Att6_Sample Research Outlines.docx

Sample Research Outlines I. In Vitro Screening Studies to Evaluate Immune Suppression

A. Background Immunotoxicity is used to describe the potential of an agent to alter the normal structure and/or function of the immune system as determined by established immunological techniques. In cases of immunomodulation, a chemical or physical agent may alter hematopoietic or immunological function in humans and animals, resulting in changes in the ability to recognize and eliminate threats such as self-reactive or cancerous cells. To evaluate whether chemical or therapeutic agents can modulate the immune responses, a panel of in vitro assays is used to provide functional and quantitative information on humoral, cell-mediated, and innate immunity.

As an example of how in vitro immunotoxicity screening for immune suppression may be conducted offerors are referred to human whole blood culture systems such as the commercially available TruCulture® (Myriad RBM) closed culture tube (see figure below). This culture system incorporates a medium optimized for maintenance of human whole blood together with a simple approach for separation of cells from media using a plunger. The system is closed and does not require an incubator with CO2/humidity control.

This sample research outline is written as a list of objectives rather than specific requirements, and offerors shall review the background material referenced here and in Section C, along with any other related and relevant literature. The TruCulture® system described above is provided as an example only and is not a requirement. Offerors may propose other assay systems that achieve the objectives outlined in Section D below. For Section D, offerors shall provide a detailed technical approach to accomplish the goals of the proposed research.

B. Project Management and Government Furnished Materials Offerors shall prepare technical approach and estimated budget proposals detailing how the work described in this research outline will be performed, including staffing plan, milestones/deliverables, schedule, budget, level of effort, and oversight.

This in vitro work need not be conducted under Good Laboratory Practice (GLP) regulations, however, offerors shall reference and utilize the Guidance Document on Good in Vitro Method Practices (https://www.oecd.org/env/guidance-document-on-good-in-vitro-method-practices-givimp-9789264304796-en.htm) for the performance of these studies.

Study-specific Government-furnished materials will be specified in a study-specific research outline. For the purposes of this sample research outline, offerors shall assume that the below materials will be provided by the Government:

· Government-furnished Materials:

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

· DTT templates for raw data deliverables

For the purposes of this sample research outline, offerors shall assume that all test agents will be purchased under the contract.

C. In Vitro Assays Offerors shall describe, in detail, their approach to conducting the evaluations described in their proposed culture system. Offerors should propose assay systems that achieve the objectives outlined in Section D below and have the ability to investigate the effects of chemicals on human primary leukocytes. The system should have the flexibility to assess both stimulated and unstimulated cells. Offerors should use a minimum of 8 donors per chemical assessment and at least three concentrations of test article as well as appropriate controls for each donor. All test agents shall be purchased under the contract, and offerors shall outline health and safety and chemical handling considerations in their technical approach.

Offerors shall propose examples of human whole blood assays that address the endpoints required as described in the Statement of Work. Human whole blood shall be purchased from a commercial vendor and should be collected from fully consented Institutional Review Board (IRB) approved donors that are tested negative for HIV1/2, HBV, HCV, and syphilis and collected under conditions that enable use in cell culture.

D. Evaluations and Endpoints Endpoints to be evaluated shall include:

· A panel of ten proinflammatory and growth promoting cytokines

· Enumeration of leukocyte subpopulations including B cells, T cells, T cell subsets, monocytes, natural killer (NK) cells, and polymorphonuclear leukocytes

· Assessment of chemical-induced cytotoxicity

· Functional evaluation of NK cell activity

· Functional assessment of cell-mediated immunity in control (unstimulated) cells and after stimulation with one human-relevant antigen

· Potential stimulants include:

· Lipopolysaccharide (LPS)

· Anti-CD3 antibody

· Anti-CD3 antibody + anti-CD28 antibody

· Staphylococcal enterotoxin type B

· Zymosan The use of stimulants such as viral peptides (cytomegalovirus, Epstein-Barr virus, influenza, measles) is encouraged and should be optimized for the assay system in the offeror’s laboratory to provide greater human health relevance endpoints and the questions to be addressed. The offeror should propose methods to evaluate neutralizing antibodies when viral peptides are used to stimulate the immune response.

