Amend_1-Att24_SampleProtocolOutlines.pdf

RFP-75N96023R00009 Attachment 24

Sample Protocol Outlines

I. Screening Study of 35 Test Agents Using a Hepatocyte Spheroid Model

II. Developing a Responsive Research Program for Harmful Algal Blooms

III. Conduct of Studies to Evaluate the Respiratory Toxicity of 3,3’ Dichlorobiphenyl (PCB 11) by Short- Term Nose-Only Inhalation in Rats and In Vitro Exposure of Air-Liquid Interface (ALI) Rat and Human Test Systems

IV. Conduct of Studies to Evaluate the Subchronic Toxicity of Test Agent X in Rats and Mice Following Whole Body Inhalation Exposure

V. Conduct of Studies to Evaluate Chronic Toxicity of Test Agent Y Administered in Feed to Rats (Following Perinatal Exposure) and Adult Mice

VI. Class Evaluation of 8 Test Agents in 28-Day Gavage Studies in Rats

I. Screening Study of 35 Test Agents Using a Hepatocyte Spheroid Model

A. Background One of the goals of DTT is to implement in routine toxicology testing, tools and approaches that have been developed and refined in-house by DTT staff. This is a research area that DTT has extensive experience (see Part B) and would like to transfer from in-house research to a contracted mechanism. This sample Protocol Outline is written as an outline of study objectives rather than specific requirements.

Offerors shall review the background material in Part B (References), along with any other related and relevant literature. For Part D (Spheroid Culture and Test Agent Exposure), Offerors shall provide a detailed study protocol and technical approach to accomplish the goals of the study.

B. References

• Ramaiahgari SC, Ferguson SS. Organotypic 3D HepaRG Liver Model for Assessment of Drug-

Induced Cholestasis. Methods Mol Biol. 2019;1981:313-323. doi: 10.1007/978-1-4939-9420-

5_20. PMID: 31016663.

• Clayton NP, Burwell A, Jensen H, Williams BF, Brown QD, Ovwigho P, Ramaiahgari S, Hermon T, Dixon D. Preparation of Three-dimensional (3-D) Human Liver (HepaRG) Cultures for Histochemical and Immunohistochemical Staining and Light Microscopic Evaluation. Toxicol Pathol. 2018 Aug;46(6):653-659. doi: 10.1177/0192623318789069. Epub 2018 Aug 8. PMID:

30089414; PMCID: PMC6117202.

• Ramaiahgari SC, Waidyanatha S, Dixon D, DeVito MJ, Paules RS, Ferguson SS. Three-Dimensional (3D) HepaRG Spheroid Model With Physiologically Relevant Xenobiotic Metabolism Competence and Hepatocyte Functionality for Liver Toxicity Screening. Toxicol Sci. 2017 Sep 1;159(1):124-

136. doi: 10.1093/toxsci/kfx122. Erratum in: Toxicol Sci. 2017 Nov 1;160(1):189-190. PMID:

28633424; PMCID: PMC5837526.

• Rowan-Carroll A, Reardon A, Leingartner K, Gagné R, Williams A, Meier MJ, Kuo B, Bourdon- Lacombe J, Moffat I, Carrier R, Nong A, Lorusso L, Ferguson SS, Atlas E, Yauk C. High-Throughput Transcriptomic Analysis of Human Primary Hepatocyte Spheroids Exposed to Per- and Polyfluoroalkyl Substances as a Platform for Relative Potency Characterization. Toxicol Sci. 2021 May 27;181(2):199-214. doi: 10.1093/toxsci/kfab039. PMID: 33772556.

• Proctor WR, Foster AJ, Vogt J, Summers C, Middleton B, Pilling MA, Shienson D, Kijanska M, Ströbel S, Kelm JM, Morgan P, Messner S, Williams D. Utility of spherical human liver microtissues for prediction of clinical drug-induced liver injury. Arch Toxicol. 2017 Aug;91(8):2849-2863. doi: 10.1007/s00204-017-2002-1. Epub 2017 Jun 13. PMID: 28612260;

PMCID: PMC5515971.

C. Project Management As applicable, studies shall be conducted in accordance with the Specifications for the Conduct of Toxicity Studies for the Division of Translational Toxicology at NIEHS (https://ntp.niehs.nih.gov/go/DTT_toxicologyspecs) when applicable. These studies do not have to be in accordance with GLPs but should utilize facility SOPs.

D. Spheroid Culture and Test Agent Exposure The goal of this study is to develop high quality screening data to inform prioritization for additional toxicity evaluations. A suite of 35 test agents shall be run in vitro using hepatocyte spheroids with endpoints evaluating various aspects of cellular architecture, functionality, and responsiveness to exposures that may include histopathology, high content imaging, cytotoxicity, reactive oxygen species, metabolomic, and transcriptomic analysis.

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

Based on the above citations, related culture models, and industry standards, spheroids shall be cultured and used in a toxicity screening study. Describe in detail how spheroids will be cultured and exposed to a set of 35 test agents at nine concentrations each, over a 10,000-fold concentration curve. Offerors should assume the test agents will be provided by the Government in the form of DMSO stock solutions (highest DMSO soluble concentration for each agent), along with test agent identity and purity reports.

Describe how and at what frequency spheroids will be evaluated. Endpoints for consideration include:

• Histopathology

• High content imaging

• Cytotoxicity and reactive oxygen species

• Transcriptomic analysis performed using the TempOSeq human S1500+ transcript system o The Offeror shall describe the data analysis approach to be used for all proposed evaluations except transcriptomic data; S1500 analysis shall be delivered as raw data files with the curation and delivery of all appropriate metadata.

