`
`
`
`
`
`FINAL REPORT
`Volume 1 of 5
`
`Testing Facility Study No. EBA00066
`Sponsor Study No. P00012-05-05
`
`BG00012: An 11-Month Toxicity Study of BG00012 Administered by the
`Oral (Capsule) Route to Dogs with a 1-Month Recovery Period
`
`TESTING FACILITY:
`
`Charles River Laboratories
`Preclinical Services
`640 North Elizabeth Street
`Spencerville, OH 45887
`
`SPONSOR:
`
`Biogen Idec, Inc.
`14 Cambridge Center
`Cambridge, MA 02142
`
`April 9, 2007
`
`Page 1 of 1844
`
`Page 1 of 21
`
`Biogen Exhibit 2275
`Coalition v. Biogen
`IPR2015-01993
`
`

`

`Sponsor Study No. P00012-05-05
`
`Page9
`Testing Facility Study No. EBA00066
`
`1. CO~LUNCESTATEMENT
`This study was conducted in compliance with the Good Laboratory Practice (GLP) regulations as
`described by the FDA (21 CFR Part 58); and the Organisation for Economic Cooperation and
`Development (OECD) Principles of Good Laboratory Practice, C(97)186/Fina1 with the
`following exception(s):
`
`Characterization and stability analyses of the bulk test article were conducted in compliance with
`GMP regulations.
`
`Toxicokinetic evaluation and reporting and histopathology peer review, conducted by the
`Sponsor, were not in complete compliance with GLP regulations.
`
`The lack ofGLP compliance of the above portions ofthe study was not considered to impact the
`validity of the study results.
`
`Mar A. Morse, Ph.D., DABT
`Study Director
`Charles River Laboratories
`Preclinical Services
`
`LJ!-r/o?
`Date
`
`Page 2 of21
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`Page 10
`Testing Facility Study No. EBA00066
`
`
`Sponsor Study No. P00012-05-05
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`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); and
`OECD Principles of Good Laboratory Practice, C(97)186/Final. Reports were submitted in
`accordance with Standard Operating Procedures as follows:
`
`
`
`Dates of Inspection
`09/19/05
`10/13/05
`10/16/05
`10/18/05
`10/18/05
`10/18/05
`10/18/05
`10/18/05
`10/18/05
`10/24/05
`11/21/05
`12/16/05
`12/19/05, 12/29/05
`12/30/05, 01/03/06
`01/04/06, 02/07/06
`01/13/06
`01/13/06
`01/23/06
`01/26/06
`02/07/06, 02/22/06
`03/07/06, 03/08/06
`04/11/06, 04/17/06,
`04/22/06, 10/30/06,
`11/09/06, 11/10/06
`04/14/06
`04/14/06
`04/17/06
`04/17/06
`04/24/06
`04/24/06
`05/06/06, 05/15/06
`05/08/06
`05/09/06, 05/12/06
`05/20/06
`06/02/06, 06/05/06,
`10/09/06
`06/27/06
`07/17/06
`07/17/06
`
`QA INSPECTION DATES
`
`
`Phase(s) Inspected
`Protocol Review
`Protocol Amendment Review
`Data Audit
`Dose Preparation
`Dosing
`Blood Collection – Bioanalytical (4 hour)
`Bioanalytical Sample Processing (4 hour)
`Blood Collection – Bioanalytical (4.5 hour)
`Bioanalytical Sample Processing (4.5 hour)
`Bioanalytical Sample Shipping
`Protocol Amendment Review
`Data Audit
`Data Audit
`Data Audit
`Data Audit
`Blood Collection – Clinical Pathology
`Hematology
`Dose Preparation
`Dosing
`Data Audit
`Data Audit
`Data Audit
`
`Date Findings Submitted to:
`
`Study Director
`Study Director
`Management
`10/11/05
`10/11/05
`11/23/05
`11/23/05
`10/21/05
`10/21/05
`10/21/05
`10/21/05
`11/23/05
`11/23/05
`10/21/05
`10/21/05
`10/21/05
`10/21/05
`11/23/05
`11/23/05
`11/23/05
`11/23/05
`11/23/05
`11/23/05
`04/14/06
`04/14/06
`12/20/05
`12/20/05
