FINAL REPORT
`
`Guideline: OECD (451)
`
`Testing Facility Study No. EBA00124
`
`Sponsor Ref. No. P00012-05-03
`
`Two-Year Oral (Gavage) Carcinogenicity Study in Mice 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
`
`30 December 2008
`
`Page 1 of 5083
`
`Page 1 of 32
`
`Biogen Exhibit 2377
`Coalition v. Biogen
`IPR2015-01993
`
`

`

`Sponsor Ref. No. P00012-05-03
`
`Page 9
`Testing Facility Study No. EBA00124
`
`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 (MHLW) Ordinance No. 21 with the following
`exception(s):
`
`Animals were received (07 June 2005) before the protocol was signed (16 June 2005).
`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 of32
`
`

`

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`Sponsor Ref. No. P00012-05-03
`
`
`
`
`Page 10
`Testing Facility Study No. EBA00124
`
`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:
`
`QA INSPECTION DATES
`
`
`
`
`
`Phase(s) Inspected
`
`
`Protocol Review
`
`Animal Receipt
`Dose Preparation
`Dosing
`Blood Collection – Bioanalytical
`Bioanalytical Sample Processing
`Protocol Amendment Review
`Bioanalytical Sample Shipping
`Protocol Amendment Review
`Blood Collection – Bioanalytical –
`5 and 10 minutes
`Bioanalytical Sample Processing –
`5 and 10 minutes
`Bioanalytical Sample Shipping
`Dose Preparation
`Dosing
`Protocol Amendment Review
`Blood Collection – Bioanalytical
`Bioanalytical Sample Processing
`Bioanalytical Sample Shipping
`Dose Preparation
`Dosing
`Data Audit
`
`Dose Preparation
`Dosing
`Clinical Observations
`Palpable Masses
`Data Audit
`
`Dates of Inspection
`
`24-May-2005,
`25-May-2005,
`16-Jun-2005
`07-Jun-2005
`28-Jun-2005
`28-Jun-2005
`28-Jun-2005
`28-Jun-2005
`28-Jun-2005
`05-Jul-2005
`13-Jul-2005
`25-Sep-2005
`
`25-Sep-2005
`
`27-Sep-2005
`27-Sep-2005
`04-Oct-2005
`27-Oct-2005
`24-Dec-2005
`24-Dec-2005
`27-Dec-2005
`31-Jan-2006
`01-Feb-2006
`25-Mar-2006,
`28-Mar-2006
`28-Mar-2006
`28-Mar-2006
`28-Mar-2006
`28-Mar-2006
`14-Apr-2006,
`18-Apr-2006,
`02-May-2006
`15-May-2006
`15-May-2006
`06-Jun-2006
`21-Jun-2006
`21-Jun-2006
`
`Date Findings Submitted to:
`Study Director
`Management
`
`22-Jun-2005
`
`Study Director
`
`22-Jun-2005
`
`22-Jun-2005
`28-Jun-2005
`28-Jun-2005
`28-Jun-2005
`28-Jun-2005
`28-Jun-2005
`11-Jul-2005
`13-Jul-2005
`04-Oct-2005
`
`22-Jun-2005
`28-Jun-2005
`28-Jun-2005
`28-Jun-2005
`28-Jun-2005
`28-Jun-2005
`11-Jul-2005
`13-Jul-2005
`04-Oct-2005
`
`04-Oct-2005
`
`04-Oct-2005
`
`04-Oct-2005
`04-Oct-2005
`04-Oct-2005
`27-Oct-2005
`24-Dec-2005
`24-Dec-2005
`27-Dec-2005
`01-Feb-2006
`01-Feb-2006
`28-Mar-2006
`
`28-Mar-2006
`28-Mar-2006
`28-Mar-2006
`28-Mar-2006
`02-May-2006
`
`04-Oct-2005
`04-Oct-2005
`04-Oct-2005
`27-Oct-2005
`24-Dec-2005
`24-Dec-2005
`27-Dec-2005
`01-Feb-2006
`01-Feb-2006
