`
`
`
`
`
`FINAL REPORT
`Volume 1 of 12
`
`
`Guidelines: OECD (451) and JMHLW (Ordinance No. 21)
`
`Testing Facility Study No. EBA00009
`Sponsor Ref. No. P00012-04-11
`
`A Two Year Oral (Gavage) Carcinogenicity Study in Rats with BG00012
`
`TESTING FACILITY:
`
`Charles River Laboratories
`Preclinical Services
`640 North Elizabeth Street
`Spencerville, OH 45887
`
`SPONSOR:
`
`Biogen Idec Inc.
`14 Cambridge Center
`Cambridge, MA 02142
`
`February 28, 2008
`
`Page 1 of 4817
`
`Page 1 of 30
`
`Biogen Exhibit 2274
`Coalition v. Biogen
`IPR2015-01993
`
`

`

`Sponsor Ref. No. P00012-04-ll
`
`Page 9
`Testing Facility Study No. EBA00009
`
`1. COMPLIANCE STATEMENT
`This study was conducted in compliance with the Good Laboratory Practice (GLP) regulations as
`described by the FDA (21 CFR Part 58); the Organisation for Economic Cooperation and
`Development (OECD) Principles of Good Laboratory Practice, C(97)186; and the Japanese
`Ministry of Health, Labor, and Welfare (MHL W) Ordinance No. 21 with the following
`exception(s):
`Characterization and stability analyses of the bulk test article were conducted in compliance with
`the Good Manufacturing Practice regulations. Toxicokinetic interpretation was not conducted in
`compliance with the GLP regulations.
`
`Mark A. Morse, Ph.D., DABT
`Study Director
`Charles River Laboratories
`Preclinical Services
`
`Date
`
`Page 2 of30
`
`

`

`
`
`
`Page 10
`Testing Facility Study No. EBA00009
`
`
`Sponsor Ref. No. P00012-04-11
`
`2. QUALITY ASSURANCE STATEMENT
`This study has been inspected by the Quality Assurance Unit to assure conformance with the
`Good Laboratory Practice (GLP) regulations promulgated by the FDA (21 CFR Part 58); OECD
`Principles of Good Laboratory Practice, C(97)186; and the Japanese MHLW Ordinance No. 21.
`Reports were submitted in accordance with Standard Operating Procedures as follows:
`
`
`
`Dates of Inspection
`
`10/12/04
`10/12/04
`10/14/04
`10/25/04
`10/25/04
`10/25/04
`10/25/04
`10/25/04
`10/26/04
`10/26/04
`10/26/04
`10/26/04
`11/01/04
`11/04/04
`01/18/05, 02/16/05,
`02/22/05, 09/02/05,
`09/12/05, 09/19/05,
`10/20/05
`03/07/05
`03/07/05
`03/07/05
`03/08/05
`03/15/05
`03/15/05
`03/15/05
`04/05/05
`04/05/05
`04/05/05
`04/26/05
`05/23/05
`05/23/05
`09/12/05
`09/12/05
`09/13/05
`09/13/05
`10/03/05
`10/17/05
`12/29/05
`
`QA INSPECTION DATES
`
`
`Phase(s) Inspected
`
`
`Animal Receipt Procedure
`Protocol Review
`Animal Receipt
`Animal Identification
`Randomization Procedure
`Dose Preparation
`Analytical Sampling
`Test Article Receipt Procedure
`Body Weights
`Detailed Clinical Observations
`Food Consumption
`Dosing
`Retention Sample
`Protocol Amendment Review
`Data Audit
`
`Date Findings Submitted to:
`
`Study Director
`Study Director
`Management
`
`
`10/12/04
`10/12/04
`10/15/04
`10/15/04
`10/15/04
`10/15/04
`10/28/04
`10/28/04
`10/28/04
`10/28/04
`10/28/04
`10/28/04
`10/28/04
`10/28/04
`11/01/04
`11/01/04
`10/28/04
`10/28/04
`10/28/04
`10/28/04
`10/28/04
`10/28/04
`10/28/04
`10/28/04
`11/01/04
`11/01/04
`02/11/05
`02/11/05
`11/14/05
`11/14/05
`
`Dosing
`Dose Preparation
`Analytical Sampling
`Detailed Clinical Observations
`Body Weights
`Food Consumption
`Protocol Amendment Review
`Toxicokinetic Blood Collection
`Plasma Processing
`Dosing
`Protocol Amendment Review
`Dose Preparation
`Dosing
`Dose Preparation
`Dosing
`Detailed Clinical Observations
`Palpable Masses
`Bioanalytical Sample Shipping
`Protocol Amendment Review
`Protocol Amendment Review
`
`03/08/05
`03/08/05
`03/08/05
`03/08/05
`03/15/05
`03/15/05
`05/27/05
`04/05/05
`04/05/05
`04/05/05
`05/27/05
`05/23/05
`05/23/05
`09/13/05
`09/13/05
`09/13/05
`09/13/05
`10/03/05
`10/17/05
`05/03/06
`
`03/08/05
`03/08/05
`03/08/05
`03/08/05
`03/15/05
`03/15/05
`05/27/05
`04/05/05
`04/05/05
`04/05/05
`05/27/05
`05/23/05
`05/23/05
`09/13/05
`09/13/05
`09/13/05
`09/13/05
`10/03/05
`10/17/05
`05/03/06
`
`Page 3 of 30
`
`

