Attachment_11_-_Mock_Protocol.pdf

Mock NCTR Protocol

1.0 STUDY TITLE: Two‐year carcinogenicity bioassay of in F344 rats

2.0 NCTR PROTOCOL NUMBER: 1234

3.0 SPONSOR:

4.0 TESTING FACILITY AND RESPONSIBLE PERSONNEL: National Center for Toxicological Research (NCTR), 3900 NCTR Road, Jefferson, AR 72079

4.1 Study director: John Doe

4.2 Dose preparation: Diet Prep Contractor

4.3 Dose analysis: NCTR Analytical Chemistry Support Group

4.4 Microbiology surveillance: NCTR Microbiology Surveillance Group

4.5 Animal care: Animal Care Contractor

4.6 Computer support: NCTR Computer Support Group

4.7 Pathology: Pathology Contractor

4.8 Statistical support: NCTR Statistical Support Group

4.9 Quality assurance: NCTR Quality Assurance Unit

5.0 STUDY OBJECTIVES

To determine the dose‐response relationship for the carcinogenicity of Compound X in F344 rats.

6.0 REGULATORY COMPLIANCE

This study will be conducted in compliance with the FDA’s Good Laboratory Practices requirements as specified in Part 58 “Good Laboratory Practices for Nonclinical Laboratories Studies” (Federal Register, 22 December 1978, Part II and any later interpretations published by the FDA).

NCTR’s Quality Assurance Unit will be responsible for assessing the quality of all aspects of the study.

This will include maintaining a file of quality assurance reports (inspections, audits, and periodic status) and responses to them.

7.0 BACKGROUND

Background information is not included in this mock protocol.

8.0 STUDY SCHEDULE

8.1 Proposed experimental start date: 13 July 2009 (first day of dosing)

8.2 Proposed experimental (in‐life) termination date: 14 November 2011 (last scheduled terminal sacrifice)

8.3 Proposed study completion date: 1 October 2013 (NCTR final report)

8.4 Proposed schedule of events:

Activity Date

Weight rank, tail tattoo, and allocate 6 July 2009 (Load 1) 20 July 2009 (Load 2) 3 August 2009 (Load 3) 17 August 2009 (Load 4) 31 August 2009 (Load 5) 14 September 2009 (Load 6) 28 September 2009 (Load 7) 12 October 2009 (Load 8) 26 October 2009 (Load 9) 9 November 2009 (Load 10)

Start dosing 13 July 2009 (Load 1) 27 July 2009 (Load 2) 10 August 2009 (Load 3) 24 August 2009 (Load 4) 7 September 2009 (Load 5) 21 September 2009 (Load 6) 5 October 2009 (Load 7) 19 October 2009 (Load 8) 2 November 2009 (Load 9) 16 November 2009 (Load 10)

36‐week interim sacrifice 22 March 2010 (Load 1) 5 April 2010 (Load 2) 19 April 2010 (Load 3) 3 May 2010 (Load 4) 17 May 2010 (Load 5) 31 May 2010 (Load 6) 14 June 2010 (Load 7) 28 June 2010 (Load 8) 12 July 2010 (Load 9) 26 July 2010 (Load 10)

Activity Date

60‐week interim sacrifice 6 September 2010 (Load 1) 20 September 2010 (Load 2) 4 October 2010 (Load 3) 18 October 2010 (Load 4) 1 November 2010 (Load 5) 15 November 2010 (Load 6) 29 November 2010 (Load 7) 13 December 2010 (Load 8) 27 December 2010 (Load 9) 10 January 2011 (Load 10)

104‐week terminal sacrifice 11 July 2011 (Load 1) 25 July 2011 (Load 2) 8 August 2011 (Load 3) 22 August 2011 (Load 4) 5 September 2011 (Load 5) 19 September 2011 (Load 6) 3 October 2011 (Load 7) 17 October 2011 (Load 8) 31 October 2011 (Load 9) 14 November 2011 (Load 10)

Draft pathology report 1 May 2012

In‐life statistics report 1 May 2012

Pathology working group (PWG) review 1 December 2012

Final pathology report 1 February 2013

Pathology statistics report 1 March 2013

NCTR final report 1 October 2013

9.0 TEST AND CONTROL ARTICLES

9.1 Characterization

9.1.1 Test article

Compound X (CAS ###‐###‐#; stated purity ≥99%) will be purchased as a single lot from Compound X

Manufacturing. The specific lot number will be recorded in the experimental file.