`01/26/06
`01/26/06
`02/07/06
`02/07/06
`02/10/06
`02/10/06
`01/13/06
`01/13/06
`01/13/06
`01/13/06
`01/26/06
`01/26/06
`01/26/06
`01/26/06
`11/01/06
`11/01/06
`03/08/06
`03/08/06
`11/10/06
`11/10/06
`
`Blood Collection – Clinical Pathology
`Clinical Chemistry
`Blood Collection – Bioanalytical
`Bioanalytical Sample Processing
`Bioanalytical Sample Shipping
`Dosing
`Data Audit
`Dose Preparation
`Protocol Amendment Review
`Data Audit
`Data Audit
`
`Protocol Amendment Review
`Blood Collection – Bioanalytical
`Bioanalytical Sample Processing
`
`04/14/06
`04/14/06
`04/17/06
`04/17/06
`04/27/06
`04/27/06
`05/15/06
`05/08/06
`05/12/06
`05/20/06
`10/09/06
`
`06/27/06
`07/17/06
`07/17/06
`
`04/14/06
`04/14/06
`04/17/06
`04/17/06
`04/27/06
`04/27/06
`05/15/06
`05/08/06
`05/12/06
`05/20/06
`10/09/06
`
`06/27/06
`07/17/06
`07/17/06
`
`Page 3 of 21
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`

`

`
`
`Phase(s) Inspected
`
`ECG’s
`Blood Pressures
`Dosing
`Data Audit
`
`Dose Preparation
`Data Audit
`
`Data Audit
`
`Protocol Amendment Review
`Blood Collection – Bioanalytical
`Bioanalytical Sample Processing
`Urinalysis
`Blood Collection – Clinical Pathology
`Necropsy
`Organ Weights
`Bioanalytical Sample Shipping
`Data Audit
`Data Audit
`Data Audit
`Data Audit
`Data Audit
`
`Data Audit
`Data Audit
`
`Data Audit
`Draft Report Review
`Data Audit
`Final Report Review
`
`Date Findings Submitted to:
`
`Study Director
`Study Director
`Management
`07/21/06
`07/21/06
`07/21/06
`07/21/06
`07/25/06
`07/25/06
`09/13/06
`09/13/06
`
`08/31/06
`09/28/06
`
`10/03/06
`
`09/08/06
`09/12/06
`09/12/06
`09/15/06
`09/15/06
`09/15/06
`09/15/06
`09/18/06
`10/09/06
`10/11/06
`10/23/06
`10/27/06
`11/08/06
`
`10/26/06
`11/10/06
`
`01/26/07
`03/09/07
`03/15/07
`04/06/07
`
`08/31/06
`09/28/06
`
`10/03/06
`
`09/08/06
`09/12/06
`09/12/06
`09/15/06
`09/15/06
`09/15/06
`09/15/06
`09/18/06
`10/09/06
`10/11/06
`10/23/06
`10/27/06
`11/08/06
`
`10/26/06
`11/10/06
`
`01/26/07
`03/09/07
`03/15/07
`04/06/07
`
`
`
`
`Page 11
`Testing Facility Study No. EBA00066
`
`
`Sponsor Study No. P00012-05-05
`
`
`
`
`Dates of Inspection
`07/21/06
`07/21/06
`07/25/06
`08/01/06, 08/16/06,
`08/17/06, 09/12/06,
`09/13/06
`08/29/06
`09/01/06, 09/05/06,
`09/13/06, 09/28/06
`09/06/06, 09/07/06,
`10/02/06, 10/03/06
`09/08/06
`09/12/06
`09/12/06
`09/14/06, 09/15/06
`09/14/06
`09/14/06
`09/14/06
`09/18/06
`10/06/06, 10/09/06
`10/11/06
`10/19/06, 10/23/06
`10/24/06, 10/27/06
`10/25/06, 10/31/06,
`11/08/06
`10/26/06
`10/30/06, 10/31/06,
`11/09/06, 11/10/06
`01/26/07
`03/08/07, 03/09/07
`03/12/07
`04/03/07, 04/06/07
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
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`Page 4 of 21
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`Page 14
`Testing Facility Study No. EBA00066
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`
`Sponsor Study No. P00012-05-05
`
`4. SUMMARY
`The purpose of this study was to evaluate the potential toxicity and toxicokinetics of the test
`article, BG00012, when administered in two divided doses daily for a minimum of 11 months by
`oral (capsule) administration followed by a 1-month recovery period. The study design was as
`follows:
`
`Experimental Design
`
`Group
`No.
