for publication any article, abstract or other item that would be harmful to the public welfare. The final decision on whether to submit an article for publication shall rest with the DTT COR after the procedures set forth above are followed. Similar procedures shall apply for scientific presentations and meeting abstracts.

1.13 Protection of Government and Contract Information

The Contractor shall not cite any information (e.g., contract information, pictures, locations, etc.) obtained through this contract on any hard copy or digital marketing tools to include its company website.

1.14 Non‐Disclosure Statements

The Contractor shall provide signed non‐disclosure agreements to the Government no later than five (5) days prior to commencement of work under the contract. Disclosure of information by Contractor personnel may result in removal of Contractor personnel from performance under this contract.

1.15 Safety Laws and Regulations

In addition to other requirements for accident prevention specified in this contract, the Contractor shall comply with all Federal and State requirements governing safety and health protection.

The Contractor shall indicate its plan for compliance considering conditions of the work described by the design, technical proposal, operational activities of the Government or others in the area of the work, identification of the coordinator, and implementation of all other accident prevention provisions of this contract.

The Contractor shall complete and provide safety notifications as required by national, state, and local regulatory bodies, and will provide documented proof to the CO via the COR, no later than 10 days that such notifications were made.

2 Required Capability Areas

2.1 General Requirements

The Contractor shall provide services, qualified personnel, materials (including animals), equipment, and facilities needed to evaluate the potential for test agents selected by DTT to modulate immune function using established in vitro and in vivo models.

The Contractor shall be responsible for ensuring the appropriate institutional review and safety assessment for each test article and approved protocols shall be delivered to the COR prior to the initiation of each study.

2.2 Evaluation of the Potential to Modulate Immune Function Using In Vitro Assays The Contractor shall have the capability to assess chemicals in primary cells such as those obtained from donor human peripheral blood using in vitro screening methods in a panel of assays that provide information on humoral, cell‐mediated, and innate immune function for their potential to modulate immunity. The assay panel shall include assessment of innate immune function, cell proliferation, and cytokine profiling. When assay appropriate, the test system shall include an assessment of antigen‐specific responses using human relevant antigens as stimulatory factors. In addition, the panel shall provide quantitative information on observational endpoints such as cytotoxicity, lymphocyte subpopulations, and relevant markers of cell activation. Proficiency with models to assess specific cell‐surface receptor expression, quantitate physiologic changes, and/or responses to novel disease‐specific antigens would be valuable.

2.2.1 Tier 1 In Vitro Screening

Tier 1 shall include assessment of differential cell viability, lymphocytotoxicity and identification of susceptible human immune cell population(s), cell proliferation, and cytokine/chemokine release in response to stimulation with anti‐CD3/CD28 or viral antigens, and Natural Killer (NK) cell activity with all endpoints being obtained from the same cell cultures using at least 8‐10 donors per sex per chemical treatment. In some instances, DTT may request donor pools with specific ethnic composition or lifestyle history.

2.2.2 Tier 2 In Vitro Screening

Development and usage of a second tier of assays, that would include an evaluation of humoral immunity (antibody production targeting specific viral antigens), T cell immunity (cytotoxic T lymphocyte mediated cell killing and assessment of virus‐specific CD8 memory cells), and innate immunity (respiratory burst/phagocytosis and complement activation) in human leukocytes would be data‐driven and based on the results of the findings from the first‐tier in vitro testing panel.

2.3 Evaluation of the Potential to Induce Hypersensitivity Using In Vitro Assays The Contractor shall evaluate test articles for their potential to produce dermal hypersensitivity using OECD test guideline in vitro methods including, but not limited to the assays described below.