The identity and purity of the Compound X will be assessed by the Analytical Support Group, NCTR.

These assays will include establishing the levels of contaminant(s).

9.1.2 Vehicle control article

Corn oil (CAS 8001‐30‐7; d = 0.9) will be obtained from Corn Oil, Inc. The specific lot number or numbers will be recorded in the experimental file.

The Analytical Support Group, NCTR, will assay the corn oil for the presence of peroxides; values of <10 mEq/kg will be deemed acceptable.

The Analytical Support Group will also assay the corn oil for the presence of pesticides and fumonisin B1.

9.2 Stability

Compound X dosing solutions will be prepared by the Diet Preparation Contractor, NCTR, for conducting dose‐stability studies. Samples will be provided directly to the Analytical Support Group, NCTR, by the

Diet Preparation Contractor. The Analytical Support Group will conduct dose‐stability studies, using the lowest (0.004 mg Compound X/ml corn oil) and highest (0.40 mg Compound X/ml corn oil) dose formulations. These studies will be for a period of at least two weeks and will be conducted prior to the initiation of any dosing.

9.3 Storage and handling

The bulk Compound X will be stored at room temperature and the distilled Compound X will be stored under a nitrogen atmosphere in amber‐colored bottles at ‐20°C. The corn oil will be stored at 5oC.

Dosing solutions containing Compound X will be prepared in corn oil in sealed glass vials. The dosing solutions will be stored at 5oC and transported to the animal room using a protective outer container.

Face masks, safety glasses, gloves, and laboratory coats will be worn when handling the material. To reduce personnel exposure from spills, the vials of dosing solution will not be opened but rather aliquots will be drawn through the septa.

No monitoring procedures will be necessary under normal handling conditions. Personal protective equipment, including latex or nitrile gloves, NIOSH approved N‐95 particle respirators and lab coats, should provide sufficient protection from any harmful vapors or dermal exposure while using Compound

X.

9.4 Dose formulations

Dosing solutions will be prepared weekly by the Diet Preparation Contractor, NCTR. The dosing solutions for the highest dose will be prepared by adding a specified volume of Compound X to a specified volume of corn oil. The remaining doses will be prepared by serial dilutions of the highest dose.

SOPs regarding the dose preparation will be prepared by the Diet Preparation Contractor before the initiation of dosing.

The dose formulations will be stored under a nitrogen atmosphere in amber‐colored glass bottles at 5oC for no longer than two weeks. The dosing solutions will be allowed to equilibrate to room temperature and then shaken by hand before treating the rats.

9.5 Sampling

The Analytical Support Group, NCTR, will conduct dose certification analyses of the Compound X test article according to SOPs prepared by the Analytical Support Group. Dose certification analyses will be conducted at least monthly according to a schedule prepared by the Analytical Support group and approved by the Study Director. The SOPs and sampling schedule will be in place before the initiation of dosing.

Samples for dose certification analyses will be provided directly to the Analytical Support Group by the

Diet Preparation Contractor, NCTR.

The Analytical Support Group will retain a portion of the bulk Compound X, as initially received and after distillation. The bulk Compound X will be maintained at room temperature and the distilled Compound X will be maintained at ‐20°C. A portion of each lot of corn oil will be retained by the Analytical Support

Group. This material will be maintained at 5°C.

9.6 Disposition

Except for the materials retained in Section 9.5, all remaining test article and vehicle control article will be disposed according to NCTR's Environmental Health & Safety Unit SOPs.