`

`
`
`
`Page 11
`Testing Facility Study No. EBA00009
`
`
`Sponsor Ref. No. P00012-04-11
`
`
`
`
`
`
`
`Dates of Inspection
`
`01/09/06
`01/09/06
`01/09/06
`02/03/06
`03/01/06, 03/02/06
`03/13/06
`03/14/06
`03/14/06
`04/07/06, 04/10/06,
`04/11/06
`04/19/06
`04/19/06
`05/19/06
`05/25/06
`06/19/06
`06/19/06
`06/20/06
`06/26/06
`08/31/06
`09/01/06, 09/06/06,
`09/07/06, 09/08/06,
`09/18/06, 09/19/06
`09/07/06
`09/08/06
`09/11/06
`09/11/06
`09/12/06
`09/12/06
`09/14/06
`09/18/06
`09/19/06, 09/20/06
`10/23/06
`10/24/06
`10/25/06
`10/26/06
`10/27/06
`10/27/06
`10/31/06
`11/01/06
`11/03/06
`11/06/06
`11/08/06
`11/15/06, 11/16/06
`11/22/06
`11/27/06, 11/28/06,
`11/29/06
`11/30/06
`12/04/06, 12/05/06
`
`
`Phase(s) Inspected
`
`
`Dose Preparation
`Dosing
`Analytical Sampling
`Data Audit
`Data Audit
`Dose Preparation
`Dosing
`Food Consumption
`Data Audit
`
`Sentinel Blood Collection
`Serum Processing
`Protocol Amendment Review
`Data Audit
`Dose Preparation
`Dosing
`Protocol Amendment Review
`Analytical Sampling
`Data Audit
`Data Audit
`
`Data Audit
`Data Audit
`Dose Preparation
`Dosing
`Detailed Clinical Observations
`Palpable Masses
`Data Audit
`Data Audit
`Data Audit
`Protocol Amendment Review
`Necropsy
`Clinical Pathology Blood Collection
`Data Audit
`Sentinel Blood Collection
`Serum Processing
`Data Audit
`Data Audit
`Data Audit
`Data Audit
`Protocol Amendment Review
`Data Audit
`Trimming
`Data Audit
`
`Data Audit
`Data Audit
`
`Date Findings Submitted to:
`
`Study Director
`Study Director
`Management
`
`
`01/09/06
`01/09/06
`01/09/06
`01/09/06
`01/09/06
`01/09/06
`02/16/06
`02/16/06
`03/02/06
`03/02/06
`03/14/06
`03/14/06
`03/14/06
`03/14/06
`03/14/06
`03/14/06
`04/11/06
`04/11/06
`
`04/19/06
`04/19/06
`05/26/06
`05/25/06
`06/19/06
`06/19/06
`07/07/06
`07/07/06
`08/31/06
`09/19/06
`
`09/07/06
`09/08/06
`09/12/06
`09/12/06
`09/12/06
`09/12/06
`09/15/06
`09/18/06
`09/21/06
`10/23/06
`10/27/06
`10/27/06
`10/27/06
`10/27/06
`10/27/06
`10/31/06
`11/01/06
`11/03/06
`11/06/06
`11/15/06
`11/16/06
`11/28/06
`11/29/06
`
`11/30/06
`12/05/06
`
`04/19/06
`04/19/06
`05/26/06
`05/25/06
`06/19/06
`06/19/06
`07/07/06
`07/07/06
`08/31/06
`09/19/06
`
`09/07/06
`09/08/06
`09/12/06
`09/12/06
`09/12/06
`09/12/06
`09/15/06
`09/18/06
`09/21/06
`10/23/06
`10/27/06
`10/27/06
`10/27/06
`10/27/06
`10/27/06
`10/31/06
`11/01/06
`11/03/06
`11/06/06
`11/15/06
`11/16/06
`11/28/06
`11/29/06
`
`11/30/06
`12/05/06
`
`Page 4 of 30
`
`