2.3.1 Direct Peptide Reactivity Assay (DPRA; OECD 442C)

The DPRA is an in chemico assay validated for examination of Key Event 1 in the Skin Sensitization Adverse Outcome Pathway (AOP) and represents a surrogate assay for chemical haptenization of proteins. For the DPRA, testing is performed in accordance with OECD test guideline 442C (reference SOW Section 7). Test compounds shall be mixed with a cysteine‐ containing peptide and a lysine‐containing peptide in two separate experiments and interaction of the test compound with the peptides is measured by high performance liquid chromatography (HPLC) analysis. Reactive chemicals that have the potential to cause skin sensitization bind to the peptides resulting in their depletion from the reaction mixture. If the test compound is a single molecule, the percent depletion of the peptide peaks is used to classify each test chemical as a non‐sensitizer, or as a low, moderate, or high sensitizer. If the test compound is a multi‐constituent mixture without a defined molecular weight, the percent depletion of the peptide peaks is used to classify each test mixture as a non‐sensitizer or a sensitizer. Multi‐constituent mixtures cannot be classified as low, moderate, or high sensitizers based on the prediction model provided by the OECD guideline. Acceptance criteria for assay controls and test compound results are applied in accordance with OECD 442C guideline.

Studies that do not meet the acceptance criteria as described shall be considered unacceptable and shall be repeated until an acceptable result is obtained.

2.3.2 Activation of Keap1-Nrf2-ARE (OECD 442D)

The KeratinoSens™ assay and similar methods are in vitro cell‐based reporter gene assays validated for examination of Key Event 2 in the Skin Sensitization AOP and represents a surrogate assay to test for chemical induced keratinocyte activation. Test chemicals are evaluated for activation of the Keap1‐Nrf2‐ARE‐dependent pathway using the immortalized, human‐derived keratinocyte cell lines as described in OECD test guideline 442D (reference SOW Section 7). The KeratinoSens™ cells used in this assay have been transfected with a plasmid containing the luciferase gene whose expression is under control of the AKR1C2 gene ARE sequence upstream of the SV40 promoter. Increased Keap1‐Nrf2‐ARE‐dependent luciferase expression is indicative of keratinocyte activation and is used to classify test chemicals as non‐ sensitizers or sensitizers. For the KeratinoSens™ assay, the human keratinocyte cell line, KeratinoSens™, is treated with each test compound at a range of concentrations. Activation of the Keap1‐Nrf2‐ARE‐dependent pathway is determined by measuring the amount of luminescence in each well after lysing cells and adding a luminescent luciferase substrate, using a microplate luminometer. Cell viability is measured in parallel using the MTT assay to ensure that direct cytotoxicity does not confound the interpretation of the results. Acceptance criteria for assay controls and test compound results are applied as described in the OECD 442D guideline. Studies that do not meet the acceptance criteria as described shall be considered unacceptable and shall be repeated until an acceptable result is obtained.

2.3.3 Human Cell Line Activation Test (h-CLAT; OECD 442E)

The h‐CLAT is an in vitro assay validated for examination of Key Event 3 in the Skin Sensitization AOP and represents a surrogate assay to test for chemical induced activation of dendritic cells.

The h‐CLAT measures dendritic cell (DC) activation in response to chemical exposure using the immortalized human monocytic leukemia cell line, THP‐1, as a DC surrogate. THP‐1 cells are treated with each test compound at a range of concentrations determined from a dose finding assay in accordance with the OECD 442E test guideline (reference SOW Section 7). Data from dose finding assays are used to derive the CV75 concentrations (25% cytotoxicity) which were used to calculate the CV75*1.2 starting concentration. DC activation is determined by measuring expression of CD86 and CD54 on the cell surface by flow cytometry. Increases in the relative fluorescent intensity greater than or equal to 150 for CD86 (EC150) and/or greater than or equal to 200 for CD54 (EC200) are indicative of DC activation. Cell viability is measured concurrently in the same cell population using propidium iodide staining and flow cytometry.

Acceptance criteria for assay controls and test compound results were applied as described in the OECD 442E guideline. Studies that do not meet the acceptance criteria as described shall be considered unacceptable and shall be repeated until an acceptable result is obtained.

