Attachment 1. Draft SOW.pdf

DRAFT STATEMENT OF WORK (SOW)

Assessment of Immunotoxicity ‐ In Vitro and In Vivo Assessment of

Immune Function July 17, 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 Protocol Outline (see below).

1.3 Background and Scope

The Division of Translational Toxicology (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 in vitro and in vivo models of health and disease.

Specific research tasks include:

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

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

DRAFT

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 OECD guideline test methods for skin sensitization as part of an integrated panel of tests and; 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.

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 Induce Immunotoxicity 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 object, research must be performed in accordance with OECD Guideline Test Methods as referenced in Part 7 of this 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 or 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 shall normally (but not always) consist of daily dosing of adult rats or mice for 28 days by a route of exposure specific in a protocol outline.

The Contractor shall have the capability to evaluate the potential of a test agent to modulate the immune system in the developing rat 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 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.

1.5 Policies and Procedures

For in vitro studies, the contractor shall conduct all work in accordance with the Guidelines on Good In Vitro Methods Practices (GIVIMP, OECD, 2018).

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 American Association for Accreditation of Laboratory Animal Care (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 Guide for the Care and Use of Laboratory Animals, and all local, state, or federal regulations.

c. If animal species regulated by the USDA are involved in a contract, the contractor shall be registered with the Secretary of Agriculture of the United States in accordance with 7 U.S.C 2136 and 9 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 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 (see below).

d. All studies shall be conducted in accordance with the Specifications for the Conduct of Toxicity Studies by the Division of Translational Toxicology at NIEHS

(https://ntp.niehs.nih.gov/go/DTT_toxicologyspecs), referred to hereafter as the DTT Specifications.

e. When specified by the Protocol Outline, the study shall be performed in accordance with the Good Laboratory Practice (GLP) Regulations established by the 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.

f. 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 annually. Formats and additional requirements for monthly reports, study reports, and study files are described in more detail in the DTT Specifications.

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 Contracting Officer’s Representative (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 Frequency Medium/Format Submit To

Status Reports

– This report shall include the status of all agents on test, protocols, reports, and any manuscripts in preparation for the previous 30 days.

On the 20th day of each month, the COR shall receive a brief (1‐3 page) summary of the work currently in progress.

All reports shall be submitted in an electronic format that meets the NIEHS/DTT hardware and software standards. Any electronic documents (data, word processing, page layout, interchange formats, www content) must conform to the latest version of Microsoft Office.

This report shall be submitted to the CO and the COR via electronic mail. No QAU audit of this progress report is required.

Test Article Study Report

A draft test article study

All reports shall be submitted in an

Within 60 days of receipt of the COR’s requested report shall be submitted to the COR 180 days after completion of the in‐life portion of each study.

electronic format that meets the NIEHS/DTT hardware and software standards. Any electronic documents (data, word processing, page layout, interchange formats, www content) must conform to the latest version of Microsoft Office.

revisions the Contractor shall supply a full copy of a revised report including covers and signature pages that is Section 508 compliant in PDF and MS Word format.

An electronic copy of the Title Page of each report shall be sent to the CO.

Animal Use and Safety Protocols

Draft protocols shall be provided to the COR prior to submission to the Contractor’s IACUC or Safety Officer.

Reports shall be sent to the COR electronically or at the address specified at time of award.

Animal Use Protocols shall be approved by the contractor’s Institutional Animal Care and use Committee and Health and Safety Protocols related to animal use and Chemical Hygiene Plans shall be approved by the appropriate internal review committees.

An electronic copy of the approved IACUC and related protocols shall be sent to the COR no later than one week prior to commencement of the study.

Laboratory Study Protocols

Laboratory study protocols shall be submitted to the COR for review within 3‐ 5 weeks of receipt of the protocol outline.

Reports shall be sent to the COR electronically or at the address specified at time of award.

An electronic copy of the Title Page of each report shall be sent to the CO.