`
`1
`
`No. of Main Study
`(Recovery) Animals
`Males
`Females
`
`4 (2)
`
`4 (2)
`
`Test Material
`BG12 placebo
`
`
`Necropsy Day
`(Recovery)
`
`332/333 (365)
`
`Dose
`Level
`(mg/kg)
`75/50a
`(0 mg/kg test
`article)
`332/333 (365)
`5
`
`BG12
`4 (2)
`4 (2)
`2
`332/333 (365)
`25
`
`BG12
`4 (2)
`4 (2)
`3
`75/50a
`332/333 (365)
`
`BG12
`4 (2)
`4 (2)
`4
`aBeginning on Day 7, due to adverse signs noted in Group 4 animals, the dose level for
` Groups 1 and 4 was decreased to 50 mg/kg.
`The following variables and end points were evaluated in this study: clinical signs, physical
`examinations, body weights, body weight changes, food consumption, ophthalmology,
`cardiology, blood pressure, clinical pathology (hematology, clinical chemistry, and urinalysis),
`toxicokinetics, gross necropsy, organ weights, and histopathology.
`Results:
`There were no test article-related mortalities. All animals survived until scheduled euthanasia.
`Test article-related clinical signs during the dosing phase were largely restricted to
`gastrointestinal disturbances such as an increased incidence of soft stools in 75/50 mg/kg/day
`animals, an increased incidence of mucoid stools in 25 mg/kg/day animals and 75/50 mg/kg/day
`animals, an increased incidence of vomitus, primarily in 75/50 mg/kg/day animals, and no
`feces/few feces in 75/50 mg/kg/day males. In addition, ocular discharge was observed in some
`75/50 mg/kg/day animals. There were no relevant clinical signs observed during the recovery
`phase.
`Profound test article-related effects on body weight, body weight gain, and food consumption
`were observed in 75/50 mg/kg/day animals during the dosing phase. Animals in the
`75/50 mg/kg/day dose group lost over 15% of their initial body weight over the first several
`weeks and had reduced body weight gains compared to controls throughout the dosing phase due
`to test article-induced inappetence. Because of this inappetence, dietary supplements including
`Nutrical and Science Diet® were offered to 75/50 mg/kg/day dogs. Males and females in the
`5 mg/kg/day and 25 mg/kg/day dose groups had body weight gains and food consumption values
`more comparable to controls during the dosing phase. Where present, reductions in body weight
`or body weight gain in 25 mg/kg/day or 75/50 mg/kg/day animals occurred within the first five
`
`Page 5 of 21
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`Page 15
`Testing Facility Study No. EBA00066
`
`
`Sponsor Study No. P00012-05-05
`
`weeks of dosing. There were no marked differences in body weight gain or food consumption
`during the recovery phase.
`There were no test article-related adverse findings in ophthalmic, cardiology or hematology
`evaluations.
`The most striking serum chemistry changes in test article-treated animals for most of the dosing
`period were dose-related decreases in creatinine observed in 25 mg/kg/day and 75/50 mg/kg/day
`males and females, and decreases in blood urea nitrogen in male dogs treated with 25 and
`75/50 mg/kg/day BG00012 and females predominantly at the 75/50 mg/kg/day dose level.
`Serum levels of creatinine and urea nitrogen were comparable to values in vehicle controls at the
`end of a one-month recovery period.
`Urine volumes recorded prior to the terminal necropsy were increased in 75/50 mg/kg/day males
`and in 25 mg/kg/day and 75/50 mg/kg/day female dogs, as compared to their corresponding
`control groups. A single statistically significant decrease in specific gravity was noted in the
`75/50 mg/kg/day females on Day 332/333. At the recovery necropsy collection interval, there
`were no observed differences in urine volume or specific gravity in any of the BG00012
`treatment groups as compared to the control group.