10.0 TEST SYSTEMS

10.1 Species/strain/substrain: Rat/F344/Nctr BR

10.2 Source: NCTR

10.3 Age of animals at randomization/allocation: 6 weeks of age

10.4 Body weight range at randomization/allocation: 100‐200 g

10.5 Number and sex: A total of 1,005 male and 1,005 female rats will be obtained. Thirty of these rats will be used for microbiological surveillance. Of the remaining 1980 rats, 1900 with the most uniform body weights will be used in the bioassay. The remaining 80 rats will be discarded without being treated.

10.6 Identification method: The rats will be tail‐tattooed for identification. The tail tattoo will consist of a three‐digit cage identification and a single digit animal identification (e.g., 4801, which indicates animal 1 in cage 480). The cages will be identified by eleven‐digit cage cards; the first six digits specify the test number and the last five digits specify the cage number (e.g. 12340200480, which indicates cage 480 of test 2 of experiment 1234.

10.7 Housing: Rats will be housed three per cage in polycarbonate cages with hardwood chip bedding and microisolator bonnets until they are weight‐ranked at 6 weeks of age, at which time they will be housed two per cage. The hardwood chips will meet standard NCTR guidelines.

10.8 Husbandry: Cages will be changed twice a week. Cages will be rotated every other week. Racks will be sanitized and rotated within the room every other week. NIH‐41 irradiated pellets and Millipore‐ filtered drinking water will be available ad libitum.

10.9 Environment: The bioassay will be conducted in three animal rooms. The specific room numbers will be recorded in the experimental file. The animal rooms will be cleaned prior to starting the study.

The environment of the animal rooms will be monitored. Environmental controls will be set to maintain the temperature at 22 ± 4°C, with a relative humidity of 40‐70%. A 12‐hour light/dark cycle will be maintained. The animal rooms will receive 10‐15 air changes per hour.

10.10 Diet/Water: NIH‐41 irradiated pellets and Millipore‐filtered drinking water will be used. The feed and water will be analyzed by the Analytical Support Group, NCTR, and the Microbiology Support Group, NCTR, according to standard NCTR procedures and schedules. The feed will also be analyzed for

Compound X by the Analytical Support Group, with concentrations of <50 ppb being deemed acceptable.

10.11 Inclusion/exclusion criteria: NA.

10.12 Quarantine/acclimation: Rats will be obtained at three weeks of age. Treatment will begin at seven weeks of age. No quarantine will be required.

10.13 Justification for use of the test system: The bioassay will be conducted in F344 (F344N Nctr BR) rats, the strain of rats used in the previous bioassay on Compound X.

10.14 Sick animals: Animals in obvious distress, as evidenced by ataxia, cyanosis, or respiratory depression, will be removed from the study. These conditions may be identified by a lack of coordination, a bluish or grayish discoloration of the skin, or slow or labored breathing. In addition, a

20% decrease in body weight within a one‐week period will be an indication for removal. The Study

Director or his designee will be available for consultation with animal room personnel to determine if the extent of any clinical signs or weight change is sufficient to remove the animal from the study.

11.0 EXPERIMENTAL DESIGN

11.1 Randomization: The Computer Support Group, NCTR, will prepare treatment randomization documentation, which will be certified by the Statistical Support Group, NCTR, prior to the animals being allocated to the Multigeneration Support System (MGSS). The study will be loaded in 10 replicates.

11.2 Treatment groups:

Group Number

Treatment Dose Level (mg/kg body wt/treatment)

Dose Concentration

(mg/ml)

Dose Volume (ml/kg body wt)

Number of

Animals

Frequency of Administration

1 Vehicle 0 0 5.0 380 5 days/week

(M‐F)

Compound

X

0.02 0.004 5.0 360

5 days/week (M‐F)

Compound

X

0.044 0.0088 5.0 260

5 days/week (M‐F)

Compound

X

0.092 0.0184 5.0 260

5 days/week (M‐F)

Compound

X

0.2 0.04 5.0 160

5 days/week (M‐F)

Compound

X

0.44 0.088 5.0 160

5 days/week (M‐F)

Compound

X

0.92 0.184 5.0 160

5 days/week (M‐F)

Compound

X 2 0.4 5.0 160

5 days/week (M‐F)

11.3 Route of administration: The Compound X will be administered by gavage in corn oil five days per week for two years, with interim sacrifices conducted at 9 (36 weeks) and 15 (60 weeks) months.

