EQUIVALENCY STUDY: FETAL TISSUE GENERATION FOR DISSECTION MICROSCOPY PRODUCT IDENTIFICATION

Transcription

1 Product Safety Labs EQUIVALENCY STUDY: FETAL TISSUE GENERATION FOR DISSECTION MICROSCOPY PRODUCT IDENTIFICATION Visikol Pre-treatment Solution Visikol Clearing Solution Visikol Post-treatment Solution DATA REQUIREMENT Not Applicable STUDY NUMBER PERFORMING LABORATORY Product Safety Labs 2394 US Highway 130 Dayton, New Jersey STUDY COMPLETION DATE January 11, 2017 STUDY DIRECTOR Lee Koetzner, PhD SPONSOR Visikol, Inc. 675 US Highway 1 North Brunswick, NJ Page 1 of 29

2 Product Safety Labs Page 2 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT Visikol Pre-treatment Solution Visikol Clearing Solution Visikol Post-treatment Solution This study meets the requirements of 21 CFR 58: U.S. FDA GLP Standards, 1987, 40 CFR 160: U.S. EPA (FIFRA), 1989, and 40 CFR 792: U.S. EPA (TSCA), 1989, with the following exception: Characterization of the control article was not documented according to GLP, however, the purity of the material used were certified by a reputable supplier. Specific information related to the characterization of the test articles as received and tested is the responsibility of the study Sponsor (Section 3.A).

3 Product Safety Labs Page 3 QUALITY ASSURANCE STATEMENT The Product Safety Labs Quality Assurance (QA) Unit has reviewed this final study report to assure the report accurately describes the methods and standard operating procedures, and that the reported results accurately reflect the raw data of the study. QA Activities for This Study: QA Activity Performed By Date Conducted Protocol review R. Krick; A. Adamiec Sep 12, ; Nov 8, 2016 Date Findings Reported To Study Director And Management Sep 12, 2016; Nov 11, 2016 In-process inspection: Tissue process for Visikol: Tissue transfer to pre-treatment solution In-process inspection: Tissue process for Visikol: Tissue transfer to Alizarin S Stain In-process inspection: Tissue process for Control: Tissue transfer to Alizarin S Stain A. Adamiec Sep 15, 2016 Sep 15, 2016 A. Adamiec Sep 15, 2016 Sep 15, 2016 A. Adamiec Sep 28, 2016 Sep 28, 2016 Raw data audit A. Adamiec Nov 8 & 11, 2016 Nov 11, 2016 Draft report review A. Adamiec Nov 8 & 11, 2016 Nov 11, 2016 Final report reviewed by:

4 Product Safety Labs Page 4 CERTIFICATIONS We, the undersigned, declare that the methods, results and data contained in this report faithfully reflect the procedures used and raw data collected during the study.

5 Product Safety Labs Page 5 TABLE OF CONTENTS Good Laboratory Practice Compliance Statement... 2 Quality Assurance Statement... 3 Certifications... 4 Table of Contents... 5 Study Information... 7 Key Personnel Objective Summary Test and Control Articles... 9 A. Source... 9 B. Test Article Identification... 9 C. Control Article Identification D. Analysis E. Hazards Basic Design A. Test System B. Test System Justification C. Identification Experimental Design Procedures A. Euthanasia B. Tissue Processing B.1 Control B.2 Visikol C. Tissue Evaluation D. Statistical Analysis Study Conduct A. Testing Facility B. GLP Compliance C. Test Procedure Guidelines Records to be Maintained Protocol, Protocol Amendments and Protocol Deviation Animal Welfare Act Compliance: Evaluation of Processed Fetal Tissue A. Mice and Rats... 13

6 Product Safety Labs Page 6 11.A.1 Control Group (Standard KOH Diaphonization) A.2 Visikol Group (Custom Method) B. Rabbits B.1 Control Group (Standard KOH Diaphonization) B.2 Visikol Group (Custom Method) Conclusion Appendix A: Protocol, Protocol Amendments and Protocol Deviation Appendix B: Contributing Scientist Qualitative Assessment Report... 26

