Amend 2 Attachment 2 - Sample Protocols for In Vivo.pdf

TECHNICAL EXHIBIT #A

Sample Protocol Outline for Screening Studies to Evaluate the Potential of Lycopene (CASRN: 502-65-8, Test Article Number IXXXX) to Induce

Immunotoxicity in B6C3F1/N Mice Following Oral Administration

This sample protocol has been provided for costing purposes and is meant to be an example of how Division of Translational Toxicology (DTT) studies may be organized and conducted:

Sample Protocol Outline for Screening Studies to Evaluate the Potential of Lycopene (CASRN: 502-65-8, Test Article Number IXXXX) to Induce Immunotoxictiy in B6C3F1/N Mice Following Oral Administration

Chemical formula: C40H56

MW: 536.87

Background and Study Rationale –

The included tests will evaluate humoral immunity, cell-mediated immunity, and innate immunity as well as quantitative measures of the immune system.

Animals – Female B6C3F1/N mice will be used for this study. The mouse is one major species of choice in conducting toxicological and immunological studies. Animals for these studies will be placed on test at approximately 6-8 weeks of age or approximately 20 grams. Mice will be individually identified by tail tattoo and housed 4 animals per cage in plastic shoebox cages with hardwood bedding. Mice will be maintained on the NTP- 2000 diet unless otherwise specified.

The study will consist of 4 assay groups (as described below) X 8 animals per group X 7 treatments (vehicle, positive control and 5 dose groups = approximately 250 animals with required sentinels).

Parameters Measured

Assay Group No.

1 2 3 4

1 IgM Antibody Forming Cell

Assay

Antigen specific Serum IgM

Body Weights

2 Cell Quantification

Natural Killer Cell Activity

Anti-CD3 T Cell

Body Weights

Proliferation & Mixed

Leukocyte Response

3 Mononuclear Phagocyte

Assessment

Organ Weights Body Weights Hematology

4 Cytotoxic T Lymphocyte

Assay

Treatment – Lycopene (CASRN 502-65-8) will be obtained from DTT. The compound will be administered daily for 28 days via oral gavage at concentrations to be provided by DTT. Doses, vehicle, and route of exposure will be selected based on known toxicity data.

Dosing solutions will be prepared in accordance with information specified by DTT.

Dosing volume will be 0.1 ml/20 grams of body weight. The animals will be evaluated for the appropriate immunological and toxicological parameters at the terminal sacrifice on day 29.

Positive controls, appropriate for specific immunologic endpoints will be used in this study.

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.

Following randomization, animals will be assigned to one of the following treatment groups. Each vehicle and lycopene treatment group will consist of 8 animals. The positive control groups will consist of 8 animals per group. The treatment groups and their designations are shown below in the table:

Group Treatment Designation 1 Vehicle VH 2 Lycopene D1 D1 3 Lycopene D2 D2 4 Lycopene D3 D3 5 Lycopene D4 D4 6 Lycopene D5 D5

7 PC PC

All assays will be conducted at the contract laboratory in accordance with their SOPs.

Endpoints to be Evaluated

1. General

a. Body Weight: Animals will be weighed on the first day of exposure, on day 8, 15, 22, and day 29, the day of sacrifice.

b. Observations: Animals will 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 any unexpected toxicity.

Any remarkable effects will be recorded on the log or activity sheet, present in the exposure room.

c. 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. The DTT COR will be notified of excessive morbidity or mortality and the COR may request tissues be saved for histopathology evaluation in select situations.

2. Standard Toxicology Endpoints

a. Scheduled Sacrifice: On the day of sacrifice, a necropsy will be performed by trained personnel in accordance with the DTT Specifications. Terminal body weights and organ weights (spleen, thymus, liver, lungs, and kidneys with adrenals) will be recorded.

b. Hematology (erythrocytes, leukocytes, hematocrit, hemoglobin, MCV, MCH, MCHC, platelets and reticulocytes) and leukocyte differentials.

