Sample_Study_Protocols.pdf

APPENDIX C

SAMPLE STUDY PROTOCOLS

The two Sample Study Protocols are meant to be an example of how NTP studies may be organized and conducted.

For assistance with the Technical Proposal, the sample protocols are provided for INFORMATION PURPOSES ONLY. A recapitulation of the sample protocols will not be acceptable. For use of the Sample Study Protocols to assist with preparation of the Technical Proposal, See Section J—List of Attachments, “Additional Technical Proposal Instructions”.

For the purposes of the Business Proposal, the offeror shall use these sample protocols to prepare cost estimates. For use of the Sample Study Protocols to assist with preparation of the Business Proposal, See See Section J—List of Attachments, Business Proposal Attachments for “Additional Business Proposal Instructions”.

I. Sample Protocol Outline For Evaluation Of The Contact Hypersensitivity………….Page 2

II. Sample Protocol Outline for the Evaluation of Immunosuppression…………………Page 6

SAMPLE PROTOCOL #1

Sample Protocol Outline For Evaluation Of The Contact Hypersensitivity Of 5-Hydroxytryptophan (CASRN: 4350-09-8, test article number IXXXX) In Female BALB/c Mice Following Topical

Administration

Chemical structure: C11H12N2O3

MW: 220.22

Background and Study Rationale

The studies described in this protocol will evaluate the sensitizing potential of 5- Hydroxytryptophan in a traditional paradigm using the local lymph node assay (LLNA) and mouse ear-swelling test (MEST). The LLNA is a murine model being used extensively to study chemical hypersensitivity. The acceptance of the LLNA as a stand-alone alternative to the GPMT/Beuhler Assay by ICCVAM demonstrates the commitment of using more humane testing models for screening chemicals that may elicit contact hypersensitivity in humans. Measurement of irritancy (IRR) is incorporated into the LLNA protocol used by the NTP. Inclusion of the MEST, which incorporates both the sensitization and challenge aspects of the response to contact allergens, allows for increased ability to detect and characterize weak sensitizers at non-irritating doses.

Animals -- Female BALB/c mice will be purchased from Charles River Laboratories and used for these studies. The mice will be ordered viral-pathogen free. Animals will arrive at the designated contract laboratory 45-60 days of age (approximately 18 g). Upon arrival, the mice will be quarantined for at least 7 days prior to commencement of exposure. Mice will be individually identified by tattoo and cage card in accordance with standard operating procedures at the designated contract laboratory. All mice will be randomized using a computer-generated randomization procedure. Mice will be housed 4 per cage in plastic shoe box cages with hardwood bedding and cage covers, and will be maintained on NTP-2000 diet and tap water ad libitum. In conducting this study 64 animals will be used for the combined LLNA and IRR studies (see Table 1) and 56 animals for the MEST (see Table 2).

Test Article – The 5-Hydroxytryptophan (5-HTP; purity 99.8%, lot # XXX, M.W. 220.22, CAS No.

4350-09-8) will be obtained from the NTP. Prior to commencement of the study the immunotoxicity contractor will conduct an evaluation of the solubility of the test article in a vehicle appropriate for the LLNA. ICCVAM has recommended the following organic solvents for the LLNA in order of priority, acetone-olive oil (4:1, AOO), acetone, dimethylformamide (DMF), and methyl ethyl ketone (MEK).

The exposure levels to be used in the IRR will be the same as the LLNA because a combined assay of IRR and LLNA will be performed in this study to reduce the number of animals used. Unless there is information to indicate otherwise, the standard exposure levels will be 50%, 25%, 10%, 5%, and 2.5% (w/v). The COR will review the results of the solubility evaluation and determine the appropriate vehicle to be used for the study. The exposure levels to be used in the MEST will be determined based on the results obtained in the LLNA and IRR. 2,4-Dinitrofluorobenzene (DNFB) (0.15% v/v, CAS No. 70-34-8) and Hexyl Cinnamic Aldehyde (HCA) (25% (w/v) CAS No. 101-86-0) in AOO will be used as positive controls. HCA is a moderate sensitizer that is used as a positive control for the LLNA assay. DNFB is a strong sensitizer that is used as a positive control in the LLNA and MEST.

Dosing solutions will be prepared daily.

Route of Administration – 5-HTP, DNFB, HCA or AOO vehicle control will be applied topically to the mouse as appropriate for each assay as detailed below. The volumes applied will be assay-specific.