`28-Mar-2006
`
`28-Mar-2006
`28-Mar-2006
`28-Mar-2006
`28-Mar-2006
`02-May-2006
`
`Data Audit
`Data Audit
`Data Audit
`Blood Collection – Serology
`Sample Processing – Serology
`
`06-Jun-2006
`19-Jun-2006
`19-Jun-2006
`21-Jun-2006
`21-Jun-2006
`
`06-Jun-2006
`19-Jun-2006
`19-Jun-2006
`21-Jun-2006
`21-Jun-2006
`
`Page 3 of 32
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`

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`Sponsor Ref. No. P00012-05-03
`
`
`
`
`Page 11
`Testing Facility Study No. EBA00124
`
`Phase(s) Inspected
`
`
`Dose Preparation
`Dosing
`Protocol Amendment Review
`Body Weights
`Data Audit
`
`Dose Preparation
`Dosing
`Food Consumption
`Protocol Amendment Review
`Protocol Amendment Review
`Dose Preparation
`Dosing
`Clinical Observations
`Palpable Masses
`Protocol Amendment Review
`Data Audit
`Data Audit
`
`Data Audit
`Data Audit
`
`Dose Preparation
`Analytical Sampling
`Tissue Trimming
`Data Audit
`
`Data Audit
`Data Audit
`
`Data Audit
`Data Audit
`Data Audit
`Processing and Embedding
`Protocol Amendment Review
`Data Audit
`
`Date Findings Submitted to:
`Study Director
`Management
`
`01-Aug-2006
`01-Aug-2006
`10-Aug-2006
`22-Aug-2006
`14-Sep-2006
`
`Study Director
`
`01-Aug-2006
`01-Aug-2006
`10-Aug-2006
`22-Aug-2006
`14-Sep-2006
`
`26-Sep-2006
`26-Sep-2006
`26-Sep-2006
`13-Oct-2006
`09-Nov-2006
`09-Jan-2007
`09-Jan-2007
`09-Jan-2007
`09-Jan-2007
`09-Jan-2007
`10-Jan-2007
`25-Jan-2007
`
`26-Sep-2006
`26-Sep-2006
`26-Sep-2006
`13-Oct-2006
`09-Nov-2006
`09-Jan-2007
`09-Jan-2007
`09-Jan-2007
`09-Jan-2007
`09-Jan-2007
`10-Jan-2007
`25-Jan-2007
`
`22-Feb-2007
`27-Feb-2007
`
`22-Feb-2007
`27-Feb-2007
`
`20-Mar-2007
`20-Mar-2007
`22-Mar-2007
`26-Mar-2007
`
`20-Mar-2007
`20-Mar-2007
`22-Mar-2007
`26-Mar-2007
`
`29-Mar-2007
`09-Apr-2007
`
`29-Mar-2007
`09-Apr-2007
`
`03-Apr-2007
`09-Apr-2007
`09-Apr-2007
`09-Apr-2007
`04-May-2007
`30-May-2007
`
`03-Apr-2007
`09-Apr-2007
`09-Apr-2007
`09-Apr-2007
`04-May-2007
`30-May-2007
`
`
`
`
`
`Dates of Inspection
`
`01-Aug-2006
`01-Aug-2006
`10-Aug-2006
`22-Aug-2006
`28-Aug-2006,
`29-Aug-2006,
`31-Aug-2006,
`11-Sep-2006,
`14-Sep-2006
`26-Sep-2006
`26-Sep-2006
`26-Sep-2006
`13-Oct-2006
`09-Nov-2006
`26-Dec-2006
`09-Jan-2007
`09-Jan-2007
`09-Jan-2007
`09-Jan-2007
`09-Jan-2007
`24-Jan-2007,
`25-Jan-2007
`01-Feb-2007
`22-Feb-2007,
`23-Feb-2007
`20-Mar-2007
`20-Mar-2007
`21-Mar-2007
`23-Mar-2007,
`26-Mar-2007
`23-Mar-2007
`02-Apr-2007,
`03-Apr-2007,
`09-Apr-2007
`03-Apr-2007
`03-Apr-2007
`09-Apr-2007
`09-Apr-2007
`04-May-2007
`23-May-2007,
`24-May-2007,
`25-May-2007,
`29-May-2007,
`30-May-2007
`29-May-2007,
`30-May-2007,
`31-May-2007
`
`
`Data Audit
`
`31-May-2007
`
`31-May-2007
`
`
`
`
`
`
`
`Page 4 of 32
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`

`