7 Product Safety Labs Page 7 STUDY INFORMATION Protocol No.: Test Articles: Physical Description: P999-VSK-2 1) Visikol Pre-treatment Solution Lot#: ) Visikol Clearing Solution Lot#: ) Visikol Post-treatment Solution Lot#: Liquid Date Test Substance Received: September 14, 2016 PSL ID: 1) D 2) D 3) D PSL Study Number: Sponsor: Visikol, Inc. (Amendment 4) 675 US Highway 1 North Brunswick, NJ Study Initiated-Completed: September 14, 2016 (see report cover page) In-Life Study Initiated-Completed: September 14, 2016 October 13, 2016 Notebook Page Numbers: 44006: pages 1-42

8 Product Safety Labs Page 8 KEY PERSONNEL Product Safety Labs: President: Director of Toxicology and Pathology: Study Director: Primary Scientist: Contributing Personnel: Director of Quality Assurance: Technical Writing Supervisor: Daniel J. Merkel, BS, MBA Odete Mendes, DVM, PhD, DACVP, DABT Lee Koetzner, PhD Jamie Boulet, RLATR Kathleen Quinn, BS Mithila Shitut, BVSc & AH, MS Colleen Wojenski, BS, LATG Rhonda S. Krick, BS Celeste Dunn, AS

9 Product Safety Labs Page 9 1. OBJECTIVE The purpose of this study was to compare diaphonization protocols (Visikol and a KOH reference procedure) using fetal specimens from rabbits, rats and mice. 2. SUMMARY Timed pregnant females from each of three species (rabbits, rats, and mice) were used on test. The rabbits were euthanized on GD 28; rats on GD 20; and mice on GD 18. Fetuses were harvested via cesarean section and split into two groups; those processed for skeletal evaluation using the control method (Alzarin S and KOH digestion) and those using the test process (Visikol Pre-treatment, Clearing, and Post-treatment Solutions). Overall, both processing methods resulted in the fetal bone tissue showing adequate ossification and staining for visualization and evaluation. The process of post-fixed skeletal evaluations typically requires some dissection of residual tissue and fat deposits in order to expose some of the deeper bones of the specimen. This evaluation included the clarity of the tissue surrounding the bone, the level of stain uptake by the tissue, ease of dissection and the overall ability to visualize and assess the condition of the various bones. Since both processes resulted in relatively comparable staining of the majority of bone tissue, the evaluation focused mostly on the overall clarity and resulting condition of the tissue of the fetuses for each species. Surrounding tissues were generally clear for control (Alizarin S and KOH digestion), with more opacity in rats and mice than rodents. More opacity was noted in all evaluations using the test process (Visikol Pre-treatment, Clearing, and Post-treatment Solutions). Specimen preparation using the Visikol (custom) method was faster and simpler than the control (standard KOH diaphonization). 3. TEST AND CONTROL ARTICLES A. Source The test articles were provided by the Sponsor. The control article was sourced by Product Safety Labs (PSL). B. Test Article Identification The test articles were received on September 14, 2016, and identified using the following information provided by the Sponsor and Product Safety Labs identification numbers. Test Article: Visikol Pre-treatment Solution Lot#: PSL ID: D Physical Description: Liquid Composition: Proprietary (98% H 2 O) Storage Conditions: Refrigerated Expiration Date: 9/13/2017 Test Article: Visikol Clearing Solution Lot#: PSL ID: D Physical Description: Liquid Composition: Proprietary (30% glycerol) Storage Conditions: Refrigerated Expiration Date: 9/13/2017