A primary endpoint for the assay system shall be multiplex quantitation of secreted cytokines and chemokines that may be assessed using the supernatant fraction. Cytokine levels in the culture supernatants shall be measured using a multiplex ELISA system and should evaluate cytokines associated with inflammation, lymphocyte activation, etc. Offerors should propose an appropriate cytokine panel that provides quantitative information on the levels of a minimum of ten cytokines in the cultured samples.

To facilitate interpretation of the cytokine data, the cytotoxic effects of the test compounds should be determined simultaneously from the same cultures. The cellular fraction of the culture system shall be used to determine cytotoxicity using a viability dye and flow cytometry.

Flow cytometry based immunophenotyping of the same white blood cell populations shall be conducted to determine immune cell types that are susceptible to the immunotoxicity of the test compound. The offeror shall propose an appropriate panel of antibodies to be used to quantitate lymphoid and myeloid cell populations in the cellular fraction.

The offeror shall evaluate killing activity of the NK cell population from the cultures via a direct target cell killing assay. The cellular fraction should be used as a source of the effector cell population and the offeror should propose an appropriate target and assay system to assess cell killing.

E. Study Schedule and Output Requirements

Study Raw Data1, Archival Records, and Electronic Submission of Results

1Per established templates

II. Screening Studies to Evaluate the Potential of Lycopene (CASRN: 502-65-8) to Induce Developmental Immunotoxicity in HSD:Sprague Dawley SD Rats Following Oral Administration

A. Background and Study Rationale This sample research outline is meant to be an example of how Division of Translational Toxicology (DTT) developmental immunotoxicology studies may be organized and conducted. The rat is one major species of choice in conducting toxicological and immunological studies. The included tests will evaluate humoral immunity, cell-mediated immunity, and innate immunity as well as quantitative measures of the immune system in rats following perinatal exposure to lycopene. Additional included endpoints will evaluate general reproductive outcomes following perinatal exposure to lycopene.

Lycopene:

Chemical formula: C40H56, MW: 536.87

This sample research outline is written as a list of objectives rather than specific requirements, and offerors shall review the background material referenced in this Section, along with any other related and relevant literature. For Sections C and D, offerors shall provide a detailed research plan and technical approach to accomplish the goals of the proposed research.

The offeror shall prepare technical approach and estimated budget proposals describing how the work outlined in this research outline will be performed, including staffing plan, milestones/deliverables, schedule, budget, level of effort, and oversight.

This study is to be conducted according to the "Specifications for the Conduct of Toxicity Studies by the Division of Translational Toxicology at the National Institute of Environmental Health Sciences" dated March, 2023 (DTT Specifications; https://doi.org/10.22427/NIEHS-00). The study shall be performed in accordance with the Good Laboratory Practice (GLP) regulations established by the U.S. Food and Drug Administration (FDA) for Non-clinical Laboratory Studies (21 CFR 58; https://www.ecfr.gov/current/title-21/chapter-I/subchapter-A/part-58) or as most recently updated.

Study-specific Government-furnished materials will be specified in a study-specific research outline. For the purposes of this sample research outline, offerors shall assume that the below materials will be provided by the Government:

· Government-furnished Materials:

· Dose formulations

· Pre-administration analysis chemistry reports detailing results of identity/purity screening of the test articles, preparation and analysis of dose formulations, and analysis of animal room samples

· Access, system support, and training for a Government-specified laboratory information management system (LIMS) (currently a NIEHS-leased version of Provantis)

· Templates for collection and submission of non-Provantis data C. Developmental Immunotoxicity Study

1. Perinatal Exposure and Dose Administration I. Gestation Time-mated (presumed pregnant) female Sprague Dawley (Hsd:Sprague Dawley® SD®) rats shall be 11-14 weeks of age and 200-255g at receipt (gestation day [GD] 2), housed individually, and provided NIH-07 diet (vendor specified by DTT) ad libitum. The animal vendor will be specified by DTT. In addition, four age-matched 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 treatment and control groups and uniquely identified by tail tattoo.