LLNA/IRR Experimental Design -- Prior to the first exposure, mice will be weighed, randomized and tattooed. Prior to the dose application on the first day of the study, measurements of ear thickness will be made (pre-measurement) using a modified Mitutoyo micrometer. Measurements will be read and recorded as mm x 10-2. On day 1, mice will be treated with the appropriate concentration of test substance (5-HTP, DNFB, or HCA) or vehicle (AOO) by applying a total of 25 µl to the dorsum of each ear. Identical treatments will be repeated for the next two days. On days 4 and 5, the mice will receive no treatment. On day 4, 24 (± 2) hrs after the final treatment, each mouse will be restrained and measurements of ear thickness will be made in the same manner as the pre-treatment measurements (post-measurement for IRR assay). On day 6, all animals will be injected with 0.25 ml (20 µCi) 3H-thymidine by tail vein. Approximately 5 hrs after injection, mice will be sacrificed and both the right and left cervical (auricular) lymph nodes from each mouse will be excised and pooled into a tube (1 tube/mouse) with 4 ml cold phosphate-buffered saline (PBS). Lymph node cells will be counted using a liquid scintillation counter to evaluate the amount of 3H-thymidine incorporated as a measure of cell proliferation. The stimulation index (SI) will be calculated as the ratio of cell proliferation (dpm) in the treated animals over that in the vehicle control animals. A chemical will be classified to be positive in the LLNA if at least one concentration produces an SI of ≥ 3 with the results being statistically significant and dose-responsive as the ICCVAM recommends. The assay time line and treatment groups are shown below.

Major Events of the Combined Local Lymph Node Assay (LLNA) and Irritancy Assay (IRR)

Table 1. Dose Groups for LLNA

GROUP TREATMENT DESIGNATION NO. OF MICE

1 VEHICLE (AOO) VH 8

2 2.5% 5-HTP D1 8

3 5% 5-HTP D2 8

4 10% 5-HTP D3 8

5 25% 5-HTP D4 8

6 50% 5-HTP D5 8

7 0.15% DNFB PC1 8

8 25% HCA PC2 8

Sentinels 4

Total 68

MEST Experimental Design –The selection of 5-HTP exposure levels to be used for sensitization and challenge in the MEST will be made by the COR after the LLNA and the IRR are conducted. The highest dose for the MEST will be the lowest dose found to be sensitizing in the LLNA. If none of the 5-HTP doses induce sensitization, the highest dose will be based on maximum non-irritating dose within the limits of solubility. Two additional lower doses will be selected, based on the LLNA and IRR results.

Prior to the first exposure, the dorsal lumbar surface of each animal will be shaved. Exposure will be accomplished by a direct application of 50 µl of the selected concentrations of 5-HTP, 15 % DNFB solution (positive control), or vehicle to the prepared site with a pipette. After exposure, the mice will be restrained long enough to allow the vehicle to volatilize. The sensitization procedures of day 1 will be repeated on days 2 and 3. Mice will rest for days 4-7. On day 8, the thickness of the right ear of each mouse will be measured as described above for the irritancy study. After measurement on day 8, mice will be challenged on the dorsal side of the right ear pinna with a total of 25 µl of vehicle, 5- HTP at the selected concentrations, or 15% DNFB solution. Post-treatment measurements of ear thickness will then be made at 24 (± 2) hrs and 48 (± 2) hrs after challenge in the same manner as the pre-treatment measurements. The assay time line and treatment groups are shown below.

Major Events of the Mouse Ear Swelling Test (MEST)

Table 2. Experimental Groups for the MEST

GROUP TREATMENT

SENSITIZATION

TREATMENT

CHALLENGE

DESIGNATION NO. OF MICE

1 VEHICLE VEHICLE VH 8

2 VEHICLEa 5-HTP TBD d VHICb 8

3 5-HTP TBD 5-HTP TBD D1 8

4 5-HTP TBD 5-HTP TBD D2 8

5 5-HTP TBD 5-HTP TBD D3 8

6 VEHICLE 0.15%DNFB PCCOc 8

7 0.15% DNFB 0.15% DNFB PC 8

Sentinels 4 Total 60 a Because the test substance may have the potential to induce an irritant effect, which will confound the measurement of mouse ear swelling, both VH and VHIC are included.

b VHIC, vehicle, irritancy control.

c PCCO, positive control, challenge only.

d Concentration to be determined.

At the end of the MEST animals will be humanely euthanized. No tissues will be collected.

Additional Endpoints to be evaluated

1. General Toxicity

Animals will be weighed on the first day of treatment and on the day of sacrifice. In addition, animals will be monitored for moribundity during the study period, and any moribund animals will be humanely euthanized.

a. Body Weight. Animals will be weighed on the first day of treatment and on the day of sacrifice.

b. Observations. Animals will be observed twice daily, once in the morning and then again in the afternoon for any pharmaco-toxicological signs. Any remarkable effects will be recorded on the study log sheet, present in the exposure room.

Data Analysis

The data obtained in this study will first be tested for homogeneity of variances using Bartlett’s Chi Square Test. Homogeneous data will be evaluated by a parametric one-way analysis of variance.