Offerors shall describe in detail their technical approach for all aspects of the study design. In addition, a detailed data management plan shall be provided outlining the availability and delivery of all raw, reduced, and summarized data in electronic and machine-readable formats that align with FAIR+ standards.

E. Study Schedule and Output Requirements

OUTPUT FREQUENCY DUE DATES

Data Management Plan 1 Prior to study start

Study Protocol 1 Prior to study start

Study Report 1 Within 3 months of test completion

Study Raw Data Archival Records and Electronic Submission of Results 1

Within 30 days of submission of Report (send to NTP Archives/CEBS per DMP)

II. Developing a Responsive Research Program for Harmful Algal Blooms

Public surface waters, including those used for recreational and commercial activities and those that serve as sources of drinking water, can experience harmful algal blooms (HABs) due to conditions that may be favorable to HAB development, including warm temperatures and rich nutrient conditions. There are several types and species of organisms that produce numerous toxins, many of which are poorly characterized for other than acute toxicity (i.e., for potential to result in short- and long-term human health effects).

B. Problem Formulation Offerors shall outline a plan to work with a group of DTT staff to conduct problem formulation in a relatively short time (e.g., 1-3 months from start to finish depending on needs). For the purposes of demonstrating approach to this Protocol Outline, Offerors should indicate key areas (topics/milestones) where interaction with DTT staff would be needed. The result of problem formulation will be to identify a discrete set of research questions to address human health concerns due to exposure to HAB toxins and outline a holistic approach that addresses each question.

Objectives and considerations:

• Research questions should be oriented toward generating added value for protecting human health. What new information will have the broadest potential utility and impact?

• What are the most impactful research questions to address health effects, and for what key areas would DTT staff be engaged to refine these questions during problem formulation?

• Who are the stakeholders that should be consulted in identifying and refining these research questions and implementing this evaluation approach, and how will they be engaged?

• What will be the major challenges for this research project and how will the proposed approach overcome these issues?

• The research approach should articulate the rationale for selection of specific test agents, biological models, and endpoints and discuss key decision points at different phases of project execution.

• Proposed experimental evaluations should consider potential procurement, logistics, and health and safety issues and determining their concentrations in relevant matrices.

• A detailed technical approach to accompany the research plan is not required; the focus of this Task is to demonstrate scientific understanding and capabilities related to problem formulation and application of the DTT strategic framework. The total response for this Protocol Outline, including Part C should not exceed 10 pages.

C. Implementing a Responsive Research Program A large algal bloom is occurring in an area where a large population is potentially affected, resulting in human exposure to multiple HAB toxins by various routes of exposure. Based on an approach developed in Section B, offerors shall outline a plan for how the project would be implemented and managed to completion. The proposed plan should balance time and level of effort required, with the complexity and content of information being generated.

The overall goal is to deliver actionable research products in an aggressive timeframe; assume completion in less than two years for this task. Actionable products should provide definitive data that can feed immediately into decision making. Offerors should provide a project schedule and outline the major outputs that can be provided at 6-month, 12-month, and 18-month milestones. Major assumptions that could impact schedule should be clearly articulated for each proposed study and study output.

III. Conduct of Studies to Evaluate the Respiratory Toxicity of 3,3’ Dichlorobiphenyl (PCB 11) by Short-term Nose-only Inhalation in Rats and In Vitro Exposure of Air-liquid Interface (ALI) Rat and Human Test Systems

Although no longer manufactured or used, inhalation exposure to polychlorinated biphenyls remains a concern. This project aims to develop high quality short term in vivo and in vitro data on the selected test agent that would facilitate in vivo to in vitro extrapolation within the rat, and in vitro species-species extrapolation from rat to human, in the context of adverse pulmonary effects.

B. General Information

1. Project Management All studies shall be conducted in accordance with the Specifications for the Conduct of Toxicity Studies for the Division of Translational Toxicology at NIEHS (https://ntp.niehs.nih.gov/go/DTT_toxicologyspecs) when applicable. These studies do not have to be in accordance with GLPs but should utilize facility SOPs.

2. Phases Phase 1. During this phase the contractor shall perform those work activities associated with development of exposure generation systems and monitoring methods, health and safety concerns that must be addressed before study start, and submission of a prestart inhalation report.

Phases 2 and 3: Upon completion of Phase 1, conduct of Phases 2 and 3 may continue in sequence or parallel. Phase 2 is for the conduct of short-term nose-only exposures in rats; Phase 3 is for the conduct of in vitro ALI exposures in human and rat lung tissue cultures.

3. Government Furnished Materials

• Test article, identity and purity information, and bulk chemical reanalysis methodology

• Access, system support and training for Provantis, a NIEHS-leased electronic data collection system

• VITROCELL® 48 Plus system

C. Phase 1- Prestart Activities

1. Development of Exposure System(s)

• Develop methods for generation and online monitoring for VITROCELL® 48 Plus and nose-only inhalation systems at the protocol required concentrations (see Phase 2 and 3 below).

A single parallel exposure generation system may be designed such that nose-only and VITROCELL® 48 Plus exposure occur in parallel, or they may be performed as separate experiments.

• Preliminary results of generation and characterization shall be reported to the COR for approval as soon as results are available.

• Validate the methods for generation and monitoring at the protocol required concentrations.