`Test article-related gross pathology findings included enlarged kidneys in 25 mg/kg/day and
`75/50 mg/kg/day males and enlarged adrenals in 75/50 mg/kg/day males, while test
`article-related changes in organ weights included increased absolute and relative (to brain
`weight) kidney weights in 25 mg/kg/day and 75/50 mg/kg/day males and females, increased
`absolute and relative (to brain weight) adrenal weights in 75/50 mg/kg/day males and females,
`and decreases in the absolute and relative (to brain weight) weights of the testis, and epididymis,
`of 75/50 mg/kg/day males. The increases in kidney weights and gross size at necropsy correlated
`with microscopic findings of hypertrophy of tubular epithelium (males only) and diffuse cortical
`tubular dilation in males and females. The increase in absolute and relative adrenal weights and
`gross size at necropsy correlated with microscopic findings of hypertrophy of the zona
`fasciculata of the adrenal gland. The decrease in testes weight had a microscopic correlate of
`degeneration of the seminiferous tubules, and the decrease in weight of the epididymides had a
`microscopic correlate of hypospermia.
`Test article-related microscopic findings in the kidney included hypertrophy of the cortical
`tubular epithelium, dilation of cortical tubules, regeneration of the cortical tubular epithelium,
`atrophy of the cortical parenchyma, infiltration of mixed inflammatory cells in the renal papilla,
`and hyperplasia of papillary urothelial cells. Microscopic findings in the kidney were observed
`at all doses, and were still evident in one or more animals per test article-treated group at the end
`of the recovery period with the exception of the 5 mg/kg/day females, but were mostly decreased
`in incidence and/or severity.
`Test article-related microscopic findings in the testis included degeneration of the seminiferous
`tubular epithelium and the presence of spermatid giant cells in the lumen of the seminiferous
`tubules. These findings were noted in male dogs in the 25 mg/kg/day and 75/50 mg/kg/day
`groups at the end of the dosing phase and at the end of the recovery phase, although the findings
`were decreased in incidence and/or severity.
`
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`Page 16
`Testing Facility Study No. EBA00066
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`Sponsor Study No. P00012-05-05
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`The only microscopic finding in the epididymis was hypospermia, which was observed only in
`males administered 75/50 mg/kg/day at the end of the dosing phase.
`The only microscopic finding in the adrenal gland was minimal to moderate hypertrophy of the
`zona fasciculata in males and females treated with 25 and 75/50 mg/kg/day. A single dog in the
`recovery placebo control group demonstrated similar findings as was observed in some of the
`dogs at the terminal necropsy interval. Hypertrophy in the adrenal gland was not considered to
`be test article related at the recovery euthanasia because the incidence was equivalent between
`the control and high-dose groups.
`Toxicokinetic evaluation of DMF, through its primary metabolite MMF, was performed on
`Study Days 1, 91, 182, 273, and 330. Exposure was confirmed in all animals throughout the
`11-month study interval. The plasma concentrations generally peaked at 0.5 to 4 hours
`post-each-dose. Both AUC and Cmax increased with dose, mostly close to dose-proportional. In
`a majority of the animals, AUC and Cmax decreased between Day 1 and Day 182 of dosing, but
`remained consistent between Days 182 and 330. The mechanism for this observed decrease is
`unknown. There was no apparent gender effect on exposure.
`Conclusion:
`Administration of BG00012 in divided daily doses by oral capsule administration resulted in test
`article-related effects at all dose levels evaluated (5, 25, and 75/50 mg/kg/day). Clinical
`observations of soft and/or mucoid stools, vomiting and ocular discharge were predominantly
`observed in the high-dose animals. The vomiting appeared most often in the first 60 days of
`treatment then decreased, suggesting adaptation to BG00012 administration. Consistent with the
`clinical observations, mean body weights were significantly reduced throughout the majority of
`the dosing period in the high-dose animals (75/50 mg/kg/day) and occasionally in the female
`dogs administered BG00012 at 25 mg/kg/day. Dosing holidays due to decrements in body
`weight gain occurred primarily in male dogs from the highest dose group. Food consumption
`was also significantly decreased, as compared to control groups in dogs of both sexes in the
`high-dose group, but actual food consumption values are confounded by the introduction of
`canned food and Nutrical in this dose group.