2.3.4 Data Analysis and Integration Using Defined Approaches (OECD 497) As none of these test methods are considered sufficient stand‐alone tests to draw conclusions on the skin sensitization potential of chemicals, data generated with these in chemico and in vitro methods addressing multiple key events of the skin sensitization AOP are proposed to be used together to define sensitization potential. In addition, information sources such as in silico and read‐across predictions from chemical analogues should be considered using integrated approaches to testing and assessment (IATA) or defined approaches (DAs). Contract laboratories that conduct the methods described above or other validated methods used to assess sensitization potential shall integrate the results from multiple information sources or data streams using established models for data interpretation as outlined in OECD guideline 497 (reference SOW Section 7).

2.4 Evaluation of the Potential to Induce Immunotoxicity Using In Vivo Rodent Models The Contractor shall assess the potential of a test agent to modulate the immune system in the developing rat and/or adult mouse or rat using protocol‐specific in vivo screening assays described below. Studies shall be conducted in accordance with the DTT Specifications (https://doi.org/10.22427/NIEHS‐00), and any exceptions shall be reviewed and approved by the COR prior to study conduct.

2.4.1 Study Design

2.4.1.1 Immunotoxicity Screening in Adult Mice or Rats

The Contractor shall evaluate the potential of a test agent to modulate the immune system in adult mice or rats using the screening assays described in Section 2.4.3. A minimum of eight animals per sex evaluated per treatment group shall be employed for the tests described. In most instances female rodents will be evaluated for consistency with previous DTT studies and ease of housing, however, the COR may specify that both males and females or males only be evaluated upon assignment of the activity. The exposure regimen for adult mice or rats shall normally (but not always) consist of daily dosing for 28 days by a route of exposure specified in a research outline. For studies including perinatal exposure, the exposure regimen shall normally (but not always) consist of daily dosing of time‐mated female rats beginning on day 6 of gestation (GD 6) and continuing through postnatal day (PND) 4. Offspring (F1) may continue to be dosed daily until PND 42 or other age as specified by the COR.

2.4.1.2 Evaluation of the Potential to Induce Immunotoxicity in the Developing Immune System The Contractor shall evaluate the potential of a test agent to modulate the immune system in the developing rat using the screening assays described in Section 2.4.3. The exposure regimen shall normally (but not always) consist of daily dosing of time‐mated female rats beginning on day 6 of gestation and continuing through PND 4. Pups shall be dosed daily when dosing of the dams is discontinued on PND 4 and continued generally through PND 42. Dosing of the pups may be delayed until weaning at PND 28 depending on the bioavailability of the chemical via lactation in which case maternal dosing shall continue through weaning. The COR shall specify the dosing regimen and route of exposure. Routes of administration may include oral gavage, drinking water, or dosed feed at three (3) dose levels in addition to a vehicle control. A minimum of 20 pups per treatment group (10 male and 10 female) shall be employed for the tests described in Sections 2.4.3.1 to 2.4.3.4. As a litter is considered a single unit, all treatment groups shall be composed of pups from different litters. Contractors shall consult the source of their laboratory animals to ascertain current estimates of successful pregnancy in plug positive dams to ensure enough pups to successfully populate the immune function testing.

2.4.2 General Endpoints

The endpoints outlined below shall generally be included in all in vivo studies, regardless of design.

2.4.2.1 In‐life Procedures and Evaluations

Exposure to test agents by various in vivo routes [e.g., dosed water, dosed feed, gavage

(including direct dosing of pups PND 4 and older)]

Disease screening

Clinical observations, including evaluation of humane endpoints

Body weights

Food or water consumption

Injection or infection with challenge agents (e.g., sheep red blood cells, keyhole limpet hemocyanin to assess antigen specific antibody responses) per specific endpoint

2.4.2.2 Terminal Assessments and Collections

Necropsies including gross evaluations; organ weights; collection and storage of blood;

collection and storage of assay‐specific tissues

Blood (survival and terminal) and tissue collection, including collection for internal dose assessment

Bronchioalveolar lavage and evaluation of cytokines, LDH activity, albumin concentration, cell counts and differentials if required by the study protocol

Quantitative assessments of functional immune endpoints as described below

2.4.3 Additional Endpoints

The endpoints listed below shall be included on a study‐specific basis as defined in a research outline. All assays in Sections 2.4.3.1 through 2.4.3.4 should include appropriate positive and negative controls (vehicle), as dictated by a study‐specific research outline to ensure that the assays performed as expected and are within 10 % of the historical control values for the specific assay in the Contractor’s laboratory.