1.6.1 Periodic Reports

1.6.1.1 Monthly Progress Reports

On the 20th day of each month, the COR shall receive a 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.

1.6.1.2 Budget Summary Report

For each activity separately undertaken, and for the contract, submit a monthly Budget Summary Report. The report must include amounts of each activity budget, total expenditures on the activity for the month, summaries of current contract year and cumulative expenditures, 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 of each month.

1.6.2 Research Products

1.6.2.1 Animal Use Protocols

Protocols shall be approved by the Contractor’s or Testing Facility’s Institutional Animal Care and Use Committee. Health & Safety Summaries and Chemical Hygiene Plans 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. Submission of Approved Animal Use Protocols and Animal Study Protocols prepared by the Contractor shall be transmitted through a Government furnished program and project management (PPM) software. All substantive communication and decisions throughout the performance of work on a study shall be documented within the software.

1.6.2.2 Laboratory Study Protocols

Non‐animal laboratory study protocols shall be submitted to the COR for review within 3‐5 weeks of receipt of the protocol 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. Submission of Laboratory Study Protocols prepared by the Contractor shall be transmitted through a Government furnished program and project management (PPM) software. All substantive communication and decisions throughout the performance of work on a study shall be documented within the software.

1.6.2.3 Final Study Reports

A draft study report shall be submitted to the COR one hundred and eighty (180) days after completion 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 tasks 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 in 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 copy of a revised report including covers and 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 Section C, Article TBD of the contract for specific requirements related to IT Security.

1.6.4 508 Compliance The Contractor shall comply with Section 508 of the Rehabilitation Act of 1973 (29 U.S.C. 794d) which requires Federal agencies to purchase Electronic and Information Technologies (EIT) that meet specific accessibility standards. In order to conform to the Section 508 Electronic and Information Technology Accessibility Standards, the Contractor shall perform all work required under this contract in compliance with the provisions delineated in the Code of Federal Regulations (CFR) Title 36 (36 CFR Part 1194) and outlined below.

All electronic deliverables including reports, data entry, web pages, web applications, audio and video shall be Section 508‐compliant; drafts do not need to be compliant. Detailed data that is meant to be processed by a program and not meant to be read by people does not need to be Section 508 compliant. The following Section 508 accessibility technical standards apply to this

SOW:

E205.1 General Electronic content shall comply with E205.

The Contractor shall submit an annual Section 508 report in accordance with the schedule set forth in the ELECTRONIC AND INFORMATION TECHNOLOGY ACCESSIBILITY Article in SECTION H of the contract. Additional information about testing documents for Section 508 compliance, including guidance and specific checklists, by application, can be found at:

https://www.hhs.gov/web/section‐508/index.html and at:

https://www.section508.gov/create/documents , "Create Accessible Documents."

Remediation of any materials that do not comply with the applicable provisions of 36 CFR Part 1194 as set forth in the SOW, shall be the responsibility of the Contractor.

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 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 Contracting Officer. The designation letter states the responsibilities and limitations of the COR.

1.9 Personnel

In accordance with HHSAR 352.237‐75, the following personnel are considered key personnel by the Government as outlined in the DTT Specifications: Principal Investigator. The following personnel are considered critical personnel by the Government as outlined in the DTT Specifications: Deputy Principal Investigator, 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. Acceptance of the CV by the CO is required before these individuals are permitted to perform any task on this contract. 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 personnel will be based on education, training, and experience with immunotoxicity studies to include:

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) 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 Principal Investigator, Deputy Principal Investigator, and technical staff shall be employees of the offeror (i.e., not consultants or subcontractors). 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 and organizationally 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 below. 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 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 PI 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) 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

The Contractor shall comply with FAR 52.236‐13, Accident Prevention (November 1991), 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 text agents selected by the DTT to modulate immune function using established in vitro and in vivo models.