Dams

Groups

Total

Total

CPS = cyclophosphamide.

aNumber of pregnant dams listed is the minimum number of dams and litters needed to populate the study. Based on experience of pregnancy rates and litter sizes, the Contractor shall order the appropriate number of time-mated dams.

bTen dams will not be treated and will be used to provide age- and gender-matched positive controls.

cThree dams per group will be utilized for biosampling at GD 18 and PND 4.

dFour additional pregnant dams will be used to provide untreated pups (5M/5F) for disease screening at PND 28 and at study termination.

Doses, vehicle, and route of exposure will be selected based on known toxicity data. Dosing solutions will be formulated and provided by DTT. For the purposes of this research outline, assume the route of exposure will be oral gavage. At three doses [D1, D2, and D3 mg/kg (see below); 0.1 mL/20 g dosing volume] and a vehicle control group (0 mg/kg), dams shall be dosed daily via oral gavage beginning on GD 6 and continuing through postnatal day (PND) 4.

Cyclophosphamide (CPS) will be given as a positive control for all assays unless otherwise specified. The positive control animals will receive 50 mg/kg of CPS on the last 4 days of the exposure period by intraperitoneal injection. Positive control animals will receive 0.1 ml/20 grams of body weight. Dams shall be weighed on GD 5 prior to exposure, and then on GD 6, 9, 12, 15, 18, and 21.

All dams 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 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.

For any unscheduled deaths, gross necropsy examination of major organs (brain and thoracic and abdominal viscera) will be performed in accordance with the DTT Specifications to assess for cause of death or morbidity. Typically, no histopathology will be conducted on these animals; however, the Government may request tissues be saved for histopathology evaluation in select situations.

On GD 18, randomly selected dams from the vehicle control and dosed groups (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, 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 125 uL, up to 5 aliquots/matrix/animal.

· 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 then stored at -70°C. Tissues and plasma shall be shipped to a DTT-designated analytical chemistry contractor. The resulting data will not be returned and are not to be included in the study reports.

II. 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, 13, 21, and 28. The number of live and dead pups, the sex, and the weight of individual pups shall be recorded on PND 0 (number of live and dead and sex of pups only), 1, 4, 7, 13, 21, and 28; litter weights by sex and total shall also be recorded for PND 1. 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. Formal clinical observations shall be performed in dams and pups daily throughout the lactation period starting on PND 1.

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 and pup plasma shall be collected from five pups/sex/litter; blood shall be collected in K3 EDTA and kept on wet ice until plasma isolation, within 60 minutes of collection. 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. Plasma shall be saved in aliquots of 125 uL, up to 5 aliquots per dam and/or pups; no aliquot less than 125 uL should be saved. Plasma shall be flash frozen within 2 hours of collection in liquid nitrogen and stored at -70°C.

Plasma samples from pups and dams shall be shipped to a DTT-designated analytical chemistry contractor. The resulting data will not be returned and are not to be included in the study reports.

2. PND 4 Cohort Assignment and Weaning On PND 4, F1 pups shall be assigned to a cohort (assay cohort 1-4, 10/sex/dose group). Within a dose group, no more than 1 pup/sex/litter shall be selected for each cohort. Beginning on PND 4 (when dosing of dams is discontinued) F1 pups shall be dosed daily through PND 42 (PND 4 – PND 42).

Each vehicle and lycopene treatment group will consist of ten F1 animals/sex/assay cohort. The positive control group will consist of 8 animals. The treatment groups and their designations are shown below in the table.

TOTAL

aMale and female pups from the vehicle control group that are not assigned to specific assay cohorts may be used for health surveillance and will be housed in accordance with the study research outline; samples shall include blood, fur wipe, and feces within 7 days of study termination.