`

`
`Sponsor Ref. No. P00012-04-11
`
`
`
`
`
`Page 12
`Testing Facility Study No. EBA00009
`
`
`Phase(s) Inspected
`
`
`
`
`Data Audit
`Data Audit
`Data Audit
`
`Data Audit
`Protocol Amendment Review
`Microtomy
`Quality Control
`Protocol Amendment Review
`Protocol Amendment Review
`Draft Report Review
`
`Date Findings Submitted to:
`
`Study Director
`Study Director
`Management
`
`
`12/08/06
`12/08/06
`12/11/06
`12/11/06
`12/12/06
`12/12/06
`
`12/21/06
`01/11/07
`01/12/07
`01/29/07
`05/01/07
`06/19/07
`07/20/07
`
`12/21/06
`01/11/07
`01/12/07
`01/29/07
`05/01/07
`06/19/07
`07/20/07
`
`
`
`
`Dates of Inspection
`
`12/05/06
`12/06/06, 12/07/06
`12/07/06, 12/08/06,
`12/11/06
`12/21/06
`01/09/07
`01/11/07
`01/26/07
`04/21/07
`06/19/07
`07/06/07, 07/10/07,
`07/11/07, 07/12/07,
`07/16/07, 07/17/07,
`07/18/07, 07/19/07
`10/16/07
`01/28/08
`02/19/08, 02/20/08
`
`Protocol Amendment Review
`Protocol Amendment Review
`Final Report Review
`
`10/29/07
`01/31/08
`02/20/08
`
`10/29/07
`01/31/08
`02/20/08
`
`
`QA statement(s) provided by the following test site(s) have been reviewed:
`Test Site(s)
`Phase
`QA Statement Location
`Charles River Laboratories,
`Preclinical Services
`Charles River Laboratories
`Charles River Laboratories
`Charles River Laboratories,
`Pathology Associates
`Charles River Laboratories,
`Pathology Associates
`
`Analytical Chemistry Report
`Pretest Health Screen (Serology)
`Sentinel Animals (Serology)
`
`Histopathology Report
`
`Appendix 3
`Appendix 4
`Appendix 5
`
`Appendix 13
`
`Histopathology Peer Review
`
`Appendix 13
`
`BioSTAT Consultants, Inc.
`
`Charles River Laboratories,
`Preclinical Services
`Biogen Idec Inc.
`
`Statistical Analysis of Survival
` and Tumor Incidence
`
`Bioanalytical Report
`
`Appendix 14
`
`Appendix 18
`
`Toxicokinetic Interpretive Report
`
`Appendix 19
`
`
`
`
`
`
`
`
`
`
`
`Page 5 of 30
`
`

`

`Sponsor Ref. No. P00012-04-ll
`
`Page 13
`Testing Facility Study No. EBA00009
`
`The final report has been reviewed to assure that it accurately describes the materials and
`methods, and that the reported results accurately reflect the raw data.
`
`Richard J. <tlarke, B.S.
`Quality Assurance Auditor I
`Charles River Laboratories
`Preclinical Services
`
`Date
`
`Page 6 of30
`
`