`Treatment-related decreases in BUN and serum creatinine were observed consistently in the
`75/50 mg/kg/day male and female dogs, and male dogs at the 25 mg/kg/day dose level. At the
`terminal necropsy interval, urine volumes were consistently higher in the 75/50 mg/kg/day dose
`groups (male and female). Urine specific gravity was reduced in female dogs at the terminal
`necropsy interval. All of these changes in BUN, creatinine and urine volume had returned to
`control values following a 1-month treatment-free period, suggesting recovery of the
`BG00012-induced changes. Histopathologic evaluation of protocol-specified tissues identified
`the kidney, testis and epididymides as target organs of BG00012 toxicity.
`Microscopic findings in the kidney included: hypertrophy of the tubular epithelium and
`regeneration of the tubular epithelium (male dogs only), tubular dilation and hyperplasia of the
`papillary urothelium (observed in both sexes in a dose proportional manner); and mixed cell
`infiltration into the papilla and atrophy of the cortical parenchyma (observed primarily in
`high-dose animals). Findings in the testis included degeneration of the seminiferous epithelium
`
`Page 7 of 21
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`Page 17
`Testing Facility Study No. EBA00066
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`Sponsor Study No. P00012-05-05
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`in mid- and high-dose male dogs. Incidence and/or severity of findings recorded at the terminal
`necropsy interval were generally reduced following a 1-month treatment-free period, suggesting
`a trend towards recovery.
`In conclusion, BG00012 administration twice daily by capsule was poorly tolerated at a dose
`level of 75/50 mg/kg/day. At the 25 mg/kg/day dose level, treatment-related changes in serum
`BUN and creatinine, together with histopathologic changes in the kidney supported that this dose
`had exceeded a NOAEL for chronic exposure in the dog. Doses of 5 mg/kg/day were considered
`well tolerated by both male and female dogs as determined by evaluation of in-life observations
`and clinical pathology evaluations. Histopathologic evidence of minimal tubule dilation in male
`and female dogs, a renal papillary hyperplasia (minimal) in a single male dog at the 5 mg/kg/day
`dose level, and hypertrophy of the tubular epithelium (in male dogs only) was not associated
`with any observed changes in in-life observations, including body weights, food consumption,
`clinical observations, clinical chemistry, and urinalysis parameters. Therefore, the NOAEL for
`chronic BG00012 capsule administration is considered to be 5 mg/kg/day in the
`dog.
`
`Page 8 of 21
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`

`

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`Page 18
`Testing Facility Study No. EBA00066
`
`
`Sponsor Study No. P00012-05-05
`
`5. INTRODUCTION
`The purpose of this study was to evaluate the potential toxicity and toxicokinetics of the test
`article, BG00012, when administered in two divided daily doses for a minimum of 11 months by
`oral (capsule) administration to
` dogs followed by a 1-month recovery period.
`The protocol was signed by the Study Director on October 4, 2005 (GLP initiation date). The
`experimental start date of the study was September 15, 2005, and the experimental completion
`date was April 4, 2007, the in-life phase of the study was initiated on October 18, 2005, and the
`in-life completion date was October 17, 2006.
`
`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
`Batch Number
`Assigned Testing Facility ID
`Purity
`Receipt Dates
`Expiration Date
`Physical Description
`Storage Conditions
`Supplier
`6.1.2. Control Article
`Identification
`Lot Number
`Assigned Testing Facility ID
`Purity
`Receipt Dates
`
`Expiration Date
`Physical Description
`Storage Conditions
`Supplier
`
`
`
`
`
`
`
`BG12
`25700
`S05.007.EBA
`None provided
`October 13, 2005; February 22, 2006
`November 2007
`Capsule with white body and blue cap
`Room temperature
`Vifor SA
`
`
`
`BG12 placebo
`25484
`V05.001.EBA
`None Provided
`October 13, 2005; February 22, 2006;
`September 6, 2006
`September 2007
`Capsule with white body and blue cap
`Room temperature
`Vifor SA
`
`
`
`Page 9 of 21
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`Page 19
`Testing Facility Study No. EBA00066
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`
`Sponsor Study No. P00012-05-05
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`6.1.3. Capsules
`Identification
`Lot Number
`Receipt Dates
`Expiration Date
`Storage Conditions
`Supplier
`
`Identification
`Lot Number
`Receipt Date
`Expiration Date
`Storage Conditions
`Supplier
`
`Identification
`Lot Number
`Receipt Date
`Expiration Date
`Storage Conditions
`Supplier
`
`Identification
`Lot Number
`Receipt Date
`Expiration Date
`Storage Conditions
`Supplier
`
`Identification
`Lot Number
`Receipt Date
`Expiration Date
`Storage Conditions
`Supplier
`
`6.1.4. Test and Control Article Characterization
`Certificates of Analysis for the test article, control article, and capsules are presented in
`Appendix 2.