3. Immunology Assays

a. Spleen IgM antibody response to a T-dependent antigen, sheep erythrocytes (Day 4 response).

b. Cell Quantification: T cell and T subsets & B cell enumeration, NK cells and monocytes.

c. Natural killer cell assay.

e. Spleen cell proliferative response to anti-CD3-mediated T cell proliferation.

f. Mouse serum IgM ELISA using sRBC Antigen. Serum from mice used for the

AFC assay will be evaluated for the primary IgM levels.

g. Cytotoxic T-cell response.

h. Mixed leukocyte response to DBA/2 spleen cells (day 5).

i. Mononuclear phagocyte assessment (phagocytosis, cytokine secretion)

TECHNICAL EXHIBIT #B

Sample #2: Protocol Outline for Screening Studies to Evaluate the Potential of

Lycopene (CASRN: 502-65-8, Test Article Number IXXXX) to Induce Developmental Immunotoxicity in HSD Rats Following Oral Administration

This sample protocol has been provided for costing purposes and is meant to be an example of how developmental immunotoxicology studies may be organized and conducted:

Sample Protocol Outline for Screening Studies to Evaluate the Potential of Lycopene (CASRN: 502-65-8, Test Article Number IXXXX) to Induce Developmental Immunotoxictiy in HSD Rats Following Oral Administration

Chemical formula: C40H56, MW: 536.87

Background and Study Rationale –

The included tests will evaluate humoral immunity, cell-mediated immunity, and innate immunity as well as quantitative measures of the immune system. Additional assessments will evaluate general reproductive outcomes following perinatal exposure to lycopene.

Animals – Harlan Sprague Dawley rats will be used for this study. The rat is one major species of choice in conducting toxicological and immunological studies. 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 post-natal day (PND) 4.

Pups shall be dosed daily when dosing of the dams is discontinued on PND 4 and continued generally through PND 42. The F1 study will consist of 4 assay groups (as described below) X 20 animals per group (10 males and 10 females) X 5 treatments (vehicle, positive control and 3 dose groups = approximately 400 animals with required sentinels).

Parameters Measured

Group No.

1 2 3 4

1 IgM Antibody Forming Cell

Assay

Antigen specific Serum IgM

Body Weights

2 Cell Quantification

Natural Killer Cell Activity

Anti-CD3 T Cell Proliferation

3 Mononuclear Phagocyte

Assessment

Organ Weights Body Weights Hematology

4 Cytotoxic T Lymphocyte

Treatment – Lycopene (CASRN 502-65-8) will be obtained from the DTT. The compound will be administered daily for 28 days via oral gavage at concentrations to be provided by DTT. Doses, vehicle, and route of exposure will be selected based on known toxicity data.

Dosing solutions will be prepared in accordance with information specified by DTT.

Dosing volume will be 0.1 ml/20 grams of body weight. The animals will be evaluated for the appropriate immunological and toxicological parameters at the terminal sacrifice on

PND 42.

Positive controls will be used in this study. 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.

Following randomization, dams will be assigned to one of the following treatment groups.

Each vehicle and lycopene treatment group will consist of 10 animals. The positive control groups will consist of 8 animals per group. The treatment groups and their designations are shown below in the table.

All assays will be conducted at the contract laboratory in accordance with their SOPs.

Endpoints to be Evaluated

1. General

a. Body Weight: Animals will be weighed on the first day of exposure, on day 8, 15, 22, and day 29, the day of sacrifice.

b. Observations: Animals will 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 any unexpected toxicity.

Any remarkable effects will be recorded on the log or activity sheet, present in the exposure room.

Group Treatment Designation 1 Vehicle VH 2 Lycopene D1 D1 3 Lycopene D2 D2 4 Lycopene D3 D3

5 PC PC

c. 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. The DTT COR will be notified of excessive morbidity or mortality and the COR may request tissues be saved for histopathology evaluation in select situations.

2. Standard Toxicology Endpoints

a. Scheduled Sacrifice: On the day of sacrifice, a necropsy will be performed by trained personnel in accordance with the DTT Specifications. Terminal body weights and organ weights (spleen, thymus, liver, lungs, and kidneys with adrenals) will be recorded.

b. Hematology (erythrocytes, leukocytes, hematocrit, hemoglobin, MCV, MCH, MCHC, platelets and reticulocytes) and leukocyte differentials.

3. Reproductive Outcomes

a. Evaluation of pregnant animals (signs of parturition and numbers of implantations/resorptions; determinations of litter parameters such as survival, size, weight, abnormalities; determination of fertility/fecundity).

b. Evaluation of pups (developmental landmarks such as anogenital distance, balanopreputial separation, vaginal opening) may be requested on a study specific basis.

4. Immunology Assays

b. Spleen IgM antibody response to a T-dependent antigen, sheep erythrocytes (Day 4 response).

b. Cell Quantification: T cell and T subsets & B cell enumeration, NK cells and monocytes.

c. Natural killer cell assay.

j. Spleen cell proliferative response to anti-CD3-mediated T cell proliferation.

k. Mouse serum IgM ELISA using sRBC Antigen. Serum from mice used for the

AFC assay will be evaluated for the primary IgM levels.

l. Cytotoxic T-cell response.

m. Mononuclear phagocyte assessment (phagocytosis or respiratory burst).