`
`Sponsor Ref. No. P00012-05-03
`
`
`
`
`Page 12
`Testing Facility Study No. EBA00124
`
`
`
`
`
`Dates of Inspection
`
`18-Jun-2007,
`19-Jun-2007,
`20-Jun-2007,
`21-Jun-2007
`26-Jun-2007
`27-Jun-2007
`27-Jun-2007
`28-Jun-2007
`28-Jun-2007
`28-Jun-2007
`17-Jul-2007
`19-Jul-2007
`07-Aug-2007
`21-Aug-2007,
`22-Aug-2007,
`23-Aug-2007,
`08-Oct-2007
`22-Aug-2007,
`24-Aug-2007
`23-Aug-2007,
`24-Aug-2007
`29-Aug-2007,
`30-Aug-2007
`31-Aug-2007,
`26-Sep-2007
`31-Aug-2007,
`27-Sep-2007,
`28-Sep-2007
`01-Sep-2007,
`02-Sep-2007,
`09-Sep-2007,
`21-Sep-2007,
`22-Sep-2007,
`23-Sep-2007,
`26-Sep-2007,
`28-Sep-2007,
`02-Oct-2007
`10-Oct-2007,
`11-Oct-2007
`12-Oct-2007
`12-Oct-2007,
`15-Oct-2007
`15-Oct-2007
`02-Nov-2007
`23-Jan-2008
`
`
`
`
`Phase(s) Inspected
`
`
`Data Audit
`
`Date Findings Submitted to:
`Study Director
`Management
`
`21-Jun-2007
`
`Study Director
`
`21-Jun-2007
`
`Dose Preparation
`Protocol Amendment Review
`Dosing
`Blood Collection – Clinical Pathology
`Hematology
`Necropsy
`Protocol Amendment Review
`Data Audit
`Microtomy
`Data Audit
`
`28-Jun-2007
`27-Jun-2007
`28-Jun-2007
`28-Jun-2007
`28-Jun-2007
`28-Jun-2007
`17-Jul-2007
`19-Jul-2007
`07-Aug-2007
`12-Oct-2007
`
`28-Jun-2007
`27-Jun-2007
`28-Jun-2007
`28-Jun-2007
`28-Jun-2007
`28-Jun-2007
`17-Jul-2007
`19-Jul-2007
`07-Aug-2007
`12-Oct-2007
`
`Data Audit
`
`Data Audit
`
`Data Audit
`
`Data Audit
`
`Data Audit
`
`Data Audit
`
`24-Aug-2007
`
`24-Aug-2007
`
`24-Aug-2007
`
`24-Aug-2007
`
`30-Aug-2007
`
`30-Aug-2007
`
`26-Sep-2007
`
`26-Sep-2007
`
`09-Oct-2007
`
`09-Oct-2007
`
`09-Oct-2007
`
`09-Oct-2007
`
`Data Audit
`
`12-Oct-2007
`
`12-Oct-2007
`
`Staining and Coverslipping
`Data Audit
`
`12-Oct-2007
`19-Oct-2007
`
`12-Oct-2007
`19-Oct-2007
`
`Data Audit
`Histology Quality Control
`Protocol Amendment Review
`
`
`
`
`17-Oct-2007
`02-Nov-2007
`23-Jan-2008
`
`
`
`
`17-Oct-2007
`02-Nov-2007
`23-Jan-2008
`
`
`
`
`Page 5 of 32
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`

`

`Sponsor Ref. No. P00012-05-03
`
`Dates of Inspection
`
`Phase(s) Inspected
`
`Page 13
`Testing Facility Study No. EBA00124
`
`Date Findings Submitted to:
`Study Director
`Management
`
`Study Director
`
`10-Apr-2008,
`11-Apr-2008,
`23-Apr-2008,
`24-Apr-2008,
`25-Apr-2008
`30-Apr-2008
`18-Dec-2008,
`19-Dec-2008
`
`Draft Report Review
`
`25-Apr-2008
`
`25-Apr-2008
`
`Data Audit
`Final Report Review
`
`30-Apr-2008
`19-Dec-2008
`
`30-Apr-2008
`19-Dec-2008
`
`QA statement(s) provided by the fo llowing test site(s) have been reviewed:
`
`Test Site(s)
`Charles River Laboratories,
`Preclinical Services
`Charles River Laboratories
`
`Charles River Laboratories
`Charles River Laboratories,
`Pathology Associates
`
`Charles River Laboratories,
`Pathology Associates
`
`BioSTAT Consultants, Inc.