DTT has investigated the immunotoxicity of a wide variety of environmental chemicals and therapeutics using this screening approach. Offerors should see Luster et al., 1992 for examples of previously conducted work using this testing panel and Watson et al., 2021 and Huang et al., 2020 for recent examples of how this panel has been applied (reference SOW Section 7).

2.4.3.1 Humoral Mediated Immunity

The Contractor shall assess humoral mediated immunity by quantitating antigen‐specific antibody response following intravenous administration of sheep red blood cells (SRBC) by the Antibody Forming Cell (AFC) plaque assay and Enzyme Linked Immunosorbent Assay (ELISA).

2.4.3.2 Cell Mediated Immunity

The Contractor shall quantitate cell‐mediated immunity by assessment of the lytic ability of splenic cytotoxic T lymphocytes.

2.4.3.3 Cell Quantification

The Contractor shall quantitate splenic cellularity, splenic differentials, and B cell, T cell, T‐cell subsets, NK cells, and monocyte enumeration by flow cytometric analysis.

2.4.3.4 Non‐specific Immunity

The Contractor shall assess non‐specific immunity by quantitation of NK cell activity. In some instances, the COR may require studies to evaluate macrophage function such as respiratory burst or phagocytosis as a measure of non‐specific immunity.

2.5 Development and Evaluation of Novel Tools and Methods to Assess Immunotoxicity The Contractor shall have the capability to assess novel methods that may be used to determine the potential for chemical agents to induce immune modulation or hypersensitivity with a focus on in vitro models. In consultation with the COR, the Contractor should propose studies that advance application, validation, and regulatory acceptance of new approach methodologies. Assessment of new strategies to evaluate immune perturbations in the developing immune system should be a priority. Studies to help establish cellular and/or molecular mechanisms of action for immune perturbations observed during the conduct of activities described in Sections 2.2, 2.3, and 2.4 may be appropriate. Development of novel screening strategies including the use of molecular techniques such as analysis of gene expression to assess xenobiotic‐induced immunologic alterations would be also desirable.

Activities performed under this capability area shall be no more than 10% of the base contract level of effort per year. This capability area of the contract may be exercised each year of the contract and shall be optional based on the COR’s discretion and the availability of funds.

3 Operating Procedures

3.1 Project Management

The Principal Investigator shall be responsible for the daily management of the contract and serve as point of contact for communication with the Government officials. Collectively, the Principal Investigator and Deputy Principal Investigator are expected to be knowledgeable and involved in all aspects of the contract, including project status, record keeping, reporting, and detailed analysis of budget for all contract functions.

Although the contract, as a whole, shall be for the ongoing and recurring support for DTT immunotoxicology research and testing evaluations, a research outline will be prepared and issued by the COR for each study and test agent(s) to be studied under the contract. Each research outline will include a detailed description of study objectives, the study(s) to be performed, expected deliverable(s), and anticipated timeline. In response, the Contractor shall submit a technical work plan outlining the proposed technical approach including detailed workflow, staffing plan, data management, milestone schedule, list of deliverable(s), and an estimated project budget describing the proposed costs and level of effort.

In general, a specific study shall begin after the Government has reviewed the technical work plan and budget documents, and any questions have been discussed and resolved between the Government and Contractor. As needed based on specific changes in a study, updates may be made to these documents after study initiation.

The Contractor shall use a DTT program and project management (PPM) software (i.e., Microsoft Dynamics 365 PSA) specified by the Government to document and update research activities, including, but not limited to, research outlines submitted by the Government, technical work plans and budget information, milestone schedule, work status, interim data (in the form of tables, graphs, spreadsheets, and/or other documents), and deliverables.

Submission of approved animal use protocols, animal study protocols, and laboratory study protocols prepared by the Contractor shall be transmitted through the PPM software. All substantive communication and decisions throughout the performance of work on a study shall be documented within the software.