On each litter’s respective PND 28, animals shall be weaned and continue to be dosed until PMD 42. After weaning, F1 pups shall be housed up to 5/sex/group and provided NTP-2000 diet ad libitum (vendor to be specified by DTT). Animals will be evaluated for the appropriate immunological and toxicological parameters at terminal euthanasia on PND 42.

After weaning, animals shall be observed twice daily, once in the morning and once in the late afternoon, at least six hours apart (before 10:00AM and after 2:00PM), for moribundity and mortality. Signs of toxicity noticed during these routine checks shall be recorded. Formal clinical observations shall be performed and recorded daily for the first week and twice weekly thereafter. After weaning, all rats shall be weighed individually on day one on test, twice weekly and at necropsy.

On the day of euthanasia, a necropsy will be performed by trained personnel in accordance with the DTT Specifications and gross lesions will be retained. Terminal body weights and organ weights (spleen, thymus, liver, lungs, and kidneys with adrenals) will be recorded as described in the table of measured parameters below. Tissues and carcasses will be discarded without further evaluation.

For any unscheduled deaths, gross necropsy examination of major organs (brain and thoracic and abdominal viscera) will be performed in accordance with the DTT Specifications to assess for cause of death or morbidity. Typically, no histopathology will be conducted on these animals; however, the Government may request tissues be saved for histopathology evaluation in select situations.

3. Immunology and Toxicology Assays Specific parameters to be evaluated in each assay group are described below. All assays will be conducted at the contract laboratory in accordance with their standard operating procedures (SOPs).

Activity Anti-CD3 T Cell Proliferation

Lymphocyte Assay

Additional information on the immunology assays and parameters is provided below:

· Cohort 1

· IgM Antibody Forming Cell (AFC) Assay: Rats shall be immunized with sheep red blood cells (SRBCs; 1 x 108 cells/rat) four days prior to scheduled euthanasia. At study termination, spleen single cell suspensions shall be evaluated for the impact of lycopene treatment on the AFC response specific for SRBCs. In addition, the same spleen single cell suspensions shall be evaluated for the impact of lycopene treatment on B-cell production of IgM antibodies specific for SRBC.

· Antigen-specific Serum IgM Measurement: At study termination, serum samples from individual animals used for the AFC assay shall be evaluated for anti-SRBC specific IgM antibodies via ELISA.

· Cohort 2

· Cell Quantification: At study termination, spleen single cell suspensions shall be evaluated for the impact of lycopene treatment on immune cell populations in the spleen. Leukocyte subpopulations shall be enumerated, including B cells, T cells, T cell subsets, monocytes, NK cells, and polymorphonuclear leukocytes.

· Natural Killer (NK) Cell Activity: Spleen single cell suspensions shall be evaluated for the impact of lycopene treatment on NK cell killing activity toward tumor target cells.

· Anti-CD3 T Cell Proliferation: At study termination, spleen single cell suspensions shall be evaluated for the impact of lycopene treatment on T cell proliferation in response to ex vivo treatment with anti-CD3 antibodies.

· Cohort 3

· Mononuclear Phagocyte Assessment: At study termination, all surviving animals shall be euthanized by CO2 inhalation and then weighed. The diaphragm shall be severed to confirm death. The spleen and thymus weights shall be recorded. Lungs shall be removed, weighed, and the mononuclear cell population shall be isolated via collagenase digestion. Isolated bronchoalveolar lavage fluid (BALF) mononuclear cells shall be incubated with fluorescent particles and phagocytic index calculated based on the number of particles taken up by phagocytic cells and the fluorescent intensity of ingested material.

· Organ Weights: At study termination, the spleen, thymus, liver, right kidney, and right adrenal shall be removed and weighed.

· Hematology: Blood shall be collected from the retro-orbital plexus under 70% CO2:30% O2 anesthesia at terminal sacrifice for hematology; hematology shall be analyzed according to the DTT Specifications.