• Each of the below shall be determined for both the VITROCELL® 48 Plus and nose-only exposure systems.

o Measurement limits, specificity, accuracy, precision, and linearity of the monitoring system.

o T90, T10, aerosol evaluation and stability.

o Stability of concentration must be established for a period of 3 hours plus T90 for three days. All the data must be obtained in the same exposure room and under identical conditions that will be used during the animal/cell exposures.

o Stability studies of the test agent in the generator reservoir. (Identification of possible degradation products is the responsibility of the Contractor.)

o Develop and test method(s) of effluent treatment, during generation runs at the protocol required concentrations

• Demonstrate effectiveness of the exhaust treatment unit immediately after the effluent treatment unit and at the point of exhaust from the building. Establish the percent efficiency of the exhaust treatment. The effluent exhaust treatment must be effective in removing the test agent to an acceptable concentration (i.e., greater that 90% efficiency of removal by the treatment system and less than 50% of the threshold limit value (TLV), if a TLV exists), or written documentation for a waiver from the appropriate air regulatory agencies must be provided. Determine lifetime expectancy of any proposed filtration/treatment units and amount of treatment media required. Confirm that none of the exhausted test agent is re-entrained.

• Develop and test method(s) of room air monitoring during generation runs at protocol required concentrations in all chambers under actual animal/cell exposure conditions.

Define the lower limit of detection of the monitoring method(s) and document that this level provides an adequate safety margin for personnel.

• Prepare and submit a Prestart Inhalation Report of Phase I chemistry, exposure system, and health and safety activities and procedures to include:

o Generation system description and approach o Monitoring system description and calibration o Stability of concentration over test period o Evaluation for the presence of aerosols o T90 and T10 o Test agent stability studies in generator reservoir o SOPs for generation of test agent, generation maintenance/change out, evaluation for the presence of aerosols; other SOPs as appropriate for the specific test agent o Description of effluent exhaust treatment and SOPs o Effluent exhaust treatment monitoring data and percent efficiency of the effluent exhaust treatment o Room air monitoring methodology, strategy, SOPs, frequency, and results

• Upon successful completion and approval of Phase 1 (including COR review of the Prestart Inhalation Report), the contractor shall conduct work specified as Phases 2 and 3.

D. Phase 2- Repeat Exposure (2-week) Nose-Only Inhalation Studies in Rats

1. Exposure Male and female HSD:Sprague Dawley rats shall be housed individually, and placed on NTP-2000 diet. After a 10 to 14-day quarantine period, 5-6 week old animals shall be randomly assigned to exposure and control groups and acclimated to the nose-only exposure system, for 3 hours + T90 per day for three days. At each of six concentrations and a control group [0, 1, 3, 10, 30, 100 and 300 ppm], 5 male and 5 female rats shall receive the test article via nose only inhalation, 3 hours + T90 per day, 5 days per week, for 2 weeks plus 2 days; animals shall be exposed for a minimum of 2 consecutive days prior to removal.

Animals Sexes Groupsa

Total

Core Study 5 2 7 = 70 Sentinelb 5 2 - = 10 a Six exposure groups and a control for core study b Five animals/sex shall be used for disease screening prior to quarantine release and resampled at 4 weeks after arrival. Both time points shall include Optispot, blood, fur wipe, and feces; the 4-week time point shall also include necropsy. At study termination, 5/sex in the highest surviving exposure group shall be sampled for disease screening. Sentinels shall be distributed amongst control and exposure groups.

2. Observations Rats shall be weighed individually on the first day of exposure, twice weekly thereafter, and at study termination. The animals shall be observed twice daily, once in the early morning and once in the late afternoon, at least six hours apart (before 10:00 AM and after 2:00 PM), including holidays and weekends for moribundity and mortality. Signs of toxicity noticed during these routine checks shall be recorded. Formal clinical observations shall be performed and recorded daily following exposure.

Animals shall be assessed for lung function using a technique to be proposed and described by the offeror.

3. Sample Collection for Internal Dose Assessment On the last day of exposure, all animals shall be removed from the chambers immediately following exposure and a survival blood collection performed via the jugular vein without anesthesia. Following blood collection, animals shall be returned to their home cages and necropsied as described in section 4 below.

Blood shall be processed to plasma and stored in 250µL aliquots at ≤ -70°C. Blood shall be stored on wet ice prior to processing, and all samples processed to plasma within 1 hour of collection.

The actual time of blood collection, and time relative to end of exposure shall be recorded for each animal.

Plasma samples shall be shipped frozen to the sponsor-designated facility; data will not be returned or included in the study report.

4. Necropsy and Histopathology Necropsy shall occur on the day following the last exposure.

Moribund animals and animals surviving to terminal sacrifice shall be deeply anesthetized via 70% CO2:30% O2 inhalation, humanely terminated via exsanguination, and death confirmed by a secondary method such as thoracotomy.

a) Blood Collection Blood shall be collected at the time of scheduled euthanasia and as much blood as possible collected. Blood shall be processed to serum and saved in aliquots of 1mL at ≤ -70°C. Blood shall be stored on wet ice prior to processing, and all samples processed to serum within 1 hour of collection. The time of blood collection shall be recorded for each animal.

b) Lung Processing and Frozen Tissue Collection

Following terminal blood collection, the lungs shall be removed from the chest cavity, weighed, and the weights recorded.

Bronchoalveolar Lavage (BAL) shall be performed on the right lung. The lungs shall be lavaged with phosphate buffered saline (PBS). The BAL fluid shall be centrifuged at 200 x g for 10 min.

The cell-free supernatant from the lavage shall be saved and stored at ≤ -70°C until analysis as directed by the Government. The cell pellet shall be resuspended in PBS, cells shall be counted (after red blood cell lysis) and cells shall be spun onto a glass slide using a cytocentrifuge (Cryospin system or similar) and stained using a Diff Quick Stain Set or similar. Cell counts and differentials shall be reviewed and recorded by a Pathologist.