10 Product Safety Labs Page 10 Test Article: Visikol Post-treatment Solution Lot#: PSL ID: D Physical Description: Liquid Composition: Proprietary (98% H 2 O) Storage Conditions: Refrigerated Expiration Date: 9/13/2017 Documentation of the methods of synthesis, fabrication, or derivation of the test articles is retained by the Sponsor. C. Control Article Identification The control article was identified using the following information: Test Article: Potassium hydroxide Lot#: None provided Physical Description: Liquid Composition: Potassium hydroxide (CAS ) in water Storage Conditions: Refrigerated Expiration Date: 9/13/2017 D. Analysis The test and control articles, as received, were expected to be stable for the duration of the study. E. Hazards Appropriate routine safety precautions were exercised in the handling of the test and control articles during the study. 4. BASIC DESIGN A. Test System Rabbit, rat and mouse fetuses were the test systems for this study. B. Test System Justification These test systems were chosen to produce data compatible with the Sponsor s requirements and existing data. C. Identification Each container was marked using study number, species, processing method (control or Visikol) and date of collection. 5. EXPERIMENTAL DESIGN Two timed pregnant animals of each species (rabbits, rats and mice) were received on October 6, 2016, from Charles River Laboratories, Inc. Rabbits were received on GD 20 from Quebec City, Canada. Rats and mice were received from Raleigh, NC (USA), on GD Days 12 and 10, respectively. Following eight days of acclimation and a general health check, animals were euthanized as described in Section 6.A. The rabbits were euthanized on GD 28; rats on GD 20; and mice on GD 18. Fetuses were divided and used for Control and Test Article processing. This, combined with the number of fetuses collected (cf. Section 6.B.), was designed to control bias.

11 Product Safety Labs Page 11 The test system was exposed to the test article (or control) by immersion as per the standard technique for this process. 6. PROCEDURES A. Euthanasia Rabbit dams were euthanized using Fatal-Plus (IV). Rat and mouse dams were euthanized by carbon dioxide inhalation. Fatal-Plus via intraperitoneal injection (IP) was used to euthanize fetuses. B. Tissue Processing Fetal specimens were harvested via Caesarean section from both animals of each species and processed as described below. Rabbits: Eleven fetuses were collected, with five used for control and six for Visikol. Rats: Sixteen fetuses were collected, with half used for control and half for Visikol. Mice: Fourteen fetuses were collected, with half used for control and half for Visikol. 6.B.1 6.B.2 Control Rabbit fetuses were skinned. All fetuses were processed according to the diaphonization procedure used for skeletal visualization with Alzarin S stain and KOH digestion as follows (times approximate): Dehydrated in 70% ethanol for 2 weeks and the eviscerated (Amendment 1) Transferred to Alizarin S stain for 24 hours Transferred to 1% KOH solution for 9 hours (mice), one day (rats) or three days (rabbits) all times ± 20% Transferred to 70% ethanol-glycerin mixture (2:1) for 1-2 Days Transferred to 70% ethanol-glycerin mixture (1:1) for at least 1 Day (Amendment 3) Visikol Rabbit fetuses were skinned. All fetuses were processed according to the proprietary Visikol skeletal visualization technique. The technique below was used as a default (times approximate): Eviscerated and placed whole in 70% ethanol and left overnight (>16 hours; Amendment 2) Placed whole in pretreatment solution for 2 hours, but not exceeding 3 hours. Transferred directly to Alizarin S stain for 1-2 Days Transferred to post treatment solution for 1-2 Days Transferred to clearing treatment until clear ( 1 Day) Any remaining animals were sent to the Sponsor for possible future analyses (Amendment 5).