· Cohort 4

· Cytotoxic T Lymphocyte (CTL) Assay: Rats shall be immunized with rat-adapted influenza virus (RAIV; approximately 2 x 105 plaque forming units) eight days prior to scheduled euthanasia. At study termination, lung single cell suspensions shall be evaluated for the impact of lycopene treatment on CTL killing activity towards target cells.

4. Dose Formulation Analysis Duplicate samples (1 mL) of vehicle and test article dose formulations will be collected on the first and last day of test article administration. One aliquot will be stored at by the Contractor as a backup and the remaining samples will be shipped to a DTT-designated chemistry contractor(s) for concentration analysis. Positive control dose formulations will be analyzed for concentration by the same DTT designated chemistry contractor that performed the dose formulation analysis for the positive control. A 1 mL sample of the positive control dose formulations will be collected from an aliquot used during the last week of dosing and stored at the contract laboratory as a backup. The remaining volume (at least 1 mL) will be shipped, in the original shipping container, to the appropriate DTT chemistry contractor(s) for concentration analysis. The samples will be shipped via same-day or overnight courier on ice packs. A copy of the signed final chemistry contributor phase report (including dose formulation procedures, dose concentration analysis procedures, dose concentration analysis results, and animal room sample analysis results) will be provided to the Contractor to be included as an appendix to the main test article report.

D. Study Schedule and Output Requirements

Study Raw Data, Archival Records, and Electronic Submission of Results

III. Proof-of-Concept: Development and Evaluation of Novel Tools and Methods to Assess Immunotoxicity In Vitro

A. Background Part of DTT’s mission is to lead the transformation of toxicology through the development and application of innovative tools and methods. In support of that mission, DTT is interested in expanding the use of novel approaches in in vitro assessments of immunotoxicity that advance the application, validation, and regulatory acceptance of new approach methodologies. Assessment of new strategies to evaluate perturbations in the developing immune system is a DTT priority. Studies to help establish cellular and/or molecular mechanisms of action for immune perturbations observed following test article exposure may be appropriate. Development of novel screening strategies including the use of molecular techniques such as analysis of gene expression to assess xenobiotic-induced immunologic alterations would be also desirable.

This sample research outline is written as a list of objectives rather than specific requirements, and offerors shall review the background material referenced in this document and the SOW, along with any other related and relevant literature. For Section B, offerors shall provide a detailed research plan and technical approach to accomplish the goals of the proposed research.

B. Proof-of-Concept Research Project The goal of this research outline is to describe a proof-of-concept approach to developing and evaluating novel tools and methods to assess immunotoxicity in vitro. With the ongoing effort to reduce the use of animals in toxicity testing, the development of in vitro models to assess all aspects of toxicity is extremely important to the field of toxicology. Offerors shall describe, in detail, their proposed research plan and technical approach to accomplish the following goals:

· Identify areas within in vitro immunotoxicity testing where novel approaches could improve efficiency and reduce the use of animals in toxicity testing.

· Propose methods for evaluating, developing, and implementing novel approaches for in vitro immunotoxicity assessments.

· Propose steps for method validation and verification.

· Describe how quality control would be ensured.

C. Research Schedule and Output Requirements

IV. Screening Studies to Evaluate the Potential of Lycopene (CASRN: 502-65-8) to Induce Immunotoxicity in B6C3F1 Mice Following Oral Administration

This sample research outline is being included solely for the purpose of allowing offerors to demonstrate technical approach. Costing of this sample research outline is not required as part of the business proposal preparation.

A. Background and Study Rationale This sample research outline has been provided for technical purposes only and is not part of the business proposal instructions. It is meant to be an example of how DTT studies may be organized and conducted. The mouse is one major species of choice in conducting toxicological and immunological studies. The included tests will evaluate humoral immunity, cell-mediated immunity, and innate immunity as well as quantitative measures of the immune system in mice orally administered lycopene.

Lycopene:

Chemical formula: C40H56

MW: 536.87

This sample research outline is written as a list of objectives rather than specific requirements, and offerors shall review the background material referenced in this Section, along with any other related and relevant literature. For Sections C and D offerors shall provide a detailed research plan and technical approach to accomplish the goals of the proposed research.