The left lung shall be fixed in formalin and processed to slides as described in the Specifications.

The right lung shall be cut into approximately 4-5 mm3 pieces and divided into two samples to be placed into separate cryotubes, flash frozen and stored at ≤ -70°C.

Following lung collection, samples of liver (5 x 200 mg) shall be collected into separate cryotubes, flash frozen and stored at ≤ -70°C.

Blood, lung, and liver samples shall be shipped frozen to the sponsor-designated facility; data will not be returned or included in the study report.

c) Additional Necropsy Requirements

Organ weights shall be determined for all animals surviving until terminal sacrifice. Those organs to be weighed are: liver, left and right kidney, heart, and lungs. Bilateral organs shall be weighed and recorded separately.

A necropsy shall be performed on all exposure and control group animals that either die or are sacrificed. The tissues listed below shall be collected for archival and histopathological evaluation. All gross lesions shall be collected for histopathological evaluation.

Specified tissues (see below table) shall be trimmed, embedded, sectioned, and stained with hematoxylin and eosin for possible histopathologic evaluation. Gross lesions shall be examined microscopically in all animals in all exposure and control groups. A complete histopathologic evaluation shall be done on all control animals, all animals in the highest exposure group with at least 60% survivors at terminal sacrifice, plus all animals in higher exposure groups. Exposure-related lesions (target organs) shall be identified and examined in lower exposure groups to a no-effect level. A complete histopathologic evaluation shall be performed for all natural death/moribund sacrifice animals.

Adrenal glands Gross lesions Heart and aorta Kidney Larynx Liver (2 sections including left lobe and median lobe) Lungs and mainstem bronchi Lymph nodes

- mandibular and mesenteric

- bronchial and mediastinal

E. Phase 3- Repeat (Three-day) In Vitro Exposure of ALI Human and Rat Airway Test

Systems

1. Biological Test System Selection The contractor shall select ALI airway culture test systems that are representative of human lung and rat lung. The test systems may be commercially available or developed by the contractor. If an in-house model is proposed, sufficient information shall be provided to assess comparability to other models (publications, SOPs, all appropriate donor information for human cultures, etc.).

Any proposed model must be compatible with the Government furnished VITROCELL® 48 Plus exposure system.

2. Exposure The biological test system shall be developed or received and allowed to acclimate to the test environment sufficiently prior to exposure. The tissues shall be exposed to the test agent for 3 hours + T90 per day for 3 consecutive days at each of six concentrations plus a control group [0, 1, 3, 10, 30, 100 and 300 ppm].

The proposed exposure paradigm should allow adequate assessment of the below endpoints.

3. Observations The tissues shall be observed after exposure and cilia activity and any other morphological changes to the apical surface documented. Environmental conditions of cell incubation (e.g., temperature, humidity) shall be automatically recorded for the duration of the experiment.

4. Assessments Assays to address the following endpoints shall be proposed. Assay shall be assessed in apical rinse, basal media, and/or tissue.

• Cell health (barrier function/TEER measurements, cytotoxicity) at multiple timepoints to inform dose response and time course

• Biomarkers of effect (e.g., multiplex assay) appropriate based on the test agent to facilitate IVIVE and species-extrapolation modeling

• Internal concentration measurements to inform cell uptake and transfer of parent test agent/metabolites to basal media

• Histopathology (including immunohistochemistry)

• RNA isolation from tissue for gene expression analysis (transcriptomics or qRT-PCR)

F. Study Schedule and Output Requirements

PRESTART SUBMISSIONS FREQUENCY DUE DATES

Submissions of Preliminary Chemistry and Generator and Chamber Characterization 1/exposure system TBD

Submissions of Prestart Inhalation Report 1/exposure system TBD, ~within 3 months of assignment

DTT Review and Approval to Proceed to Next Phase NA Within 4 weeks of receipt of report

2-WEEK STUDIES FREQUENCY DUE DATES

Data Management Plan 1 each for Phases 2 and 3 Prior to study start

Study Protocol (IACUC approved for Phase 2) 1 each for Phases 2 and 3 Prior to study start

Start of Study(s) NA 4 Weeks from approval to proceed

Serum Samples for Disease Screening See Specs As collected

Internal Dose Samples (Phases 2 and 3) Shipped as soon as feasible

Study Report 1 each for Phases 2 and 3 Within 5 months from last day of necropsy

Slides, Blocks, Wet tissues, and Inventory 1 each for Phases 2 and 3 Within 30 days of submission of 2- Week Reports

Study Raw Data Archival Records for 2-Week Study and Electronic Submission of Results

1 each for Phases 2 and 3 Within 30 days of submission of 2- Week Reports (send to NTP Archives)

IV. Conduct of Studies to Evaluate the Subchronic Toxicity of Test Agent X in Rats and Mice Following Whole Body Inhalation Exposure

Test Agent X is used in manufacturing with most potential exposures occurring in an occupational setting.

The primary concern for human exposure is related to occupational exposure by inhalation. For the purposes of this Protocol Outline, Offerors shall prepare Prestart technical approaches with the assumption that Test Agent X is a vapor for Phase 1A or a particle for Phase 1B.

B. General Information

1. Project Management All studies shall be conducted in accordance with GLPs and the Specifications for the Conduct of Toxicity Studies for the Division of Translational Toxicology at NIEHS (https://ntp.niehs.nih.gov/go/DTT_toxicologyspecs).