`
`Charles River Laboratories,
`Preclinical Services
`
`Phase
`
`QA Statement Location
`
`Analytical Chemistry Report
`
`Appendix 3
`
`Pretest Health Screen (Serology)
`
`Sentinel Animals (Serology)
`
`Histopathology Report
`
`Appendix 5
`
`Appendix 6
`
`Appendix 14
`
`Histopathology Peer Review
`
`Appendix 14
`
`Statistical Analysis of Survival
`and Tumor Incidence
`
`Appendix IS
`
`Bioanalytical Report
`
`Appendix 19
`
`Biogen Idee Inc.
`
`Toxicokinetic Interpretive Report
`
`Appendix 20
`
`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.
`
`~ Matthew D. Bruns, B.S.
`
`..2~ -i::>EC--20:.8
`Date
`
`Quality Assurance Auditor I
`Charles River Laboratories
`Preclinical Services
`
`Page 6 of32
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`

`

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`Sponsor Ref. No. P00012-05-03
`
`
`
`
`Page 15
`Testing Facility Study No. EBA00124
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`4. SUMMARY
`
`The purpose of this study was to evaluate the potential carcinogenicity of BG00012 following
`once daily oral gavage to CD-1 mice for at least 104 weeks and toxicokinetics following once
`daily oral gavage for 180 days. The study design was as follows:
`
`Experimental Design for the Carcinogenicity and Toxicokinetic Phases
`
`No. of Animals
`(Toxicokinetic Phase)
`
`Males
`
`Females
`
`75 (12)
`
`75 (12)
`
`Dosage
`Material
`HPMCa
`
`75 (30)
`
`75 (30)
`
`BG00012
`
`75 (30)
`
`75 (30)
`
`BG00012
`
`Group
`No.
`
`1
`
`2
`
`3
`
`BG00012
`Dose Level
`(mg/kg/day)
`
`BG00012
`Dose Volume
`(mL/kg)
`
`BG00012
`Dose Conc.
`(mg/mL)
`
`0
`
`25
`
`75
`
`10
`
`10
`
`10
`
`0
`
`2.5
`
`7.5
`
`75 (30)
`
`BG00012
`
`10
`
`4
`
`75 (30)
`
`20
`200
`60/40b
`600/400b
`5
`75 (30)
`75 (30)
`BG00012
`10
`aHydroxypropylmethylcellulose or Hypromellose (3,500-5,600 cps), 0.8% w/v in reverse osmosis
` deionized water.
`bDue to mortality observed in the high-dose group, the dose level for Group 5 was decreased to
` 400 mg/kg/day (40 mg/mL) beginning on Day 9.
`
`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 microscopic evaluation.
`
`Results:
`
`Test article-related mortality was observed in the 600 mg/kg/day dose group (Group 5) during
`the first week of treatment. Fifteen males and thirteen females in this dose group were found
`dead during Days 5-8. As a result, Group 5 animals were not dosed on Days 6-8. Dosing
`resumed on Day 9 in Group 5 animals (60 males and 62 females) at a dose of 400 mg/kg/day.
`Survival was generally comparable to controls at the lower dose of 400 mg/kg/day. Group 5
`(600/400 mg/kg/day) animals were euthanized 3 weeks earlier than the scheduled necropsy
`because the number of survivors had declined to ten animals/sex. Group 4 (200 mg/kg/day)
`males had slightly greater mortality compared to controls, whereas the survival of Group 4
`(200 mg/kg/day) females was comparable to controls. Survival was comparable in controls and
`in animals of Group 2 (25 mg/kg/day) and Group 3 (75 mg/kg/day).
`
`There were no clear clinical signs associated with BG00012 administration. Body weights of the
`Group 5 (600/400 mg/kg/day) animals were significantly lower (5.0 to 7.3%) than those of
`controls by Week 2, with a corresponding decrease in food consumption in this group from
`Weeks 1 to 2. Following the brief dosing holiday and the resumption of dosing at the lower dose
`(400 mg/kg/day), body weights and food consumption of the high-dose animals were more
`comparable to those of controls. No effects on body weights or food consumption were observed
`in other BG00012-treated groups.
`
`Page 7 of 32
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`

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`Sponsor Ref. No. P00012-05-03
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`
`
`
`Page 16
`Testing Facility Study No. EBA00124
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`The Group 4 (200 mg/kg/day) and Group 5 (600/400 mg/kg/day) males had lower erythrocytes,
`hemoglobin, and hematocrit compared to controls, and Group 5 (600/400 mg/kg/day) males and
`females had higher total leukocytes, lymphocytes, segmented neutrophils, monocytes, and
`eosinophils compared to controls. There were no test article-related differences in red cell
`morphology.