11.4 Dosing equipment: Dosing will be conducted with syringes (1 or 3 ml) equipped with 3‐inch, 18‐ gauge stainless steel feeding tubes. MGSS will calculate the dosing volumes based upon the weight of the rats. The dose volumes will be rounded to the nearest 0.01 ml when using a 1‐ml syringe and 0.05 ml when using a 3‐ml syringe.

11.5. Justification for the route of administration: Dosing by gavage was used in the previous bioassay on Compound X.

11.6 Sacrifice schedule:

Group Number

Doses of Compound X

(mg/kg body wt)

Total Number of Rats per Group

Number of Rats Examined at 9‐ month Sacrifice

Number of Rats Examined at 15‐ month Sacrifice

Number of Rats for Terminal Sacrifice

1 0 380 40 20 300

2 0.02 360 40 10 300

3 0.044 260 40 10 200

4 0.092 260 40 10 200

5 0.2 160 40 10 100

6 0.44 160 40 10 100

7 0.92 160 40 10 100

8 2 160 40 10 100

Total 1900 320 180 1400

11.7 Surveillance Diagnostic Program: Thirty rats will be sent to the Surveillance/Diagnostic Program, Microbiology Support Group, NCTR. These rats will be submitted at approximately 3 month intervals during the course of the study. Feed, water, and bedding will be analyzed by the Analytical Support

Group, NCTR, and the Microbiology Support Group, NCTR, according to standard NCTR procedures and schedules.

12.0 PARAMETERS TO BE MEASURED

12.1 Mortality and morbidity checks: Observations for mortality and moribundity will be conducted twice daily (in the a.m. and p.m.) and recorded on the MGSS.

12.2 Clinical observations: Abnormal observations including the appearance of lesions on the skin of the rats will be entered daily. Clinical observations will be conducted weekly and the findings recorded on the MGSS. Digital images will be taken monthly until the occurrence of skin tumor, at which time digital images will be taken weekly. The findings will be recorded on the MGSS. The digital images will be evaluated by the Study Director for skin assessment and the findings recorded on an Excel spreadsheet.

12.3 Body weights: Body weights will be measured daily (Monday through Friday) to determine the doses to be administered. The weights will be recorded on the MGSS. A "weight and observation" session shall be conducted weekly for the purpose of monitoring trends in body weights. The weights will be recorded on the MGSS.

12.4 Feed and water consumption: Feed and water consumption will not be monitored.

12.5 Neurological examinations: Neurological examinations will not be conducted.

12.6 Eye examinations: Eye examinations will not be conducted.

12.7 Clinical pathology blood sampling: Blood will be collected at the 9‐month interim sacrifice for serum chemistries and hematology by heart puncture. Hematology measurements will include erythrocyte (RBC) count, hemoglobin, packed cell volume, erythrocyte morphologic assessment, leukocyte count, leukocyte differential count, and platelet count and morphological assessment. Clinical chemistry measurements will include total protein, albumin, amylase, urea nitrogen, creatinine, alanine aminotransferase, creatine phosphokinase, aspartate aminotransferase, glucose, cholesterol, triglycerides, sodium, potassium, chloride, calcium, and phosphorus. Serum samples in excess of volume needed for clinical chemistry will be stored at ‐20oC for the Principal Investigator.