Phase 1. During this phase the contractor shall perform those work activities associated with identity and purity determination of the bulk test agent, development of exposure generation and monitoring methods, health and safety concerns that must be addressed before study start, and submission of a prestart inhalation report.

Phase 2: Upon completion of Phase 1, the subchronic (3-month) studies shall be conducted.

• Access, system support and training for Provantis, a NIEHS-leased electronic data collection system

C. Phase 1A- Prestart Activities (Vapor) For the below, offerors shall assume the test agent is a vapor and describe the technical approach, where possible without knowing the specific identity of the test agent.

1. Test Agent Analyses

• Procure the test agent.

• Develop identity and purity methods for the test agent.

• Conduct identity and purity analysis of the test agent using the methods developed.

Preliminary results shall be reported to the DTT for approval as soon as results are available.

2. Development of Exposure System

• Offerors shall describe all the necessary information they would need to design the exposure system and perform health and safety assessments.

• Develop methods for generation and chamber monitoring at the protocol required concentrations. Preliminary results of generator and chamber characterization shall be reported to the COR for approval as soon as results are available.

• Validate the methods for generation and chamber monitoring at the protocol required concentrations.

• Conduct detailed 3-day chamber characterization studies.

• Determine measurement limits, specificity, precision, and linearity of the chamber concentration monitoring system.

• Determine T90, T10, chamber homogeneity, aerosol evaluation and stability of the chamber concentration in all chambers. The stability of chamber concentration must be established for a period of 6 hours plus T90 for three days. All the data must be obtained in the same exposure room and under identical conditions that will be used during the animal exposures.

• Conduct stability studies of the test agent in the generator reservoir and chamber.

(Identification of possible degradation products is the responsibility of the Contractor.)

• Develop and test method(s) of effluent treatment, during generation runs at the protocol required concentrations in all chambers under actual animal exposure conditions.

Demonstrate effectiveness of the exhaust treatment unit immediately after the effluent treatment unit and at the point of exhaust from the building. Establish the percent efficiency of the exhaust treatment. The effluent exhaust treatment must be effective in removing the test agent to an acceptable concentration (i.e., greater that 90% efficiency of removal by the treatment system and less than 50% of the threshold limit value (TLV), if a TLV exists), or written documentation for a waiver from the appropriate air regulatory agencies must be provided. Determine lifetime expectancy of any proposed filtration/treatment units and amount of treatment media required. Confirm that none of the exhausted test agent is re-entrained.

• Develop and test method(s) of room air monitoring during generation runs at protocol required concentrations in all chambers under actual animal exposure conditions. Define the lower limit of detection of the monitoring method(s) and document that this level provides an adequate safety margin for personnel.

• Prepare and submit a Prestart Inhalation Report of Phase I chemistry, exposure system, and health and safety activities and procedures to include:

o Description of bulk test agent identity and purity method development o Confirmatory data on identity, purity and if necessary, stability of the bulk test agent using methods developed, as well as copies of the SOPs for the bulk reanalysis methods o Description of inhalation exposure chamber o Generation system description and approach o Chamber monitoring system description and calibration o Homogeneity of test atmosphere in chambers o Stability of chamber concentration over 3 days o Evaluation for the presence of aerosols o Chamber T90 and T10 o Test agent stability studies in generator reservoir and chamber o SOPs for generation of test agent, generation maintenance/change out, chamber monitoring system including calibration approach, bulk test agent identity and purity and reanalysis, chamber homogeneity determinations, evaluation for the presence of aerosols; other SOPs as appropriate for the specific test agent o Description of effluent exhaust treatment and SOPs o Effluent exhaust treatment monitoring data and percent efficiency of the effluent exhaust treatment o Room air monitoring methodology, strategy, SOPs, frequency, and results

D. Phase 1B- Prestart Activities (Particle) For the below, offerors shall assume the test agent is a particle and describe the technical approach, where possible without knowing the specific identity of the test agent.

1. Test Agent Analyses

• Procure the test agent.

• Develop identity and purity methods for the bulk test agent; methods shall include, but may not be limited to, SEM, TEM with test agent specific detection.

• Conduct identity and purity analysis of the test agent using the methods developed.

2. Development of Exposure System

• This Protocol Outline (PO) is written assuming the exposure would be conducted by whole-body inhalation. Offerors also offer a framework or considerations for which a nose-only exposure would be more appropriate than a whole-body exposure.

• Develop methods for generation and monitoring at the protocol required concentrations.

Monitoring methods shall include, but may not be limited to, SEM, TEM with test agent specific detection.

• Validate the methods for generation and monitoring at the protocol required concentrations.

• Conduct detailed 3-day characterization studies.

• Determine measurement limits, specificity, precision, and linearity of the concentration monitoring system.

• Determine T90, Tl0, homogeneity, aerosol evaluation, and stability of the concentrations in all chambers. The stability of chamber concentrations must be established for a period of 6 hours plus T90 for three days. All the data must be obtained in the same exposure room and under identical conditions that will be used during the animal exposures.

• Perform thorough physical and chemical characterization of the particles/fibers in the bulk and exposure atmosphere.

• Conduct stability studies of the test agent in the generator reservoir and chambers.

• Develop and test method(s) of effluent exhaust treatment, during generation runs at the protocol required concentrations in all chambers under actual animal exposure conditions. Demonstrate effectiveness of the exhaust treatment unit immediately after the effluent treatment unit and at the point of exhaust from the building. Establish the percent efficiency of the exhaust treatment. The effluent exhaust treatment must be effective in removing the test agent to an acceptable concentration (i.e. greater than 90% efficiency of removal by the treatment system and less than 50% of the TLV, if a TLV exists), or written documentation for a waiver from appropriate air regulatory agencies must be provided. Determine lifetime expectancy of any proposed filtration/treatment units and amount of treatment media required. Confirm that none of the exhausted test agent is re-entrained.