`
`Gross necropsy findings were observed primarily in the kidneys and stomach of test
`article-treated animals. In the kidney, findings included enlargement, roughened surface, and
`discoloration and were generally higher in incidence at dose levels of 75 mg/kg/day and above
`(Groups 3-5) in males, whereas these findings were generally not present at a higher incidence in
`test article-treated females compared to control females. In the stomach, prominent epithelial
`surface was observed at a low incidence in control males and females, and was observed at an
`increased incidence in test article-treated animals at doses of 75 mg/kg/day and above
`(Groups 3-5). These findings corresponded to microscopic findings of squamous epithelial
`hyperplasia, squamous cell papilloma, and squamous cell carcinoma of the nonglandular
`forestomach.
`
`BG00012-induced non-neoplastic microscopic findings included a dose-dependent increased
`severity of nephropathy, particularly in males, as well as an increased incidence of nonglandular
`stomach lesions such as squamous cysts, hyperkeratosis, chronic-active inflammation, erosions,
`and ulcers and inflammation that extended into the submucosa and the serosa. In addition, a
`dose-related increase in incidence and/or severity of retinal degeneration was observed in both
`sexes at the two highest doses evaluated (200 and 600/400 mg/kg/day).
`
`BG00012 administration also resulted in a dose-related increased incidence of squamous cell
`papillomas and squamous cell carcinomas of the nonglandular stomach (i.e., the forestomach) in
`all dose groups. There were statistically significant dose-related increasing trends in the onset of
`leiomyosarcoma, papilloma, squamous cell carcinoma, and the combination papilloma/squamous
`cell carcinoma in males and females. In pairwise comparisons with controls, there were
`statistically significant increases in the onset of papilloma and the combination
`papilloma/squamous cell carcinoma in both the 200 mg/kg/day (Group 4) and
`600/400 mg/kg/day (Group 5) groups. There was also a statistically significant increase in the
`onset of squamous cell carcinoma in the 600/400 mg/kg/day group.
`
`In the kidney of male mice, there were statistically significant dose-related increasing trends in
`the onset of adenoma, carcinoma, and the combination adenoma/carcinoma. In pairwise
`comparisons with controls, there were increases in the onset of carcinoma and the combination
`adenoma/carcinoma in both the 200 mg/kg/day (Group 4) and 600/400 mg/kg/day (Group 5)
`animals. In the kidney of female mice, there was a statistically significant dose-related
`increasing trend in the onset of adenoma and thus, for the combination adenoma/carcinoma.
`Only one kidney carcinoma was found in any of the treated females; this carcinoma was found in
`a 600/400 mg/kg/day (Group 5) female that also had an adenoma. In pairwise comparisons with
`controls, there was an increase in the onset of adenoma (and in the combination
`adenoma/carcinoma) in the 600/400 mg/kg/day group.
`
`Page 8 of 32
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`Sponsor Ref. No. P00012-05-03
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`
`Page 17
`Testing Facility Study No. EBA00124
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`Together with the observation of increased incidence of renal tumors was an exacerbation of
`age-related nephropathy in the mouse, most prominently described in male mice. The multiple
`complex changes in the kidney were given the summary diagnosis of nephropathy; however, it is
`noted that changes in the outer stripe and medullary ray of the kidney may be BG00012-related
`alterations. Thus, renal injury eventually leading to increased renal tumor incidence in this study
`may be due to BG00012 exacerbation of nephropathy, BG00012 specific renal injury, or a
`combination of the two factors. It is apparent, however, that BG00012 elicits an exacerbation of
`nephropathy in both the mouse and also in the rat carcinogenicity assessment (Biogen Idec Study
`No. P00012-04-11, Charles River Study No. EBA00009). In female mice, where the
`incidence/severity of nephropathy was less apparent than in males, the dose of BG00012
`required to significantly increase renal tumor incidence was higher, at 600/400 mg/kg/day, as
`compared to the male mouse, that had increased incidence at both 200 and 600/400 mg/kg/day.
`
`Exposures associated with doses in males and females that did not significantly increase renal
`tumor incidence are approximately 1X and 4X by AUC over the highest intended clinical dose of
`BG00012.