12.8 Pharmacokinetic blood sampling: Blood samples will not be collected for pharmacokinetic studies.

12.9 Urine sampling: Urine samples will not be collected.

13.0 PATHOLOGY

13.1 Moribund and early death animals

13.1.1 Moribund animals: All animals removed before a scheduled sacrifice will be removed from MGSS and assigned a CID number [the CID number will consist of the experiment number, the test number, a four‐digit cage number, and a single‐digit animal identification number within the cage (e.g., 12340204801, which indicates animal 1 from cage 480 of test 2 of experiment 1234)]. They will then be sent to Pathology to undergo a gross examination. Whenever possible these examinations will be conducted under the supervision of a pathologist. All gross lesions will be described completely and recorded in the Gross Pathology System (GPS). Tissues will be fixed and histopathologic analyses will be conducted on the organs and tissues listed on the pathology protocol.

13.1.2 Early deaths: All animals dying before a scheduled sacrifice will be removed from MGSS and assigned a CID number. They will then be sent to Pathology to undergo a gross examination. Whenever possible these examinations will be conducted under the supervision of a pathologist and cause of death will be determined. All gross lesions will be described completely and recorded in GPS. Tissues will be fixed and histopathologic analyses will be conducted on the organs and tissues listed on the pathology protocol.

13.2 Pathology

13.2.1 Euthanasia: On the afternoon before the scheduled sacrifice, the animals will be removed from

MGSS, assigned a CID number, and then delivered to the necropsy holding area. They will continue to receive water but not food. On the following day, the animals will be weighed, assigned a CID number, and euthanized by exposure to carbon dioxide. Verification of death will be in accordance with the SOPs of the Pathology Contractor, NCTR.

13.2.2 Necropsy: Gross examinations of animals euthanized at a scheduled sacrifice will be conducted under the supervision of a pathologist. All gross lesions will be recorded including, when appropriate, organ, site, morphology, size, distribution, and color. In addition, digital images will be taken of all gross lesions of the skin. If the gross lesion cannot be represented in a digital image (for example “flaking”), a digital image will not be required.

13.2.3 Tissue collection list: The specific tissues to be fixed are listed below:

Adrenal glands Brain Clitoral glands Esophagus Eyes Femur Gross lesions Harderian glands Heart and aorta Intestine, large (cecum, colon, rectum) Intestine, small (duodenum, jejunum, ileum) Kidneys Liver Lungs and mainstem bronchi Lymph nodes ‐ mandibular and mesenteric ‐ bronchial and mediastinal Mammary gland with adjacent skin Muscle, thigh Nerve, sciatic Nasal cavity and nasal turbinates Oral cavity, larynx and pharynx Ovaries

Pancreas Parathyroid glands Pituitary gland Preputial glands Prostate Salivary glands Seminal vesicles Skin (all lesions; if none, a 1 cm2 site determined by Study Director or his designee) Spinal cord Spleen Stomach (forestomach and glandular) Testes, epididymides and vaginal tunics of testes Thymus Thyroid gland Tissue masses Tongue Trachea Urinary bladder Uterus Vagina Zymbal glands

13.2.4 Wet tissue handling: Tissues will be fixed in 10% neutral buffered formalin, with the exception of the eyes and testes, which will be fixed in Modified Davidsons. Fixation of the liver and gross lesions will be restricted to 96 hours; fixation in Modified Davidsons will be restricted to 24‐48 hours. All tissues except the skin will be processed to paraffin blocks. Skin will be processed and embedded in plastic polymer blocks.

13.2.5 Organ weights: Organ weights will be obtained at the 9‐month interim sacrifice with paired organs weighed together.

13.2.6 Histopathology: Histologic sections of less than 6 microns in thickness will be prepared, fixed, and stained with hematoxylin and eosin. Histopathologic analyses will be conducted on the organs and tissues listed below. When applicable, non‐neoplastic skin lesions will be graded for severity.

Immunohistochemistry will be performed on liver using the following antibodies: alpha‐fetoprotein

(AFP), polyclonal carcinoembryonic antigen (pCEA), CD10, and CD34. After completion of all microscopic evaluations, the tissue slides, paraffin blocks, and residual wet tissues will be stored in the NCTR pathology archives.