`
`
`Torpac “Lock Ring” Capsules, size 13
`1571
`February 9, 2005
`October 2009
`Room Temperature
`Torpac, Inc.
`
`Torpac “Lock Ring” Capsules, size 13
`1687
`December 8, 2005, January 20, 2006
`July 2010
`Room Temperature
`Torpac, Inc.
`
`Torpac Gelatin, size 13
`1776
`May 5, 2006
`January 2011
`Room Temperature
`Torpac, Inc.
`
`Empty Porcine Hard Gelatin Capsules, size 13
`1805
`July 5, 2006
`March 2011
`Room Temperature
`Torpac, Inc.
`
`Empty Porcine Hard Gelatin Capsules, size 13
`1835
`September 1, 2006
`June 2011
`Room Temperature
`Torpac, Inc.
`
`Page 10 of 21
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`

`

`Sponsor Study No. P00012-05-05
`
`Page 20
`Testing Facility Study No. EBA00066
`
`6.1.5. Reserve Sample
`Ten capsules of each lot of the bulk test and control articles were collected as a reserve sample
`and maintained at room temperature by the Testing Facility.
`
`6.1.6. Inventory and Disposition
`An inventory of the test and control articles supplied by the Sponsor was maintained. With the
`exception of the reserve samples, the bulk test and control articles will be returned to the Sponsor
`following completion of all scheduled studies, unless otherwise instructed by the Sponsor.
`Empty and partially used containers of test materials were discarded according to proper disposal
`procedures, unless instructed otherwise.
`
`6.1. 7. Preparation of Dose Formulations
`The appropriate number of BG 12 drug product capsules were opened an~
`transferred to size 13 torpac - latin ca sules. Each BG 12 drug product c~
`120 mg of test article. Each
`tablet contained approximately 3 mg of test article.
`The capsules were placed in an am er a e ed pill vial. The vials were then placed in a labeled
`amber ziplock bag. Doses were prepared, verified, and dispensed daily.
`
`6.1.8. Dose Sample Analysis
`Dose sample analysis was not performed for this study.
`6.2. Test System
`
`6.2.1. Receipt and Description
`A total of 28 male and 28 female
`
`was received on September 15, 2005 . .
`All animals were examined and wcighed on the
`to the laboratory environment for a
`minimum of 25 days prior to the first day of dosing. One female dog, #D476, had decreased
`food consumption and diarrhea the day following receipt. These signs did not resolve and
`#D4 76 was examined by the Facility Veterinarian. At the recommendation of the Facility
`Veterinarian, this animal was euthanized on September 19, 2005. The vendor provided a
`replacement female-
`dog which was received on September 22, 2005.
`
`6.2.2. justification of Test System/Route/Frequency
`
`dog was chosen as the animal model for this study as it is a preferred species for
`The-
`prec~ oral toxicity testing by regulatory agencies. The oral route of exposure was selected
`since this is the intended route of human exposure. The test article was administered twice daily
`in order to more closely approximate intended clinical dosage frequency.
`
`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,
`
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`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
`72 ± 7ºF (22 ± 4ºC)
`Humidity
`50 ± 20%
`Light Cycle
`12-hour light/12-hour dark cycle
`Air Changes
`Ten or more air changes per hour with 100% fresh air
`Actual room temperature and relative humidity were recorded a minimum of once daily and
`ranged from 62 to 83°F (17 to 28°C) and 14 to 87%, respectively.