`

`
`
`
`
`Sponsor Ref. No. P00012-04-11
`
`4. SUMMARY
`The purpose of this study was to evaluate the potential oncogenicity of BG00012 following once
`daily oral gavage to Sprague Dawley rats for up to 2 years and toxicokinetics following once
`daily oral gavage for 180 days. The study design was as follows:
`
`Page 15
`Testing Facility Study No. EBA00009
`
`Experimental Design for the Carcinogenicity and Toxicokinetic Phases
`No. of Animals
`Dosage
`Dosage
`Dosage
`(TK Satellite Animals)a
`Dosage
`Group
`Level
`Conc.
`Volume
`(mg/kg/day)
`(mg/mL)
`(mL/kg)
`Material
`No.
`Male
`Female
`HPMCb
`10
`0
`0
`75 (15)
`75 (15)
`1
`10
`2.5
`25
`BG00012
`75 (15)
`75 (15)
`2
`10
`5
`50
`BG00012
`75 (15)
`75 (15)
`3
`10
`10
`100
`BG00012
`75 (15)
`75 (15)
`4
`10
`15
`150
`BG00012
`75 (15)
`75 (15)
`5
`aToxicokinetic phase animals began dosing during Week 24 of the carcinogenicity phase. The first day of
` dosing was designated Day 1. The duration of the toxicokinetic phase was a minimum of 180 days.
`bHydroxypropylmethylcellulose or Hypromellose (3,500-5,600 cps), 0.8% w/v in reverse osmosis
`deionized water.
`The following variables and end points were evaluated in this study: survival, clinical signs,
`tumor incidence, body weights, body weight changes, food consumption, hematology
`parameters, toxicokinetic parameters, gross necropsy findings, and histopathology findings.
`Results:
`There were dose-related decreases in the survival of males administered BG00012 at 50, 100, or
`150 mg/kg/day. The 50 mg/kg/day (Group 3) males had a survival percentage of 17.3% at
`termination versus 30.7% in controls, while the 150 mg/kg/day (Group 4) males were terminated
`during Week 86 and the 100 mg/kg/day (Group 4) males were terminated during Week 88
`because the number of survivors was only 10 of 75. Termination of a study group once the
`number of survivors reached 10 or fewer was implemented consistent with FDA guidance to the
`Sponsor (Protocol Amendment No. 6.) Survival of BG00012-treated females was similar to that
`of controls and ranged from 26.6-37%. All female groups were terminated at scheduled
`euthanasia.
`There were few clinical signs associated with BG00012 administration, but there were clear
`dose-related effects on body weight gain throughout the study in both males and females that
`were not associated with differences in food consumption. Males administered BG00012 at 50,
`100, or 150 mg/kg/day had lower body weights than controls throughout the study. The
`50 mg/kg/day males had body weights that were generally within ±10% of controls, whereas the
`100 mg/kg/day males had lower body weights that differed by more than 10% compared to
`controls from Weeks 61 through 85, and the 150 mg/kg/day males had lower body weights that
`differed by more than 10% relative to controls from Weeks 37 through 85. Test article-related
`effects on body weight gains were less pronounced in females. Only females administered
`
`Page 7 of 30
`
`

`

`
`
`
`Page 16
`Testing Facility Study No. EBA00009
`
`
`Sponsor Ref. No. P00012-04-11
`
`150 mg/kg/day had body weight gains that were 10% lower than controls; these differences in
`body weight occurred during the latter half of the study (Weeks 69-104).
`There were few treatment-related effects on hematology parameters in males, although the
`150 mg/kg/day males and females had elevated leukocytes, lymphocytes, and segmented
`neutrophils compared to controls. Test article-treated females had lower erythrocytes and mean
`corpuscular hemoglobin concentration and higher reticulocytes and mean corpuscular volume
`compared to controls, with a clear dose-response, and one or more test article-treated females in
`each group had slight to moderate macrocytosis, polychromasia, and anisocytosis.
`Gross necropsy observations were recorded primarily in the kidneys, stomach and testes. In the
`kidney, gross findings included enlargement, roughened or rough surface, and discoloration that
`were generally observed in higher incidence with increasing dose of BG00012. In the stomach,
`the principal gross finding was prominent epithelial surface, which was observed in a
`dose-related manner in test article-treated animals. These gross findings corresponded to
`microscopic findings of squamous epithelial hyperplasia, squamous cell papilloma, and
`squamous cell carcinoma of the nonglandular forestomach. In addition, test article-treated males
`had dose-related increases in the incidence of small and soft testes, which were associated with
`microscopic changes of degeneration and atrophy of the testicular tubular epithelium as well as
`an increased incidence of interstitial cell hyperplasia and adenoma in the 100 mg/kg/day and
`150 mg/kg/day males.
`Evaluation of the neoplastic findings demonstrated that there were test article-related findings
`including squamous cell papilloma and squamous cell carcinoma of the nonglandular stomach,
`renal tubule adenomas and/or carcinomas, an increased incidence of testicular interstitial cell
`adenomas. The mucosa of the nonglandular stomach was the primary site of test article-induced
`neoplasia, with hyperplasia of the squamous epithelium, squamous cell papilloma, and squamous
`cell carcinoma observed in test article-treated animals. The incidence of squamous cell
`carcinomas in males or females administered BG00012 at 100 or 150 mg/kg/day ranged from
`40.0% to 77.3%.
`A dose-related exacerbation of chronic progressive nephropathy (CPN) was observed in the
`kidneys of test article-treated males and females, and was the most common cause of death of
`test article-treated animals, particularly males. A low incidence of renal tubular adenomas was
`observed in males and a low incidence of renal tubular adenomas and carcinomas was observed
`in females administered BG00012 at 100 or 150 mg/kg/day.
`As a consequence of the increased severity of CPN induced by BG00012, an increased incidence
`of parathyroid hyperplasia was observed in test article-treated males and females, as well as a
`low incidence of parathyroid adenomas in males administered BG00012 at 50, 100, or
`150 mg/kg/day. In males, increases in the incidence and severity of interstitial cell hyperplasia
`and an increase in the incidence of interstitial cell (Leydig cell) adenoma were observed in
`animals administered BG00012 at 100 and 150 mg/kg/day. In addition, degeneration and
`atrophy of the testicular tubular epithelium were also observed at 100 and 150 mg/kg/day.
`
`
`Page 8 of 30
`
`