`
`Conclusion:
`
`Under the conditions of this study, administration of BG00012 by gavage resulted in little effect
`on body weight gain and overall survival in mice, after an early dose reduction from
`600 mg/kg/day to 400 mg/kg/day based upon acute mortality. Non-neoplastic microscopic
`findings in treated animals were observed in the kidney and nonglandular stomach, and a
`dose-related increase in incidence and/or severity of retinal degeneration of the eyes was
`observed in both sexes at the two highest doses. There were dose-related increases in
`hyperplasia of the squamous epithelium, squamous cell papilloma, and squamous cell carcinoma
`of the nonglandular stomach, accompanied by the presence of leiomyosarcoma and fibrosarcoma
`at the high dose, although lesions of the rodent nonglandular stomach are not generally
`considered relevant in human risk assessment. Increased incidence of renal tumors was observed
`in male mice at the 200 and 600/400 mg/kg/day dose levels and in female mice at the
`600/400 mg/kg/day dose level, as evaluated by pairwise statistical comparisons to vehicle control
`groups. Together with this finding were a BG00012-mediated exacerbation of an age-related
`nephropathy, most prominently observed in male mice, and changes in the medullary ray and
`outer stripe of the kidney that may have been BG00012-related. Renal injury eventually leading
`to increased renal tumor incidence in this study may have been due to BG00012 exacerbation of
`nephropathy, BG00012 specific renal injury or a combination of the two factors. Exposures
`associated with doses in males and females that did not significantly increase renal tumor
`incidence are approximately 1X and 4X by AUC over the highest intended clinical dose of
`BG00012.
`
`Page 9 of 32
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`

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`Sponsor Ref. No. P00012-05-03
`
`
`
`
`Page 18
`Testing Facility Study No. EBA00124
`
`5.
`
`INTRODUCTION
`
`The purpose of this study was to evaluate the potential carcinogenicity of BG00012 following
`once daily oral gavage to CD-1 mice for at least 104 weeks and toxicokinetics following once
`daily oral gavage for 180 days.
`
`The protocol was signed by the Study Director on 16 June 2005 (GLP initiation date). The
`experimental start date for the study was 07 June 2005 (animal receipt) and the experimental
`completion date for the study was 16 December 2008. The in-life phase of the study was
`initiated on 28 June 2005 and the in-life completion date was 11 July 2007.
`
`6. MATERIALS AND METHODS
`
`The study protocol, protocol amendments, 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%
`14-Oct-2004
`21-Jul-2007
`White crystalline powder
`Room temperature, protected from light
`Sponsor
`
`1102643 33004999
`S06.005.EBA
`99.8%
`12-Jul-2006
`21-Jul-2007
`White crystalline powder
`Room temperature, protected from light
`Sponsor
`
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`Page 19
`Testing Facility Study No. EBA00124
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`6.1.2. Control Article
`
`The control material, HPMC (hydroxypropylmethylcellulose or hypromellose), was received
`from Sigma-Aldrich Inc., stored at room temperature, and identified as follows:
`
`Control
`Article
`Identification
`
`Lot
`Number
`
`Assigned
`Testing
`Facility ID
`
`Receipt
`Date
`
`Testing
`Facility
`Expiration
`Date
`
`Apparent
`Viscositya
`(cps)
`
`HPMC
`
`HPMC
`
`HPMC
`
`HPMC
`
`HPMC
`
`HPMC
`
`122K0149
`
`V04.088
`
`16-Aug-2004
`
`31-Dec-2014
`
`013K0621
`
`V04.117
`
`18-Nov-2004
`
`31-Dec-2014
`
`122K0149
`
`V05.010
`
`23-Feb-2005
`
`31-Dec-2015
`
`013K0621
`
`V05.042
`
`19-May-2005
`
`19-May-2015
`
`103K0135
`
`V05.066
`
`04-Aug-2005
`
`31-Dec-2015
`
`103K0135
`
`V05.080
`
`09-Sep-2005
`
`31-Dec-2015
`
`5163
`
`4508
`
`5163
`
`4508
`
`5153
`
`5153
`
`Physical
`Description
`
`White powder
`
`White powder
`
`White powder
`
`White powder
`
`White powder
`
`White powder
`
`HPMC
`
`HPMC
`
`HPMC
`
`HPMC
`
`HPMC
`
`HPMC
`
`045K0051
`
`V05.092
`
`06-Oct-2005
`
`31-Dec-2015
`
`103K0135
`
`V05.093
`
`06-Oct-2005
`
`31-Dec-2005
`
`045K0051
`
`V05.101
`
`19-Oct-2005
`
`31-Dec-2015
`
`045K0054
`
`V06.001
`
`03-Jan-2006
`
`31-Dec-2016
`
`045K0054
`
`V06.047
`
`09-May-2006
`
`31-Dec-2016
`
`045K0054
`
`V06.051
`
`12-May-2006
`
`31-Dec-2016
`
`4616
`
`5153
`
`4616
`
`4104
`
`4104
`
`4104
`
`4104
`31-Dec-2016
`20-Nov-2006
`V06.121
`045K0054
`HPMC
`aVendor’s stated apparent viscosity range: 3,500 – 5,600 cps (2% aqueous solution at 20°C).