Administrative activities shall be conducted by the Contractor as described in the administrative research outline. These activities include general management, periodic reports, or other Government‐approved activities not directly attributable to a specific study.

3.2 Data Management

Data management plans for each study shall be reviewed and approved by the Government prior to any data collection. For in vivo data, the Contractor shall use an electronic data capture system specified by the Government for collection of study data, unless the Government has determined that collection of a specific endpoint(s) is/are not feasible. The current toxicology data collection system is a NIEHS‐leased system (i.e., Provantis); however, other systems, such as a Government‐owned system, may be specified by the Government. Study data shall be entered directly into the system. The Contractor shall not record data on paper (or other means) for which a NIEHS electronic data capture system is available. NIEHS will provide access, system support, and training for the system. In vitro data shall be provided to NIEHS in a format reviewed and approved by the Government and shall be deposited in NIEHS’s database “Chemical Effects in Biological Systems (CEBS)” with appropriate metadata. Additional data management requirements are included in the DTT Specifications, Chapter 11 (https://doi.org/10.22427/NIEHS‐00).

The Contractor shall provide a data management plan for the management, sharing, backup, storage, and security of all paper and electronic files and information collected and generated as part of completing the products outlined in this SOW. This data management plan shall adhere to HHS Office of the Chief Information Officer (OCIO) policy and be approved by the COR. The Contractor shall keep all data and documents used or prepared confidential until Government publication. All software and databases used or secured must be PC and MAC compatible unless approved by the COR. All electronic deliverables must be PC and MAC compatible unless approved by the COR. Once projects are completed, all paper and electronic files will be transferred to the Government upon the COR’s request for long term storage.

All data derived or collected by the Contractor shall reside in a data management system approved by the Government after acceptance by the COR. The Contractor shall use IDs and metadata interoperable with those used in the NIEHS/DTT data ecosystem for interoperability, accuracy, and reliability across the NIEHS/DTT data ecosystem. Metadata shall meet DTT standards for discoverability and re‐use. Sensitive data shall be stored securely in accordance with patient agreements. Code shall be stored in Government repositories such as but not limited to BitBucket or in GitHub as directed by COR. All research data products shall be registered in the DTT Research Data Catalog with metadata to facilitate discoverability.

The Government will have unlimited data rights ownership of all data and information generated during the contract. The Government retains the right to access all information and data generated under this contract at any time.

The Contractor shall plan for or acquire any subscriptions needed for accessing databases and/or information resources upon the COR’s approval. All software and databases used to carry out the project objectives and requirements for scientific data management shall be approved by the COR.

3.3 Subcontractors and Consultants

Performance of all work shall occur at the prime contractor’s facility, however, if required, the Contractor, as directed by the COR, may identify, and propose qualified personnel to conduct the work as subcontractor(s) and present to the CO and the COR for approval. A detailed written subcontract plan shall be provided to the CO and COR. The subcontract plan shall include the area of expertise of the proposed consultant relevant to the request, as well as cost and a detailed timeline of accomplishing this project. Only upon approval by the CO shall work by the subcontractor begin. The Contractor shall ensure that no institutional or individual COI issues exist.

The Contractor shall summarize subcontractor performance and include the summary in the Contractor’s monthly progress report to the COR. Information included in this summary shall consist of work performed by the subcontractor including deliverables, level of effort, and costs expended during the past month, etc. In addition, the Contractor shall provide a description of how the subcontract aided the Contractor in completing or nearing completion of a specific project within the monthly progress report. Once the specified project is completed, no further work shall be given to the subcontractor until a new detailed project plan is presented to the

COR.

The Contractor shall ensure that the employees of the prime contract and any subcontractors work together as a unified team. It should be transparent to the Government whether a contract employee works for the prime or a subcontractor. The Contractor and any subcontractors shall conduct or provide to their employees detailed instruction on Government statutes, regulations, policies, and guidelines in areas such as employee conduct, ethics, safety, security, health, fire prevention, and the environment as they pertain to this contract.