`

`
`
`
`Page 17
`Testing Facility Study No. EBA00009
`
`
`Sponsor Ref. No. P00012-04-11
`
`Conclusion:
`Under the conditions of this study, administration of BG00012 by gavage resulted in dose-related
`decreases in body weight gain and overall survival, especially in male rats. Reduction in mean
`body weight and body weight gains were observed consistently at the 150 mg/kg/day dose level
`in females; however, overall survival was not affected by BG00012 administration. There were
`dose-related increases in hyperplasia of the squamous epithelium, squamous cell papilloma, and
`squamous cell carcinoma of the nonglandular stomach, although such lesions are not generally
`considered relevant in human risk assessment. A dose-related exacerbation of chronic
`progressive nephropathy (CPN) was observed in test article-treated males and females. The
`increased incidence and severity of CPN was considered the probable cause of early deaths in
`males, and likely contributed to the low incidence of renal tubular adenomas that was observed
`in male animals administered BG00012. The renal carcinoma incidence was significantly
`greater in the 150 mg/kg/day treated female rats, as compared to the respective control group.
`As BG00012 has demonstrated both exacerbation of the rat specific renal finding of CPN and a
`BG00012-specific regeneration of the renal tubular epithelium, it is difficult to determine
`whether either or both of these processes contributed to the tumor development. The relevance
`of these renal tumors to human risk assessment is unknown, as CPN is not apparent in humans.
`If CPN is required for ultimate tumorigenicity, then the human relevance of the rat renal tumors
`would be suspect. In addition, there was an increased incidence of parathyroid hyperplasia in
`test article-treated males and females, and a low and non-statistically significant incidence of
`parathyroid adenomas in males at 50, 100, or 150 mg/kg/day. These findings were described as
`secondary to severe CPN and renal failure, and not a direct test article-related effect on the
`parathyroid gland. In males, an increased incidence of interstitial cell hyperplasia and interstitial
`cell (Leydig cell) adenomas was also observed in animals administered BG00012 at 100 and
`150 mg/kg/day. The relevance of rat interstitial cell adenomas has been reviewed extensively
`and found to have no relevance to human risk.1
`
`
`
`Page 9 of 30
`
`

`

`
`
`
`Page 18
`Testing Facility Study No. EBA00009
`
`
`Sponsor Ref. No. P00012-04-11
`
`5. INTRODUCTION
`The purpose of this study was to evaluate the potential oncogenicity of BG00012 following once
`daily oral gavage to Sprague Dawley rats for up to 2 years and toxicokinetics following once
`daily oral gavage for 180 days.
`The protocol was signed by the Study Director on October 12, 2004 (GLP initiation date). The
`experimental start date for the study was October 12, 2004 (animal receipt), and the experimental
`completion date for the study was February 19, 2008. The in-life phase of the study was initiated
`on October 26, 2004, and the in-life completion date was October 27, 2006.
`
`6. MATERIALS AND METHODS
`The study protocol, protocol amendment(s), and protocol deviations are presented in
`Appendix 1.
`
`6.1. Test Materials
`
`6.1.1. Test Article
`Identification
`
`Lot Number
`Assigned Testing Facility ID
`Purity
`Receipt Date
`Expiration Date
`Physical Description
`Storage Conditions
`Supplier
`
`Lot Number
`Assigned Testing Facility ID
`Purity
`Receipt Date
`Expiration Date
`Physical Description
`Storage Conditions
`Supplier
`
`
`
`
`
`
`BG00012 (dimethyl fumarate)
`
`1102642 33004998
`S04.003.EBA
`100.2%
`October 14, 2004
`None provided
`White crystalline powder
`Room temperature, protected from light
`Sponsor
`
`1102643 33004999
`S06.005.EBA
`99.8%
`July 12, 2006
`July 21, 2007
`White crystalline powder
`Room temperature, protected from light
`Sponsor
`
`Page 10 of 30
`
`