2.2 Task 1 – Evaluation of the Potential to Modulate Immune Function Using an In Vitro Testing Panel

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 should 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 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 Task 2 – Evaluation of the Potential to Induce Hypersensitivity Using In Vitro Test Methods

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)

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 Part 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

The KeratinoSens™ assay and similar me‐too 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 Part 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)

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 Part 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 (RFI) 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 (PI) 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

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 Part 7).

2.4 Task 3 – Evaluation of the Potential to Induce Immunotoxicity in 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.

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)]

Disease screening

Clinical observations, including evaluation of humane endpoints

(https://ntp.niehs.nih.gov/go/DTT_toxicologyspecs).

Body weights

Food or water consumption

Injection or infection with challenge agents per specific endpoint

Terminal Assessments and Collections

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

Blood (survival and terminal) and tissue collection

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.

2.4.1 Immunotoxicity Screening in Adult Mice or Rats

A minimum of 8 animals 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 task. All assays in section 2.4.2 through 2.4.5 should include appropriate positive and negative controls (vehicle) per the sample protocol outlines (Attachment XXX) 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 Part 7).

2.4.2 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 Cell Mediated Immunity

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

2.4.4 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.

2.4.5 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.

2.4.6 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 Sections 2.4.2 through 2.4.5. 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 Section 2.4.2‐2.4.5. As a litter is considered a single unit, all treatment groups shall be composed of pups from different litters. Contractors will need to 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.

3 Operating Procedures

3.1 Project Management

The Principal Investigator and Program Manager shall be responsible for the daily management of the contract and serve as points of contact for communication with the Government officials.

Collectively, the Principal Investigator and Program Manager 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 toxicology research and testing evaluations, a Protocol Outline will be prepared and issued by the Government for each study and test agent(s) to be studied under the contract. Each Protocol Outline will include a detailed description of the study(s) to be performed, or an outline of study objectives.

In response, the Contractor shall submit a technical work plan outlining the proposed technical approach and a project budget describing the proposed costs and level of effort. Information to be included in the technical work plan and budget are included in Technical Exhibit B of this

SOW.

Submission of Protocol Outlines by the Government and technical work plans and budgets prepared by the Contractor shall be transmitted through a Government furnished program and project management (PPM) software. All substantive communication and decisions throughout the performance of work on a study shall be documented within the software.

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.

Administrative activities shall be conducted by the Contractor as described in the administrative Protocol 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; 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. Additional data management requirements are included in the DTT Specifications, Chapter 11 and can be located via the following weblink:

(https://ntp.niehs.nih.gov/go/DTT_toxicologyspecs).

3.3 Contractor Travel

Contractor travel is not anticipated as a requirement in direct support of this contract. Travel expenditures must be approved in advance by the CO.

3.4 Contract Management and Monitoring

Contract performance will be monitored through periodic site visits, meetings with the Principal Investigator, review of laboratory study protocols and study reports, electronic mail, and telephone discussion, as required. The COR will monitor laboratory performance.

DTT site visits may be conducted to inspect facilities, study data, and records. DTT Laboratory Animal Medicine site visits may be conducted to ensure that all facilities, practices, and procedures of husbandry, care, and maintenance of animals used in the performance of this contract are in accordance with the latest issue in effect of the Guide for the Care and Use of Laboratory Animals. DTT may also perform health and safety inspections.

4 Government Furnished Property and Services

4.1 Supplies and Services

DTT will provide the test articles to be evaluated as described in individual work assignments.

DTT will provide the information on test article identity and purity and methods for dose formulation, storage, and handling when the test article is provided. In general, there will be a desire to maintain consistency with other DTT testing efforts and thus, the Government will furnish the test material. Compounds used as positive controls and test articles that can be purchased through commercial sources, where there is no requirement for consistency with other DTT studies, shall be purchased by the Contractor. Study‐specific Government‐furnished materials will be specified in a study‐specific Protocol Outline. In general, the below materials will be provided by the Government:

Test agents

Documentation of test agent identity and purity determination

Methods for dose formulation and analysis

Information on storage and stability for test agents and dose formulations

Access to Government‐specified laboratory information management system (LIMS) (currently a NIEHS‐leased version of Provantis)

Information on government‐designated suppliers for laboratory animals and disease screening

Government‐owned rodent models

4.2 Facilities

All work shall be conducted at the Contractor’s facility.