• Develop and test method(s) of room air monitoring during generation runs at protocol required concentrations under actual animal exposure conditions. Define the lower limit of detection of the monitoring method(s) and document that this level provides an adequate safety margin for personnel.

• Prepare and submit a Prestart Inhalation Report of Phase I chemistry, exposure system, and health and safety activities and procedures to include:

o Description of bulk test agent identity and purity method development o Confirmatory data on identity, purity and, if necessary, stability of the bulk test agent using methods developed, as well as copies of the SOPs for the bulk reanalysis methods o Generation system description and approach o Monitoring system description and calibration o Homogeneity of test atmosphere o Stability of concentration over 3 days o Evaluation of the particles/fibers o T90 and Tl0 times o Test agent stability studies in generator reservoir and chamber o SOPs for the following: generation of test agent; generation maintenance/changeout; monitoring system including calibration approach;

homogeneity determinations; evaluation of particles/fibers; and other as appropriate for the specific test agent o Description of effluent exhaust treatment and SOPs; effluent exhaust treatment monitoring data and percent efficiency of the effluent exhaust treatment o Room air monitoring methodology, strategy, SOPs, frequency, and results

E. Phase 2- Subchronic (3-month) Studies and Reporting

1. Exposure Male and female HSD:Sprague Dawley SD rats and B6C3F1 mice shall be housed individually, placed on NTP-2000 diet, and acclimated to the exposure chambers, at least 6 hours + T90 a day for three days. After a 10 to 14-day quarantine period, 5-6 week old animals shall be randomly assigned to exposure and control groups. At each of six concentrations and a control group [0, 3, 10, 30, 100 and 300 ppm], ten male and female rats and mice shall receive the test article via whole body inhalation, 6 hours + T90 per day, 5 days per week, for up to 3 months. All animals shall be exposed for a minimum of 2 consecutive days prior to scheduled removal.

Animals Species Sexes Groupsa

Core Study 10 2 2 6 = 240 Internal Dose 3 2 2 6 = 72 5-day Interim 5 2 2 6 = 120 Sentinelb 5 2 2 - = 20 a Five exposure groups and a control for core study bFive animals/sex/species shall be used for disease screening prior to quarantine release and resampled at 4 weeks after arrival. Both time points shall include dried blood spot, fur wipe, and feces; the 4-week time point shall also include necropsy. At study termination, 5/sex/species in the highest surviving exposure group shall be sampled for disease screening. Sentinels shall be distributed amongst control and exposure chambers.

Rats and mice shall be weighed individually on the first day of exposure, twice weekly for the first 2 weeks of exposure, at weekly periods thereafter, and at study termination. The animals shall be observed twice daily, once in the early morning and once in the late afternoon, at least six hours apart (before 10:00 AM and after 2:00 PM), including holidays and weekends for moribundity and mortality. Signs of toxicity noticed during these routine checks shall be recorded. Formal clinical observations shall be performed and recorded twice weekly for the first 2 weeks of exposure and weekly thereafter, following exposure.

3. Sample Collection for Internal Dose Assessment Animals assigned to the internal dose cohort shall have blood collected immediately following exposure on the 5th day of the study (survival blood collection in unanesthetized animals via jugular vein) and in the final week of the study.

On each collection day, as soon as feasible following exposure, animals in the internal dose assessment cohort shall be removed from the chambers and blood collected. On the 5th day of the study, following blood collection, animals shall be placed in metabolism cages for a 24-hour urine collection. Urine collection method shall be compatible with volatile constitutes and described in detail. Urine shall be stored in 250µL aliquots at ≤ -70°C.

Blood shall be processed to plasma and stored in 250µL aliquots at ≤ -70°C. Blood shall be stored on wet ice prior to processing, and all samples processed to plasma within 1 hour of collection.

The actual time of blood collection, and time relative to the end of exposure shall be recorded for each animal.

Following terminal blood collection, samples of liver (5 x 200 mg) shall be collected and flash frozen. Blood, urine, and liver samples shall be shipped frozen to a sponsor-designated facility;

data will not be returned or included in the study report.

4. 5-Day Interim Assessment Moribund animals and animals surviving to study termination shall be humanely terminated via exsanguination while under 70% CO2:30%O2 anesthesia. Necropsies for each study block shall be completed by noon.

The liver and right kidney shall be weighed.

Two samples from each the left liver lobe (same lobe for all animals) and right kidney shall be collected within five minutes of termination for toxicogenomic evaluation. One sample shall be processed as appropriate for RNA isolation. The second sample shall be cut into smaller pieces (approximately 5 mm3), aliquoted into screw-cap tubes, flash frozen in liquid nitrogen, and stored at ≤ -70°C. Frozen tissues shall be labeled to indicate animal identification number, study number, tissue type, and date of storage collection.

Quality and quantity of RNA shall be determined. Extracted RNA shall be sequenced using the S1500+ TempOseq platform including 1.5 million reads/sample (500 mapped reads/transcript).

Fastq files shall be delivered electronically to NIEHS; data are not to be included in the study report.

5. Core Assessments

a) Clinical Pathology Blood shall be collected from the retro-orbital plexus or sinus at terminal sacrifice for clinical pathology [clinical chemistry (rats only) and hematology (mice and rats)].