`
`White powder
`
`White powder
`
`White powder
`
`White powder
`
`White powder
`
`White powder
`
`White powder
`
`6.1.3. Test Article Characterization
`
`Certificates of Analysis for the test and control articles 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.
`
`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.
`
`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
`
`Page 11 of 32
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`
`Page 20
`Testing Facility Study No. EBA00124
`
`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
`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 the 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 a pre-calibrated beaker and the mortar was rinsed with vehicle. An
`appropriate quantity of the vehicle was then added to achieve the desired concentration. A
`magnetic stir bar was added and the dosing formulation was stirred for a minimum of
`30 minutes. The beaker was wrapped in aluminum foil and the dosing formulation was stirred
`continuously. Each formulation was dispensed into individual amber glass containers for daily
`dosing. The dosing formulations were prepared 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 (approximately 30 minutes). 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 cloudy white suspensions, and the Group 4 and 5 test article formulations
`were white suspensions.
`
`To accommodate the dose preparation schedule, the dose formulations prepared on Day 50 were
`utilized for 8 days of dosing. On Day 99, the formulations were found to be frozen due to a
`technical failure in the cold room; these formulations were dosed on Day 99; however, new
`formulations were prepared for the following day. Due to insufficient quantity of the dose
`formulations prepared on Day 505, additional dose formulations were prepared and dispensed on
`Day 512 and for the following week.
`
`Page 12 of 32
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`Sponsor Ref. No. P00012-05-03
`
`
`
`
`Page 21
`Testing Facility Study No. EBA00124
`
`6.1.7. Analysis of Dose Formulations
`
`Dose formulation samples were collected for analysis as indicated in the following table:
`
`Dose Formulation Samples for Analysis
`
`Time Point
`
`Concentration
`
`Homogeneity
`
`Stability
`
`Week 1
`
`Week 2
`
`3 Months
`
`6 Months
`
`9 Months
`
`12 Months
`
`15 Months
`
`18 Months
`
`21 Months
`
`24 Months
`
`Groups 1-5
`Groups 1-5
`N/A
`Group 5 (40 mg/mL)a Group 5 (40 mg/mL)a Group 5 (60 mg/mL)b
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-4
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-5
`
`Groups 1-4
`
`N/A
`
`N/A
`
`N/A
`
`N/A
`
`N/A
`
`N/A
`
`N/A
`
`N/A
`
`Note: N/A = not applicable.
`aAfter 5 days of dosing, the 600 mg/kg/day dose (60 mg/mL) was discontinued due to excessive
` mortality. The Group 5 dose level was changed to 400 mg/kg/day on Day 9. The 400 mg/kg/day
` dose level (40 mg/mL) was analyzed for concentration verification and homogeneity on Week 2.
`bThe Sponsor has stability data for concentration ranges encompassing the 400 mg/kg/day dose
` level (40 mg/mL). Stability was only conducted on the 600 mg/kg/day dose level (60 mg/mL).
`
`6.1.7.1. Concentration
`
`Four 1-mL samples were collected from the dosing formulations for Groups 1-5 on Week 1 and
`Months 3, 6, 9, 12, 15, 18, and 21; Groups 1-4 on Month 24; and Group 5 (40 mg/mL) on
`Week 2 for concentration analysis.
`
`6.1.7.2. Homogeneity
`
`Four 1-mL samples were collected from the top, middle, and bottom of the dosing formulations
`for Groups 1-5 on Week 1 and Months 3, 6, 9, 12, 15, 18, and 21; Groups 1-4 on Month 24; and
`Group 5 (40 mg/mL) on Week 2 for homogeneity analysis.