4.3 Software/Training

The Government will provide licensed access to a Government‐specified laboratory information management system (LIMS; currently an NIEHS‐leased version of Provantis) for collection of study data. DTT will provide contract staff with the necessary training in person or via videocast prior to accessing the Provantis system and annual refresher training thereafter. All contract staff who use the Provantis system must complete the training prior to use.

The Government will provide licensed access to a Government‐specified program and project management software (currently Microsoft Dynamics 365). DTT will provide contract staff with the necessary training prior to accessing the system.

4.4 Equipment

The Contractor is not expected to need a government furnished computer or other equipment.

If access to a system requires use of government furnished computer equipment, the

Government shall work with the Contractor to ensure the appropriate network account and equipment are procured.

The Government will provide access to the Virtual Private Network (VPN) in use at NIEHS to the Contractor, given the proper security clearances are processed for the Contractor staff using the equipment. Contractor staff who are granted access to the NIEHS VPN will be required to comply with all NIH training requirements for the maintenance of these accounts.

5 Contractor Furnished Property and Services

5.1 General

The Contractor shall provide all other chemicals, materials, animals, computing and laboratory equipment. Information on DTT‐designated suppliers such as those for test animals and disease screening will be provided after award.

5.2 Facilities

The recipient of this contract shall have the facilities and suitably trained personnel to function under the Food and Drug Administration (FDA) GLP regulations. 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 late interpretations published by the FDA) and to conduct Quality Assurance audits.

The Specifications for the Conduct of Toxicity Studies by the Division of Translational Toxicology at the National Institute of Environmental Health Sciences (https://ntp.niehs.nih.gov/go/DTT_toxicologyspecs; February 2023, Appendix A) apply to work performed under this contract, including the use of serology testing to establish the health status of the animals, use of a DTT‐approved diet and animal vendor, and a minimum 4‐day quarantine period for all animals prior to use in any study. Animal care facilities shall be of a design and operation (as specified in section V of Appendix A) such that an appropriate barrier minimizes the risk of infection influencing the outcome of experiments.

This SOW has been reviewed by the NIEHS Animal Care and Use Committee and the head of the DTT Comparative Medicine Group. 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.

6 Definitions and Acronyms

6.1 Definitions

Although not inclusive of every term used within this SOW, the following provides a list of definitions used throughout this SOW and commonly used in the acquisition field.

Contracting Officer (CO) – means a person with authority to enter into, administer, and or terminate contracts, and make related determinations and findings on behalf of the Government. Note: The only individual who can legally bind the Government.

Contracting Officer's Representative (COR) – As defined in DFARS 202.101, means an individual designated and authorized in writing by the Contracting Officer to perform specific technical or administrative functions. Any individual delegated responsibilities pursuant to subpart FAR 1.602‐2 of Reference (e), regardless of local terminology, must be certified in accordance with this instruction. For example, local terminology can be COR, Contracting Officer’s Technical Representative, Technical Point of Contact, Technical Representative, Alternate COR, Administrative COR, Assistant COR, Line Item Manager, Project Manager, Quality Assurance Personnel, Quality Assurance Evaluator, or COR management. These other surveillance support personnel may serve as on‐site representatives of the COR in performance of actual contract surveillance if they meet all COR requirements and have been appointed by the Contracting Officer as alternate CORs.

Contractor – means a supplier or vendor awarded a contract to provide specific supplies or service to the Government. The term used in this contract refers to the prime.

Contractor‐Acquired Property – means property acquired, fabricated, or otherwise provided by the Contractor for performing a contract, and to which the Government has title.

Day – means, unless otherwise specified, a calendar day.

Deliverable – means anything that can be physically delivered, but may include non‐ manufactured things such as meeting minutes or reports.

Governmen…