HEMATOLOGY CLINICAL CHEMISTRY

Erythrocyte count Total protein Hemoglobin concentration Albumin, Globulin and A/G ratio Hematocrit (packed cell volume; Urea nitrogen (BUN) spun and automated) Creatinine Mean corpuscular volume Alanine aminotransferase (ALT) Mean corpuscular hemoglobin Sorbitol dehydrogenase (SDH)

Mean corpuscular hemoglobin concentration Alkaline phosphatase (ALP) Leukocyte count and differential Aspartate aminotransferase (AST) Reticulocyte count Total bile acids Platelet count Glucose Morphologic assessment of erythrocytes, Creatine kinase (CK) leukocytes and platelets Cholesterol Nucleated erythrocyte count5 Triglycerides Total Bilirubin Indirect Bilirubin Direct bilirubin

b) Necropsy and Histopathology Moribund animals and animals surviving to terminal sacrifice shall be deeply anesthetized via 70% CO2:30% O2 inhalation, humanely terminated via exsanguination, and death confirmed by a secondary method such as thoracotomy.

Organ weights shall be determined for all animals surviving until terminal sacrifice. Those organs to be weighed are: liver, thymus, left and right kidney, left and right testis, left and right epididymis, left and right ovary, heart, and lungs. Bilateral organs shall be weighed and recorded separately.

A complete necropsy shall be performed on all exposure and control group animals that either die or are sacrificed.

Tissues to be collected for histopathological evaluation are listed below. All gross lesions shall be collected for histopathological evaluation.

Specified tissues (see below table) shall be trimmed, embedded, sectioned, and stained with hematoxylin and eosin for possible histopathologic evaluation, with the exception of the testes, which shall be stained with PAS/H.

Gross lesions shall be examined microscopically in all animals in all exposure and control groups. A complete histopathologic evaluation shall be done on all control animals, all animals in the highest exposure group with at least 60% survivors at terminal sacrifice, plus all animals in higher exposure groups. Exposure-related lesions (target organs) shall be identified and examined in lower exposure groups to a no-effect level. A complete histopathologic evaluation shall be performed for all natural death/moribund sacrifice animals.

Adrenal glands Nasal cavity and nasal turbinates (3 sections) Brain (7 sections) Nerve (if neurologic signs present)6 Cervix - sciatic Clitoral glands - tibial (rat only) Esophagus - trigeminal (with ganglion) Eyes Ovaries Femur Pancreas Gallbladder (mouse) Parathyroid glands Gross lesions Pituitary gland Harderian glands Preputial gland Heart and aorta Prostate Intestine, Large Salivary glands

(cecum, colon, rectum) Seminal vesicles Intestine, Small Spinal cord (3 sections) (duodenum, jejunum, ileum) (if neurologic signs present)6 Kidneys Spleen Larynx Stomach (forestomach and glandular) Liver Testes with epididymides (2 sections including left lobe Thymus and median lobe) Thyroid gland Lungs and mainstem bronchi Tissue masses Lymph nodes Trachea

- mandibular and mesenteric Urinary bladder

- bronchial and mediastinal Uterus Mammary gland with adjacent skin Vagina Muscle, thigh (if neuromuscular signs present)6

6. Study Schedule and Output Requirements

PRESTART SUBMISSIONS FREQUENCY DUE DATES

Submissions of Preliminary Chemistry and Generator and Chamber Characterization 1 TBD

Submissions of Prestart Inhalation Report 1 TBD, ~within 3 months of assignment

DTT Review and Approval to Proceed to Next Phase NA Within 4 weeks of receipt of report

3-MONTH STUDIES FREQUENCY DUE DATES

Data Management Plan 1 Prior to study start

Study Protocol (IACUC approved) 1/Species Prior to study start

Start of 3-Month Study NA 4 Weeks from approval to proceed

Serum Samples for Disease Screening See Specs As collected

Internal Dose Samples 1 Shipped within 1 week of collection

FastQ files from 5-day Interim 1 Delivered within 12 weeks of necropsy

Blood for Micronuclei Rats and Mice (as applicable)

1 Shipped within 24 hours of collection

Clinical Pathology Data (unaudited) 2 Within 21 days of collection

3-Month Study Report 1/Species Within 5 months from last day of necropsy

Slides, Blocks, Wet tissues, and Inventory 1/Species Within 30 days of submission of 3- Month Reports

Study Raw Data Archival Records for 3-Month Study and Electronic Submission of Results

1/Species Within 30 days of submission of 3- Month Reports

V. Conduct of Studies to Evaluate Chronic Toxicity of Test Agent Y Administered in Feed to Rats (Following Perinatal Exposure) and Adult Mice

The primary concern for human exposure to test agent Y is related to contamination of food stuffs due to being a degradation byproduct of the common food additive XYZ in shelf stable foods. In addition to standard chronic toxicology and carcinogenicity endpoints, this study includes the collection and distribution of samples to multiple NIH-funded extramural investigators.

B. General Information

1. Project Management All studies shall be conducted in accordance with GLPs and the Specifications for the Conduct of

Phase 1. During this phase the contractor shall perform those work activities associated with bulk test agent reanalysis, formulation development and validation, and stability testing. Submission and approval of a prestart chemistry report is required for completion of Phase 1.

Phase 2: Upon completion of Phase 1, the chronic (6-month) studies shall be conducted.

system

• Bulk test agent

• Identity and purity analysis of the bulk test agent, and bulk reanalysis methods

C. Phase 1- Prestart Activities Receive the test agent, perform bulk reanalysis according to the methods provided.

Develop and validate formulation methods at the specified concentrations.

Prepare and submit a Prestart Chemistry Report per the DTT Specifications.