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`6.2.5. Food
`PMI Nutrition International Certified Canine Diet® #5007 was provided to each dog. An
`approximate 400 gram ration of feed was provided daily to each dog beginning on the day after
`receipt. During the acclimation and study periods, the feed was left in the dog’s cage overnight
`and then removed prior to dosing. 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.
`Beginning on Day 15, all dogs in the 75/50 mg/kg/day dose group were offered one can of
`Science Diet® per day. The consumption of Science Diet® was documented qualitatively.
`Nutrical was offered to animals when food consumption values were 150 grams or less. On
`numerous days, many animals’ food consumption value was deleted due to excessive
`contamination (most often, urine). On days when this occurred, the Study Director was
`contacted and gave verbal approval to either offer or withhold the Nutrical based upon the
`previous day’s food consumption as well as the volume of food that was left in the bowl which
`was contaminated. Nutrical was offered to the following animals twice a day:
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`Nutrical Supplements
`Animal
`Days Nutrical was
`Group
`Offereda
`No./Gender
`No.
`D454/M
`Days 16-337
`4
`D458/M
`Days 8-330
`4
`D459/M
`Days 8-331
`4
`D460/M
`Days 8-337
`4
`D462/M
`Days 8-332
`4
`D465/M
`Days 8-332
`4
`D480/F
`Days 8-331
`4
`D483/F
`Days 10-331
`3
`D486/F
`Days 8-331
`4
`D489/F
`Days 12-332
`3
`D492/F
`Days 9-332
`3
`D493/F
`Days 8-332
`4
`D494/F
`Days 8-335
`4
`D498/F
`Days 8-337
`4
`D502/F
`Days 16-329
`4
`aDay range includes first and last day of Nutrical
` supplementation. If more than 150g of Kibble
` was consumed in one day during the day range,
` Nutrical supplementation was skipped for the
` day.
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`6.2.6. Water
`Municipal tap water following treatment by reverse osmosis and ultraviolet irradiation was
`available ad libitum throughout the study. 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 Attending
`Veterinarian as warranted by clinical signs or other changes. Any veterinary examinations were
`documented in the study records. Veterinary medicinal treatments are listed in Appendix 3. The
`veterinary treatments were undertaken in an attempt to improve animal health/welfare. These
`treatments did not appear to impact the conduct of the study or the interpretation of the study
`results.
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`6.2.8. Acclimation Physical Examinations
`Physical examinations were performed on all dogs by a facility veterinarian within 7 days of
`animal receipt. In addition, fecal samples were collected and examined microscopically for
`parasites on the same day as the physical examinations. No parasites were observed in any dog
`placed on study.
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`6.2.9. Assignment to Study Groups
`Prior to randomization procedures, the animals were weighed and examined in detail. Animals
`determined to be suitable as test subjects were randomly assigned to groups by a stratified
`randomization scheme designed to achieve similar group mean body weights. The animals were
`approximately 7 months of age at the time of randomization with body weights ranging from
`7.8 to 9.9 kg for the males and 6.5 to 8.2 kg for the females.
`
`6.3. Experimental Design
`The experimental design was as follows:
`
`Experimental Design
`
`Group
`No.
`
`1
`
`No. of Main Study
`(Recovery) Animals
`Males
`Females
`
`4 (2)
`
`4 (2)
`
`Test Material
`BG12 placebo
`
`
`Necropsy Day
`(Recovery)
`
`332/333 (365)
`
`Dose
`Level
`(mg/kg)
`75/50a
`(0 mg/kg test
`article)
`332/333 (365)
`5
`
`BG12
`4 (2)
`4 (2)
`2
`332/333 (365)
`25
`
`BG12
`4 (2)
`4 (2)
`3
`75/50a
`332/333 (365)
`
`BG12
`4 (2)
`4 (2)
`4
`aBeginning on Day 7, due to adverse signs noted in Group 4 animals, the dose level for
` Groups 1 and 4 was decreased to 50 mg/kg.
`6.4. Justification of Dose Levels
`The dose levels were selected in an attempt to produce graded responses to the test article. The
`high-dose level was expected to produce toxic effects, but not excessive lethality that would
`prevent meaningful evaluation. The mid-dose level was expected to produce minimal toxic
`effects. The low-dose level was expected to produce no observable indications of toxicity.