Offerors shall prepare a technical approach proposal describing how the work outlined in this sample research outline will be performed, including staffing plan, milestones/deliverables, schedule, and oversight.

This study is to be conducted according to the "Specifications for the Conduct of Toxicity Studies by the Division of Translational Toxicology at the National Institute of Environmental Health Sciences" dated March, 2023 (DTT Specifications; https://doi.org/10.22427/NIEHS-00). The study shall be performed in accordance with GLP regulations established by the U.S. Food and Drug Administration (FDA) for Non-clinical Laboratory Studies (21 CFR 58; https://www.ecfr.gov/current/title-21/chapter-I/subchapter-A/part-58) or as most recently updated.

Study-specific Government-furnished materials will be specified in a study-specific research outline. For the purposes of this research outline, offerors shall assume that the below materials will be provided by the Government:

· Government-furnished Materials:

· Dose formulations

· Pre-administration analysis chemistry reports detailing results of identity/purity screening of the test articles, preparation and analysis of dose formulations, and analysis of animal room samples

· Access, system support, and training for a Government-specified laboratory information management system (LIMS) (currently a NIEHS-leased version of Provantis)

· Templates for collection and submission of non-Provantis data C. Immunotoxicity Study

1. Dose Administration Female B6C3F1 mice (vendor specified by DTT) shall be placed on the NTP-2000 diet (vendor specified by DTT) unless otherwise specified and housed up to 4 animals per cage. When mice are 6-8 weeks old or approximately 20 grams, they shall be randomly assigned to treatment and control groups and uniquely identified by tail tattoo.

Animals

Groups

Total

Total

CPS = cyclophosphamide.

a32 mice will not be treated and will be used to provide age- and gender-matched positive controls.

bThree mice per group will be utilized for biosampling at study termination.

c14 naïve female mice will used to provide untreated animals for disease screening (7 females 5-7 days after receipt, 7 females 5-7 days after study termination).

Doses, vehicle, and route of exposure will be selected based on known toxicity data. Dosing solutions will be formulated and provided by DTT. For the purposes of this research outline, assume the route of exposure will be oral gavage. At five doses [D1, D2, D3, D4, and D5 mg/kg (see below); 0.1 mL/20 g dosing volume] and a vehicle control group (0 mg/kg), mice shall be dosed daily via oral gavage for 28 days.

Cyclophosphamide (CPS) will be given as a positive control for all assays unless otherwise specified. The positive control animals will receive 50 mg/kg of CPS on the last 4 days of the exposure period by intraperitoneal injection. Positive control animals will receive 0.1 ml/20 grams of body weight.

2. Observations All mice shall be observed twice daily, once in the early morning and once in the late afternoon, at least six hours apart (before 10:00AM and after 2:00PM), 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 for the first week and twice weekly thereafter. All mice shall be weighed individually on the first day of exposure, on day 8, 15, 22, and day 29, the day of scheduled euthanasia.

3. Cohort Assignment Prior to the onset of dosing, mice shall be randomly assigned to a cohort (assay cohort 1-5, eight/dose group). Each vehicle, lycopene, and positive control group will consist of eight mice/assay cohort. The treatment groups and their designations are shown below in the table.

TOTAL

Animals will be evaluated for the appropriate immunological and toxicological parameters at terminal euthanasia. on day 29.

On the day of euthanasia, a necropsy will be performed by trained personnel in accordance with the DTT Specifications and gross lesions will be retained. Terminal body weights and organ weights (spleen, thymus, liver, lungs, and kidneys with adrenals) will be recorded as described in the table of measured parameters below. Tissues and carcasses will be discarded without further evaluation.

For any unscheduled deaths, gross necropsy examination of major organs (brain and thoracic and abdominal viscera) will be performed in accordance with the DTT Specifications to assess for cause of death or morbidity. Typically, no histopathology will be conducted on these animals; however, the Government may request tissues be saved for histopathology evaluation in select situations.