When significant differences occur, treatment groups will be compared to the appropriate control using Dunnett’s T Test. For the LLNA, proliferation in the lymph nodes from treatment groups will be compared to that in the vehicle control group. Both statistical significance and the stimulation index will be reported. For the MEST, changes in ear thickness in the treatment groups and the vehicle irritancy control group will be compared to changes in ear thickness in the vehicle control group (VH).

If data are non-homogeneous, they will be evaluated using a non-parametric analysis of variance.

When significant differences occur, treatment groups will be compared to the appropriate control using the Wilcoxon Rank Test. The Jonckheere’s Test will be used to determine treatment-related trends.

SAMPLE PROTOCOL #2

Sample Protocol Outline For Screening Study to Evaluate The Potential Of Lycopene (CASRN: 502- 65-8, test article number IXXXX) to Induce Immunotoxictiy In B6C3F1Mice 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 – B6C3F1 mice will be used for this study. The mouse is one major species of choice in conducting toxicological and immunological studies. B6C3F1 mice will be supplied as part of a standing NTP contract to the Immunotoxicology contractor. 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 NTP 2000 diet unless otherwise specified.

The study will consist of 7 assay groups (as described below) X 8 animals per group X 7 treatments (vehicle, positive control and 5 dose groups = approximately 400 animals).

Parameters Measured Assay Group No.

1 2 3 4

1 Organ Wgts;

Gross Pathology

Histopathology Hematology Body Weights

2 IgM Antibody Forming Cell Assay

Antigen specific Serum IgM

Body Weights

3 Cell Quantification

Natural Killer Cell Activity

Anti-CD3 T Cell Proliferation & Mixed Leukocyte Response

Body Weights

4 Delayed type Hypersensitivity responses to Candida albicans

Body Weights

5 IgM Titers to Keyhole Limpet Hemocyanin

Body Weights

6 Mononuclear Phagocyte Assessement

Organ Weights Body Weights

7 Cytotoxic T Lymphocyte Assay

Body Weights

Treatment – Lycopene (CAS No. 502-65-8) will be obtained from the NTP. The compound will be administered daily for 28 days via oral gavage at concentrations to be provided by the NTP. 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 the NTP. 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.

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

Cyclophosphamide (CPS) will be given as a positive control for several assays, including organ weights, hematological parameters, B-cell, T-cell and T-cell subset surface markers, spleen IgM antibody-forming cell response, mixed leukocyte response and delayed-type hypersensitivity response (DTH) (Assay groups 1-5 and 7). The positive control animals will receive 50 mg/kg of CPS on the last 4 days of the exposure period by i.p. injection. Positive control animals will receive 0.1 ml/20 grams of body weight. Anti-Asialo GM1 (Rabbit) antibody will be given as a positive control for the natural killer (NK) cell assay (Assay group 3). The positive control animals will receive 1 ml of a 1:10 dilution by intravenous injection 24 hours prior to evaluation of NK cell activity. Maleic vinyl ether (MVE) will be given as a positive control in the MPS assay (Assay group 6). The positive control animals will receive 50 mg/kg of MVE by intravenous injection approximately 24 hours prior to evaluation of the MPS. Anti-Asialo GM-1, CPS and MVE will be obtained from commercial sources.

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 1 PC1

8 PC 2 PC2

9 PC3 PC3

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: A gross necropsy examination of major organs (brain and thoracic and abdominal viscera) will be performed in accordance with the NTP Immunopathology Specifications (Attachment B) to assess for cause of death or morbidity. Typically, no histopathology will be conducted on these animals. The NTP 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 NTP Immunopathology specifications (Attachment B).

Terminal body weights and organ weights (spleen, thymus, liver, lungs, and kidneys with adrenals) will be recorded.

b. Gross pathology will be performed on selected organs (spleen, thymus, liver, lungs, kidneys and adrenals). Liver, kidneys, lung, spleen, bone marrow (femur), GI tract with peyer’s patches, mesenteric, submandibular, and popliteal lymph nodes and thymus will be fixed and stored for histopathology as described in the Statement of Work. Individual animal necropsy reports (IANRs) will be completed on all animals. Tissues stored for histopathology will be fixed in neutral buffered 10% formalin. The tissues will be delivered to NIEHS for processing within 72 hours after collection.

c. 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. Serum IgM response to the T-dependent antigen KLH.

d. Natural Killer assay.

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

f. Mononuclear phagocytic system (MPS) assay.

g. Delayed-Type Hypersensitivity Response to Candida albicans.

h. Mouse Serum IgM ELISA using sRBC Antigen. Serum from mice used for the AFC assay will be evaluated for the primary IgM levels.

i. Cytotoxic T-cell response.

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