12 Product Safety Labs Page 12 C. Tissue Evaluation The Visikol and KOH diaphonization specimens were compared using skeletal visualization. The endpoints included (but were not limited to): C1-C8 cervical vertebrae for clarity and ability to distinguish important anatomical features Sacral and caudal vertebrae for clarity and ability to distinguish Clarity of lower mandible Clarity of tarsals, metatarsals, carpals, metacarpals Durability of specimens to physical manipulation Flexibility of specimens to physical manipulation An overall comparison of the quality of staining, transparency of skin/tissue, and ease of visual analysis was assessed qualitatively based on PSL experience with traditional technique and results thereof. D. Statistical Analysis No statistical analyses were conducted as no quantitative results were recorded. 7. STUDY CONDUCT A. Testing Facility This test was conducted at Product Safety Labs (PSL) test facility at 2394 U.S. Highway 130, Dayton, NJ B. GLP Compliance This study was conducted in compliance with the following Good Laboratory Practice (GLP) regulations: US FDA GLP: 21 CFR 58, US EPA GLP: Pesticide Programs (FIFRA): 40 CFR 160, US EPA GLP: Toxic Substances Control Act (TSCA): 40 CFR 792, C. Test Procedure Guidelines The procedures described in this protocol were not based on regulatory guidelines. 8. RECORDS TO BE MAINTAINED Information on the test system, equipment maintenance and calibration and all other records that would demonstrate adherence to the protocol are maintained. Facility records which are not specific to the subject study are maintained by the testing facility and archived according to PSL SOP. The original signed final report, together with the protocol, associated amendments and/or deviations, if applicable, and all raw data generated at PSL are maintained in the PSL Archives. 9. PROTOCOL, PROTOCOL AMENDMENTS AND PROTOCOL DEVIATION See Appendix A for the Protocol, Protocol Amendments and Protocol Deviation.

13 Product Safety Labs Page ANIMAL WELFARE ACT COMPLIANCE: This study will comply with all applicable sections of the Final Rules of the Animal Welfare Act Regulations (9CFR). The sponsor should make particular note of the following: A. The sponsor s authorization to conduct this study provides PSL with the sponsor s assurance that the study described in this protocol does not unnecessarily duplicate previous experiments. B. Procedures used in this study are designed to avoid or minimize discomfort, distress or pain to animals. All methods are described in this test method or in written laboratory standard operating procedures. C. Animals that experience severe or chronic pain or distress that cannot be relieved will be painlessly euthanized as deemed appropriate by the veterinary staff and/or study director. The sponsor will be advised by the study director of all circumstances which could lead to this action in as timely a manner as possible. D. Methods of euthanasia used during this study will conform to the regulation cited above. 11. EVALUATION OF PROCESSED FETAL TISSUE Detailed observations are included in the contributing scientist report (Appendix B; Deviation 1). A. Mice and Rats 11.A.1 Control Group (Standard KOH Diaphonization) Rodent fetal tissue using this processing method was firm yet flexible and not damaged by the dissection and evaluation process, thus exhibiting adequate durability. 11.A.2 Visikol Group (Custom Method) B. Rabbits The residual fatty tissue was gelatinous, and required greater manipulation to remove thoroughly and residual skin and muscle was somewhat more opaque, yet still allowing visualization of stained bone tissue beneath. Rodent fetal tissue using this processing method was flexible and quite durable given the additional manipulations required to expose deeper bones. 11.B.1 Control Group (Standard KOH Diaphonization) Overall, staining was darker and more pronounced than in rodent specimens, likely due to the skinning of the fetuses prior to processing. The residual fatty tissue was firm, easily removed and residual muscle fairly translucent allowing visualization of stained bone tissue beneath. Rabbit fetal tissue using this processing method was firm yet flexible and not damaged by the dissection and evaluation process, thus exhibiting adequate durability. 11.B.2 Visikol Group (Custom Method) Overall, staining was darker and more pronounced than in rodent specimens, but slightly paler than control method rabbit fetuses.

14 Product Safety Labs Page CONCLUSION Moderate dissection of residual tissue was required to visualize deeper pelvic bones and long bones of the limbs due to somewhat opaque tissue. In some fetuses the femur did not stain completely. Overall, residual muscle was somewhat more opaque, yet still allowed visualization of stained bone tissue beneath after some dissection. Rabbit fetal tissue using this processing method was flexible and quite durable. Specimen preparation using the Visikol (custom) method was faster and simpler than the control (standard KOH diaphonization). Alizarin Red staining was effective using both methods. Tissue diaphonization was more complete using the control method; visualization was more straightforward using the control method.

15 Page 15 APPENDIX A: PROTOCOL, PROTOCOL AMENDMENTS AND PROTOCOL DEVIATION

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26 Page 26 APPENDIX B: CONTRIBUTING SCIENTIST QUALITATIVE ASSESSMENT REPORT

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