Adrenal glands Brain (3 sections including frontal cortex and basal ganglia, parietal cortex and thalamus, and cerebellum and pons) Clitoral glands Esophagus Eyes Femur, including diaphysis with marrow cavity and epiphysis (femoral condyle with epiphyseal cartilage plate, articular cartilage and articular surface) Gross lesions Harderian glands Heart and aorta Intestine, large (cecum, colon, rectum) Intestine, small (duodenum, jejunum, ileum) Kidneys Larynx Liver (2 sections including left lateral lobe and median lobe) Lungs and mainstem bronchi Lymph nodes ‐ mandibular and mesenteric ‐ bronchial & mediastinal

Mammary gland with adjacent skin Muscle, thigh (only if neuromuscular signs were present) Nasal cavity and nasal turbinates (3 sections) Ovaries Pancreas Parathyroid glands Pituitary gland Preputial glands Prostate Salivary glands Seminal vesicle Skin Spinal cord and sciatic nerve (if neurologic signs were present) Spleen Stomach (forestomach and glandular) Testes with epididymides Thymus Thyroid gland Tissue masses Trachea Urinary bladder Uterus

14.0 STATISTICAL ANALYSES

The probability of survival will be estimated by the product‐limit procedure of Kaplan and Meier.

Animals that die from reasons other than natural causes will be censored from the survival analyses.

Statistical analyses for possible dose‐related effects on survival will use Cox's method for testing two groups for equality and Tarone's life table test to identify dose‐related trends. The Poly‐K test will be used to assess neoplasm prevalence. The relationship between dose and nonneoplastic lesion severity will be analyzed by the Jonckheere‐Terpstra test. Pairwise comparisons will be conducted using

Williams' modification of Shirley's test. Analysis of continuous variables will be conducted by analysis of variance, with Dunnett's test being used to compare the dosed group means to the vehicle control means. These analyses will be conducted by Biometry Statistical Support Group, NCTR.

15.0 DATA ACQUISITION AND ELECTRONIC RECORDS

All in‐life data will be recorded and stored on the MGSS. Gross pathology examination data will be recorded using the Gross Pathology System (GPS). Histopathology data will be recorded in the

Laboratory Data Acquisition System (LDAS).

16.0 SUPPORT GROUP RESPONSIBILITIES

16.1 Support group duties:

Support Group Duty

Diet Prep Contractor Dose preparation

Analytical/Chemistry Support Group Test chemical identification, test chemical purity assessment, dose stability analyses, dose certification, accuracy of dose measurements, and feed and water analyses

Animal Care Contractor Animal dosing and care

Microbiology Support Group Microbiological surveillance

Contractor Animal room temperature and humidity monitoring

Computer Support Group In‐life computer support

Pathology Contractor Pathological assessments

Statistical Support Group Statistical analyses

16.2 Support group reports:

Support Group Report

Diet Prep Contractor Dose preparation report

Analytical/Chemistry Support Group Analytical chemistry report

Microbiology Support Group Microbiological surveillance report

Pathology Contractor Pathology report

Statistical Support Group Statistical analyses report

17.0 RECORDS TO BE MAINTAINED

The following records will be retained: 1. Protocol and any amendments, deviations, or related information; 2. Study‐related SOPs and documentation; 3. Test article accountability and characterization; 4. Raw data generated in operational areas, as defined in applicable SOP's; 5.

Computer files containing in‐life and pathology raw data; 6. Daily animal room logs; and 7. A copy of the final report.

Raw data sheets from this study will be stored in the NCTR archives. Histopathology samples collected during the course of the study will be placed in a secure storage area under the control of the Pathology

Contractor. Computer printouts and statistical analyses results will be stored in the NCTR archives.

Backup computer records will be maintained by the NCTR computer staff. All records and samples will be stored in accordance with FDA's Good Laboratory Practices.

18.0 PROTOCOL AMENDMENTS

All protocol amendments will be signed and dated by the Study Director and maintained with the study file.

19.0 PROTOCOL DEVIATIONS:

All protocol deviations will be documented as soon as possible after they occur. The Study Director will evaluate each deviation for its impact on the study. The documentation will be signed and dated by the

Study Director and maintained with the study file.