`
`6.5. Administration of Test Materials
`The test or control articles were administered via oral capsule to the appropriate dogs as two
`divided daily doses, 4 hours apart, for a minimum of 331 days, followed by a 1-month recovery
`period. The appropriate number of
`tablets or placebo
`were loaded into gelatin
`capsules. The dose volume for each animal was based on the most recent body weight
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`measurement. After dose administration, a small amount of RODI water was given to the
`animals. The first day of dosing was designated as Study Day 1.
`The following animals received a dosing holiday:
`Dosing Holiday
`Day Dosing was
`Days of Dosing
`Animal
`Group
`Resumed
`Holiday
`No./Gender
`No.
`Day 29
`Days 26-28
`D458/M
`4
`Day 35
`Days 32-34
`D462/M
`4
`Day 63
`Days 35-62
`D458/M
`4
`Day 49
`Days 38-48
`D459/M
`4
`Day 56
`Days 38-55
`D460/M
`4
`Day 49
`Days 38-48
`D462/M
`4
`Day 42
`Days 38-41
`D480/F, D486/F, D493/F
`4
`Day 91
`Days 77-90
`D458/M, D460/M
`4
`D486/Fa
`Day 92
`Days 82-91
`4
`Day 119
`Days 98-118
`D458/M
`4
`Day 140
`Days 126-139
`D458/M
`4
`Day 168
`Days 147-167
`D458/M
`4
`Day 182
`Days 175-181
`D458/M
`4
`Day 203
`Days 189-202
`D458/M
`4
`Day 217
`Days 210-216
`D458/M
`4
`Note: Prior to Study Day 35, animals were placed on dosing holidays upon the
`veterinarian’s recommendation based on the animal’s food consumption and clinical
`signs. In order to provide more objective criteria for placing animals on dosing
`holidays, effective Study Day 35, any animal with a 30% body weight loss was
`placed on a dosing holiday until the animal achieved a body weight within 10% of
`its Day 1 body weight. Group 4 females #D480, #D486, and #D493 received the
`first dose of the day on Study Day 38 before being placed on dosing holiday.
`aGroup 4 female #D486 was placed on a dosing holiday since the animal was
`treated with antibiotics due to an infection.
`7. EXPERIMENTAL PROCEDURES
`
`7.1. Mortality/Moribundity Checks
`General health/mortality and moribundity checks were performed twice daily, in the morning
`and afternoon.
`
`7.2. Clinical Observations
`Detailed clinical observations were performed once prior to treatment (Day -1) and weekly
`during the treatment and recovery periods. Cage-side observations were performed daily between
`30 minutes to 2 hours post-dose on days of dosing. Cage-side observations were also performed
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`on Day 332 (non-dosing day) for animals euthanized on Day 333. During the recovery period,
`cage-side observations were performed once daily, except on the days when detailed clinical
`observations were performed. A final detailed clinical observation was performed for each
`animal on the day of scheduled euthanasia (Days 332/333 and 365).
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`7.3. Body Weights
`Individual body weights were recorded on the day of randomization and weekly throughout the
`treatment and recovery periods. A final fasted body weight was recorded on the day of
`scheduled euthanasia (Days 332/333 and 365) for calculation of organ-to-body weight ratios.
`For the purpose of determining if a dosing holiday was necessary, 75/50 mg/kg/day males
`#D459, #D460 and #D462 were weighed on Day 37. The body weight measurement was hand
`recorded and will remain in the study file; these weights were not used to determine dosing
`amounts.
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`7.4. Food Consumption
`Food consumption measurements were recorded daily throughout the treatment and recovery
`periods and reported in weekly intervals. In addition, qualitative consumption of Science Diet®
`was documented daily for all animals in the 75/50 mg/kg/day dose group, beginning on Day 15.
`
`7.5. Physical Examinations
`Physical examinations were performed by a staff veterinarian once prior to in-life initiation and
`prior to scheduled euthanasia. The examination included a record of general condition and rectal
`body temperature (conducted by a laboratory technician).
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`7.6. Ophthalmology Examinations
`Ophthalmological examinations were performed by a board-certified veterinary ophthalmologist
`prior to in-life initiation (Day -10), during the last week of the treatment period (Da