`
`6.1.7.3. Stability
`
`As per Sponsor, stability of the test article concentrations encompassing the dosing
`concentrations utilized in this study up to 40 mg/mL has been previously established and is
`included in Appendix 3.
`
`For the 60 mg/mL concentration, four 1-mL samples were collected for 1 day at room
`temperature and for 10 days at 5 ± 3ºC from the Group 5 dosing formulations for stability
`analysis.
`
`Page 13 of 32
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`
`Sponsor Ref. No. P00012-05-03
`
`
`
`
`Page 22
`Testing Facility Study No. EBA00124
`
`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 for concentration and homogeneity analysis were maintained at the
`Testing Facility and discarded after acceptance of the analytical results. The remaining sample
`sets for stability analysis were shipped overnight on ice packs to the analytical chemistry
`laboratory and stored with the stability samples and discarded after acceptance of the analytical
`results.
`
`The 12-month concentration and homogeneity back-up samples for all groups (due to technical
`difficulties with the analytical instruments), and the 24-month homogeneity Group 4 back-up
`samples (due to out-of-specification results) were shipped to the analytical chemistry laboratory.
`
`6.2. Test System
`
`6.2.1. Receipt and Description
`
`A total of 572 male and 572 female CD-1 [Crl:CD-1®(ICR)Br] mice was received on 07 June
`2005, from Charles River Laboratories, Raleigh, North Carolina. The animals were examined
`and weighed on the day following receipt, and were all allowed to acclimate to the laboratory
`environment for 21 days prior to the first day of dosing.
`
`6.2.2. Justification of Test System/Route
`
`The CD-1 mouse was chosen as the animal model for this study as it is a preferred rodent species
`for nonclinical oral administration evaluations. The oral route of exposure was selected since
`this is the intended route of human exposure.
`
`6.2.3. Housing
`
`The animals were housed individually in suspended stainless steel cages during acclimation and
`while on study. Housing and care were as specified in the USDA Animal Welfare Act (9 CFR,
`Parts 1, 2, and 3) and as described in the Guide for the Care and Use of Laboratory Animals1.
`Targeted environmental conditions were as follows:
`
`Temperature
`Humidity
`Light Cycle
`
`Air Changes
`
`72 ± 7ºF (22 ± 4ºC)
`50 ± 20%
`12-hour light/12-hour dark cycle, except when the
`room light cycle was disrupted to accommodate study
`procedures
`Ten or more air changes per hour with 100% fresh air
`(no recirculation)
`
`Actual room temperature and relative humidity were recorded a minimum of once daily and
`ranged from 64 to 75°F (18 to 24°C) and 25 to 84%, respectively.
`
`Page 14 of 32
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`
`
`
`
`Page 23
`Testing Facility Study No. EBA00124
`
`Every 2 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
`
`Each animal was identified with a temporary identification number recorded on the cage card.
`The pretest health screen animals were identified by a tail mark using an indelible marker.
`
`
`
`
`
`
`
`
`6.2.5. Food
`
`PMI Nutrition International Certified Rodent Chow® #5002 (powdered and/or pelleted) was
`provided ad libitum throughout the study. The lot number and expiration date of each batch of
`diet used during the study were recorded. The feed was analyzed by the supplier for nutritional
`components and environmental contaminants. Results of the dietary analyses were provided by
`the manufacturer and are maintained on file at the Testing Facility. Based on these results, there
`were no contaminants that would interfere with the conduct or interpretation of the study.
`
`6.2.6. Water
`
`Municipal tap water following treatment by reverse osmosis and ultraviolet irradiation was
`available ad libitum throughout the study. Water was supplied to the animals by an automatic
`watering system (or water bottles, when clinical signs warranted). The water is periodically
`analyzed for total dissolved solids, hardness, microbiological content, and various potential
`environmental contaminants. Results of these analyses are maintained on file at the Testing
`Facility. Based on these results, there were no contaminants that would interfere with the
`conduct or interpretation of the study.
`
`6.2.7. Veterinary Care
`
`Veterinary care was available throughout the study and animals were examined by the veterinary
`staff as warranted by clinical signs or other changes. Any veterinary examinations and
`recommended therapeutic treatments were documented in the study records. Veterinary
`medicinal treatments are listed in Appendix 4. The veterinary treatments were undertaken in an
`attempt to improve animal health/welfare. These treatme