`

`
`Sponsor Ref. No. P00012-04-11
`
`6.1.2. Control Article
`
`
`
`
`Page 19
`Testing Facility Study No. EBA00009
`
`The control material, HPMC (hydroxypropylmethylcellulose or hypromellose), was received
`from Sigma-Aldrich Co., stored at room temperature, and identified as follows:
`
`Testing
`Apparent
`Facility
`Assigned
`Control
`Viscositya
`Expiration
`Receipt
`Testing
`Lot
`Article
`Physical
`Date
`Date
`Facility ID
`Number
`Identification
`Description
`(cps)
`White powder
`5163
`12/31/14
`08/16/04
`122K0149 V04.088
`HPMC
`White powder
`4508
`12/31/14
`11/18/04
`013K0621 V04.117
`HPMC
`White powder
`4508
`12/31/14
`12/13/04
`013K0621 V04.127
`HPMC
`White powder
`5163
`12/31/14
`12/27/04
`122K0149 V04.134
`HPMC
`White powder
`5163
`12/31/15
`02/23/05
`122K0149 V05.010
`HPMC
`White powder
`4508
`12/31/15
`02/25/05
`013K0621 V05.012
`HPMC
`White powder
`4508
`12/31/15
`03/25/05
`013K0621 V05.019
`HPMC
`White powder
`4508
`05/19/15
`05/19/05
`013K0621 V05.042
`HPMC
`White powder
`4508
`06/20/15
`06/20/05
`013K0621 V05.052
`HPMC
`White powder
`5153
`12/31/15
`08/04/05
`103K0135 V05.066
`HPMC
`White powder
`5153
`12/31/15
`09/09/05
`103K0135 V05.080
`HPMC
`White powder
`4104
`12/31/15
`09/13/05
`045K0051 V05.082
`HPMC
`White powder
`4104
`12/31/15
`10/06/05
`045K0051 V05.092
`HPMC
`White powder
`5153
`12/31/15
`10/06/05
`103K0135 V05.093
`HPMC
`White powder
`4104
`12/31/15
`10/19/05
`045K0051 V05.101
`HPMC
`White powder
`4104
`12/31/16
`01/03/06
`045K0054 V06.001
`HPMC
`White powder
`4104
`12/31/16
`05/09/06
`045K0054 V06.047
`HPMC
`White powder
`4104
`12/31/16
`05/12/06
`045K0054 V06.051
`HPMC
`aVendor’s stated apparent viscosity range: 3,500 – 5,600 cps (2% aqueous solution at 20°C)
`6.1.3. Test Article Characterization
`Certificates of Analysis for the test article are presented in Appendix 2.
`
`6.1.4. Reserve Sample
`A 1-gram sample of each lot of the bulk test article was collected as a reserve sample and
`maintained at room temperature, protected from light by the Testing Facility. A 1-gram sample
`of each lot of the bulk control article was collected as a reserve sample and maintained at room
`temperature by the Testing Facility.
`
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`6.1.5. Inventory and Disposition
`An inventory of the test material supplied by the Sponsor was maintained. With the exception of
`the reserve sample(s), the bulk test article will be returned to the Sponsor following completion
`of all scheduled studies, unless otherwise instructed by the Sponsor.
`
`Page 20
`Testing Facility Study No. EBA00009
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`6.1.6. Preparation of Dose Formulations
`The vehicle control material was prepared by weighing a specified amount of the HPMC into a
`calibrated container, adding an appropriate amount of heated reverse osmosis deionized (RODI)
`water (supplied by the tap in the Testing Facility’s formulations department) and stirring the
`formulation by hand. Room temperature RODI water was then added to produce a 0.8% w/v
`solution and the formulation was stirred overnight (except for one vehicle preparation that was
`not stirred overnight prior to the first day of dosing). The vehicle control material was prepared
`a minimum of weekly and stored refrigerated.
`For each dose level, an appropriate amount of the test article (BG00012) was weighed into a
`plastic weigh boat and placed in a mortar. The weigh boat was rinsed with vehicle (0.8% w/v
`HPMC in RODI water). A sufficient volume of the vehicle was added to the mortar, the mixture
`was transferred into an Erlenmeyer flask (or a beaker) and the mortar was rinsed with vehicle.
`An appropriate quantity of the vehicle was then added to achieve the desired concentration and
`the flask was inverted several times (beaker was not inverted). The flask (or beaker) was
`wrapped in aluminum foil and the dosing formulation was stirred using a magnetic stir bar. Each
`formulation was stirred continuously and dispensed into individual amber glass containers for
`daily dosing. The dosing formulations were prepared at least weekly. A sufficient quantity of
`the vehicle control material was similarly dispensed for administration to control animals. The
`dosing mixtures were stored refrigerated and allowed to warm to room temperature prior to
`administration. The formulations were stirred continuously prior to and during dosing. The
`physical description of each dosing formulation was recorded following preparation, the vehicle