4. Immunology and Toxicology Assays Specific parameters to be evaluated in each assay group are described below. All assays will be conducted at the contract laboratory in accordance with their SOPs.

Activity

Additional information on the immunology assays and parameters is provided below:

· Cohort 1

· IgM Antibody Forming Cell (AFC) Assay: Mice shall be immunized with sheep red blood cells (SRBCs; 7.5 x 107 cells/mouse) four days prior to scheduled euthanasia. At study termination, spleen single cell suspensions shall be evaluated for the impact of lycopene treatment on the AFC response specific for SRBCs. In addition, the same spleen single cell suspensions shall be evaluated for the impact of lycopene treatment on B-cell production of IgM antibodies specific for SRBC.

· Antigen-specific Serum IgM Measurement: At study termination, serum samples from individual animals used for the AFC assay shall be evaluated for anti-SRBC specific IgM antibodies via ELISA.

· Cohort 2

· Cell Quantification: At study termination, spleen single cell suspensions shall be evaluated for the impact of lycopene treatment on immune cell populations in the spleen. Leukocyte subpopulations shall be enumerated, including B cells, T cells, T cell subsets, monocytes, NK cells, and polymorphonuclear leukocytes.

· Anti-CD3 T Cell Proliferation: At study termination, spleen single cell suspensions shall be evaluated for the impact of lycopene treatment on T cell proliferation in response to ex vivo treatment with anti-CD3 antibodies.

· Hematology: Blood shall be collected from the retro-orbital plexus under 70% CO2:30% O2 anesthesia at terminal sacrifice for hematology; hematology shall be analyzed according to the DTT Specifications.

· Cohort 3

· Natural Killer (NK) Cell Activity: Spleen single cell suspensions shall be evaluated for the impact of lycopene treatment on NK cell killing activity toward tumor target cells.

· Organ Weights: At study termination, the spleen, thymus, liver, right kidney, and right adrenal shall be removed and weighed.

· Cohort 4

· Cytotoxic T Lymphocyte (CTL) Assay: Mice shall be immunized with mouse-adapted influenza virus (MAIV; approximately 4 x 104 plaque forming units) eight days prior to scheduled euthanasia. At study termination, lung single cell suspensions shall be evaluated for the impact of lycopene treatment on CTL killing activity towards target cells.

· Cohort 5

· Biosampling: Two hours after the last dose for the selected mice from the vehicle control and dosed groups (n = 3/group), animals shall be euthanized by CO2 inhalation and have plasma collected. All individual animals shall be sampled at 2 hours after the respective dose and samples from all animals shall be collected within a 2-hour time period. Blood shall be collected in K3 EDTA and kept on wet ice until plasma isolation, within 60 minutes of collection. The time of blood collection shall be recorded for each animal. Plasma shall be saved in aliquots of 125 uL, up to 5 aliquots/animal; no aliquot less than 125 uL should be saved. Plasma shall be flash frozen in liquid nitrogen within 2 hours of collection and stored at -70°C.

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

5. Dose Formulation Analysis Duplicate samples (1 mL) of vehicle and test article dose formulations will be collected on the first and last day of test article administration. One aliquot will be stored at by the Contractor as a backup and the remaining samples will be shipped to a DTT-designated chemistry contractor(s) for concentration analysis. Positive control dose formulations will be analyzed for concentration by the same DTT designated chemistry contractor that performed the dose formulation analysis for the positive control. A 1 mL sample of the positive control dose formulations will be collected from an aliquot used during the last week of dosing and stored at the contract laboratory as a backup. The remaining volume (at least 1 mL) will be shipped, in the original shipping container, to the appropriate DTT chemistry contractor(s) for concentration analysis. The samples will be shipped via same-day or overnight courier on ice packs. A copy of the signed final chemistry contributor phase report (including dose formulation procedures, dose concentration analysis procedures, dose concentration analysis results, and animal room sample analysis results) will be provided to the Contractor to be included as an appendix to the main test article report.

D. Study Schedule and Output Requirements

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