Upon successful completion and approval of Phase 1 (including COR review of the Prestart Chemistry Report), the contractor shall conduct work specified as Phase 2.

D. Phase 2- Chronic (6-month) Study (In-life)

1. Gestation Time-mated (presumed pregnant) female Sprague Dawley (Hsd:Sprague Dawley® SD®) rats shall be 11-14 weeks of age and 200-225g at receipt (gestation day 2, GD 2), housed individually, and provided NIH-07 diet, ad libitum. In addition, 10 age-matched non-pregnant females shall be received and used for disease screening prior to exposure of the time-mated animals and four weeks after arrival; samples shall include blood, fur wipe and feces at both time points and necropsy at the four-week time point. On GD 3 dams shall be randomly assigned to exposure and control groups and uniquely identified by tail tattoo.

Time-Mated Damsa Groups Total

Pregnant Dams 31 X 4b = 124 a These numbers are based on the assumption that ~85% of time-mated dams will produce acceptable litters (at least 2/sex with a total litter size of 8). If additional information is available at the time of animal order, these numbers shall be revised.

b 3 exposure groups and a control

At each of three concentrations plus a control group [0, 10, 100, 300 PPM] dams shall receive the test article via dosed feed (NIH-07) ad libitum starting on GD 6 and continuing until postnatal day (PND) 28. Dams shall be weighed on GD 5 prior to exposure and then on GD 6, 9, 12, 15, 18 and

21. Starting on GD 6 food consumption shall be measured every three days during gestation.

All dams shall be observed two times daily, once in the early morning and once in the late afternoon, at least six hours apart (before 10:00 AM and after 2:00 PM), including holidays and weekends, for signs of delivery, moribundity, and mortality. Formal clinical observations shall be performed and recorded daily from GD 6 – GD 21. During the gestation and lactation exposure periods, any unexpected toxicity (such as mortality, body weight change, clinical signs in dams or pups) that may impact the continuation of the study shall be reported immediately. Moribund dams and dams that do not deliver within 3-4 days of the anticipated delivery date shall be euthanized via 100% CO2 inhalation and death confirmed by a secondary method such as thoracotomy/exsanguination; all dams shall be examined for uterine implantations/resorptions (including early resorptions), and these findings recorded. The duration of pregnancy and number of pregnant females per group shall be recorded and reported.

a) GD 18 Internal Dose Assessment

On GD 18, randomly selected dams (n=3/group) shall be euthanized and maternal plasma isolated from trunk blood; blood shall be collected in K3 EDTA and kept on wet ice until plasma isolation.

Blood collection shall occur immediately after lights-on and all samples on a given day shall be collected within a 2-hour time-period and each sample shall be frozen within 2 hours of collection;

the time of blood collection shall be recorded for each animal.

− Plasma and amniotic fluid (pooled by litter) shall be saved in aliquots of 125µL, up to 5 aliquots/matrix/animal; no aliquot less than 125µL should be saved.

− Fetuses (entire litter, 2-3 fetuses/vial) shall also be collected. The number of fetuses and total weight for each litter shall be recorded.

Plasma, amniotic fluid and fetuses shall be flash frozen in liquid nitrogen and stored at -70°C.

Tissues and plasma shall be shipped to a DTT-designated analytical chemistry laboratory. The resulting data will not be returned and are not to be included in the study reports.

2. Lactation The lactation period includes the period from the day of delivery (PND 0) until PND 28, when the animals are weaned. Dams and individual pups shall be weighed on PND 1, 4, 7, 10, 13, 16, 19, 21, 25 and 28. Number of live and dead pups and the sex, and weight of individual pups shall be recorded on PND 0 (number of live and dead and sex of pups only), 1, 4, 7, 10, 13, 16, 19, 21, 25 and 28; litter weights by sex and total shall also be recorded for PND 1. Food consumption measurements shall be made on PND 4, 7, 10, 13, 16, 19, 21, 25 and 28 for the periods covered.

All dams and pups shall be observed two times daily, once in the early morning and once in the late afternoon, at least six hours apart (before 10:00 AM and after 2:00 PM), including holidays and weekends, for morbidity, mortality, or abnormal maternal behavior. Pups shall be uniquely identified by paw tattoo on PND 0. Anogenital distance and corresponding pup weight shall be collected on all pups on PND1.

Formal clinical observations shall be performed in dams and pups daily throughout the lactation period starting on PND 1.

On PND 4, litters of greater than 8 pups shall be culled by random selection, to 8 pups, with as close to 4 males/4 females as achievable. Dams in the control group shall be used for disease screening at terminal sacrifice.

a) PND 4 Internal Dose Assessment

On PND 4, randomly selected dams (n=3/group) and their pups shall be euthanized and used for biological sampling.

− Maternal plasma shall be isolated from trunk blood. Blood collection shall occur immediately after lights-on and all samples on a given day shall be collected within a 2-hour time frame. The time of blood collection is to be recorded for each animal. Blood shall be collected in K3 EDTA and kept on wet ice until plasma isolation, within 60 minutes of collection. Plasma shall be saved in aliquots of 125µL, up to 5 aliquots per animal; no aliquot less than 125µL should be saved. Plasma shall be flash frozen within 2 hours of collection in liquid nitrogen and stored at -70°C.

− Pup plasma shall be saved in a single aliquot per pup, target of 125µL. Plasma shall be collected and treated as described above for maternal plasma.

Plasma samples shall be shipped to an DTT-designated analytical chemistry laboratory. The resulting data will not be returned and are not to be included in the study reports.

b) PND 28 Cohort Assignment and Weaning

On PND 28, pups shall be assigned to the following cohorts as described.