`was a clear colorless solution, the Group 2 and 3 test article formulations were white or cloudy
`white suspensions, and the Group 4 and 5 test article formulations were white suspensions.
`Beginning with the Week 22 preparation, at least two individual preparations were made for
`Groups 2-5.
`A second formulation for the vehicle control and test article groups was prepared during
`Week 50 (Day 344) to replace the frozen formulation that occurred due to a refrigerator with a
`failing thermostat.
`
`6.1.7. Analysis of Dose Formulations
`Dose formulation samples were collected for analysis as indicated in the following table:
`
`
`
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`Page 21
`Testing Facility Study No. EBA00009
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`Dose Formulation Samples for Analysis
`
`Time Point
`Week 1
`Week 2
`Week 3
`Week 4
`Week 8
`Week 12
`Week 16
`Week 20
`Week 21
`Week 22
`Week 23
`Week 24
`Week 28
`Week 32
`Week 36
`Week 40
`Week 44
`Week 48
`Week 52
`Week 56
`Week 60
`Week 64
`Week 68
`Week 72
`Week 76
`Week 80
`Week 84
`Week 88
`Week 92
`Week 96
`Week 100
`Week 104
`Note: N/A = not applicable.
`
`Concentration
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 2-5
`Groups 2-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`Groups 1-5
`
`Homogeneity
`Groups 1-5
`Groups 1-5
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`N/A
`
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`Testing Facility Study No. EBA00009
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`Sponsor Ref. No. P00012-04-11
`
`6.1.7.1. Concentration
`During Weeks 1 and 2, four 1-mL samples were collected from the top, middle, and bottom of
`each dosing formulation for concentration analysis. Beginning on Week 3, four 1-mL samples
`were collected from the middle of each dosing formulation.
`Due to erroneous results for the Week 20 analysis, Group 2 and 3 formulations for Week 20 were
`re-sampled and all groups from the Week 21 formulation were also sampled for analysis to
`verify the formulation procedure.
`Two preparations for each test article group (Groups 2-5) were made for Weeks 22 and 23. Four
`samples were collected from the middle and aliquots were taken from each container for each
`group from which the preparations were made.
`For Week 24 preparations, and every 4 weeks thereafter, samples were collected from the middle
`and aliquots were taken from the containers from which the preparations were made. Four
`samples were collected from each of the preparations made for each group.
`
`6.1.7.2. Homogeneity
`Four 1-mL samples were collected from the top, middle, and bottom of each dosing formulation
`for homogeneity analysis. The mean of the homogeneity values obtained for all locations served
`as the concentration value for that preparation.
`
`6.1.7.3. Stability
`Ten-day stability of the test article in the vehicle control material at concentrations encompassing
`the dosing concentrations utilized in this study has been previously established (Biogen Idec
`Study No. P00012-04-06, Charles River Laboratories Study No. EBA000162 and Biogen Idec
`Study No. P00012-04-10, Charles River Laboratories Study No. EBA000443).
`
`6.1.7.4. Analytical Sample Storage and Shipment
`All samples were stored refrigerated (5 ± 3°C) and protected from light. Duplicate sets of
`samples were shipped overnight on ice packs to the analytical chemistry laboratory, for analysis.
`The remaining sample sets were maintained at the Testing Facility and discarded after
`acceptance of the analytical results.
`The original Week 20 back-up samples from Groups 2 and 3 were not shipped for analysis in
`case a sampling error occurred. New back-up samples for Week 20 were obtained from the
`original formulation and shipped for analysis. The original Week 20 back-up samples were
`maintained by the Testing Facility and discarded after acceptance of the analysis results.
`Two samples from each duplicate preparation (Groups 2-5) for Weeks 22 and 23 were shipped
`for analysis and two samples were retained at the Testing Facility as back-up samples. The two
`preparations for each group were labeled “A” and “B”, and all samples and aliquots taken were
`labeled “A” and “B” to reflect the duplicate preparation from which the sample or aliquot was
`taken.
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`Two samples of randomly selected triplicate preparations for Week 24 (and every 4 weeks
`thereafter) were shipped for analysis and two samples were retained at the Testing Facility as
`back-up samples. The preparations were labeled “A”, “B”, or “C”; the selections were based on
`computer-generated random numbers. The following preparations were selected for shipment
`for analysis.
`
`Page 23
`Testing Facility Study No. EBA00009
`
`Samples
`Samples
`Samples
`Selected
`Selected
`Selected
`Week
`Week
`Week
`2B, 3A, 4A, 5A
`72
`2A, 3A, 4A, 5A
`48
`2B, 3B, 4A, 5B
`24
`2B, 3A, 4B, 5B
`76
`2B, 3B, 4A, 5B
`52
`2B, 3B, 4B, 5A
`28
`2B, 3A, 4B, 5A
`80
`2B, 3A, 4B, 5B
`56
`2B, 3A, 4A, 5C
`32
`1, 2B, 3B, 4, 5
`84
`2B, 3B, 4B, 5B
`60
`2B, 3B, 4C, 5A
`36
`1, 2B, 3A, 4, 5
`88
`2A, 3B, 4A, 5B
`64
`2A, 3C, 4A, 5C
`40
`1, 2A, 3B, 4, 5
`92
`2B, 3A, 4A, 5A
`68
`2B, 3C, 4C, 5B
`44
`Note: For groups that have only one preparation, the preparation was labeled with the group number only
`and was not accompanied by a letter designation. Beginning Week 80 for Group 5 and Week 82 for
`Group 4, only one preparation was made since dosing was discontinued for males in these groups because
`the number of survivors had reached 20 animals/sex/group. By Week 96, the number of surviving animals in
`Group 2 and Group 3 was sufficiently low to allow preparation of a single dosing formulation per group per
`week.
`The duplicate back-up samples from Group 4B for Week 76 were shipped to the analytical
`chemistry laboratory for analysis.
`Due to an assay value which deviated from the required concentration by more than 15%
`(-25.3%) for Group 2 during the Week 104 analysis, the back-up samples for Group 2 were
`shipped for analysis in case an analytical error had occurred. The back-up samples were
`analyzed (-26.0%) and the original analytical results were confirmed, indicating that there were
`no apparent errors in the initial analysis. In addition, the dose formulation records were
`reviewed and there were no apparent calculation errors.
`
`6.2. Test System
`
`6.2.1. Receipt and Description
`A total of 513 male and 513 female Sprague Dawley Crl:CD(SD)IGS BR rats was received in
`three shipments. For the carcinogenicity phase, 310 males and 310 females were received on
`October 12, 2004, and 120 males and 120 females were received on October 14, 2004, from
`Portage, Michigan. For the toxicokinetic phase, 83 males and 83 females were received on
`March 29, 2005, from Charles River Laboratories, Portage, Michigan. The animals were
`examined and weighed following separation from group housing on Day -11 (first shipment for
`the carcinogenicity phase), Day -8 (second shipment for the carcinogenicity phase) or Day -4
`(toxicokinetic phase). The carcinogenicity animals were allowed to acclimate to the laboratory
`environment for a minimum of 12 days prior to the first day of dosing and the toxicokinetic
`animals were allowed to acclimate to the laboratory environment for 7 days prior to the first day
`of dosing.
`
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`Page 24
`Testing Facility Study No. EBA00009
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`6.2.2. justification of Test System/Route
`The Sprague Dawley rat was chosen as the animal model for this study as it is a preferred rodent
`species for preclinical toxicity testing by regulatory agencies. The oral route of exposure was
`selected since this is the intended route of human exposure.
`
`6.2.3. Housing
`The animals were initially group housed (two to three animals/sex/cage) to allow the animals to
`adjust to the automatic watering system. During the remainder of the acclimation period and
`while on study, the animals were housed individually in suspended stainless steel cages. Sentinel
`animals were maintained in the study room housing the carcinogenicity phase animals and
`toxicokinetic phase animals were housed in a separate room. Housing and care were as specified
`in the USDA Animal Welfare Act (9 CFR, Parts 1, 2, and 3) and as described in the Guidefor
`the Care and Use of Laboratory Animals 4
`. Targeted environmental conditions were as follows:
`
`72 ± 7°F (22 ± 4°C)
`Temperature
`Humidity
`50± 20%
`12-hour light/12-hour dark cycle
`Light Cycle
`Ten or more air changes per hour with 100% fresh air
`Air Changes
`Actual room temperature and relative humidity were recorded a minimum of once daily and
`ranged from 63 to 80°F (17 to 2rC) and 30 to 89%, respectively.
`Every 1 to 4 weeks, all racks housing the individual animal cages on one side of the animal room
`were repositioned to the opposite side of the room to equalize animal exposure to any ambient
`zone differences.
`
`6.2.4. Animal Identification
`Foll