P.O. Box 331, Millwood, VA 22646 (540) 837-1602 

June 29, 2012 

BY HAND 

Document Control Office 

Office of Pollution Prevention and Toxics 

Room 6428 

Environmental Protection Agency 

1200 Pennsylvania Avenue, NW 

Washington, D.C. 20460 

11111111111111111111111111111111111111111111111111111111 11111111 

40120000075 

Re: Testing Consent Order for Ethylene Dichloride; 

Dear Sirs: 

Study Plan for Extended One-Generation Drinking Water 

Reproductive Toxicity Study of Ethylene Dichloride in Rats 

(Docket No. OPPT-2003-0010) 

--_1. 

C . .J 

Pursuant to Section IX of the Enforceable Consent Agreement for Ethylene Dichloride, 

the HAP Task Force submits the enclosed study plan for an extended one-generation drinking 

water reproductive toxicity study of ethylene dichloride in rats. Six copies of the study plan are 

enclosed. 

Please do not hesitate to contact me if there are any questions. 

Enclosures 

cc: J. Schaeffer 

W. Caffey Norman, Esq. 

CONTP.!NS NO CBI 

3800151v1 
Extended One-Generation Drinking Water Reproductive Toxicity Study of Ethylene 

Dichloride in Rats 

Study Plan 

I. Identity of Consent Order 

This study plan is submitted pursuant to the Testing Consent Order for Ethylene 

Dichloride, 68 Fed. Reg. 33125 (June 3, 2003), as amended 74 Fed. Reg. 36196 (July 

22, 2009), as amended by Addendum Modification signed by Maria J. Doa on 

November 23, 2011 and Peter E. Voytek on December 6, 2011, Docket No. EPA-HQOPPT-2003-0010. This plan contains the information required by 40 CFR § 

790.62(b). 

II. Test Requirements Contained in Study Plan 

This study plan contains the test requirements for an extended one-generation 

drinking water reproductive toxicity study of ethylene dichloride in rats. The test 

methods are described in the attached protocol. 

III. Principal Sponsor of Test 

HAP Task Force 

P.O. Box 331 

Mill wood, VA 22646 

IV. Administrative Official and Project Manager for Principal Sponsor: 

Peter E. Voytek, Ph.D. 

Manager 

HAP Task Force 

P.O. Box 331 

Mill wood, VA 22646 

(540) 837-1602 
V. Technical Contacts 

Ann Davis 

EH&S Global Product Leader 

The Dow Chemical Company 

2020 Dow Center 

Midland, MI 48674 

(989) 638-7409 

David Goldsmith 

Principal Engineer 

Georgia Gulf Chemicals & Vinyls, LLC 

26100 Highway 405 

Plaquemine, LA 70764 

(225) 685-2677 

Heather Burleigh-Hayer, Ph.D. 

Manager, Environmental Health Sciences & Toxicology 

PPG Industries, Inc. 

4325 Rosanna Drive 

Allison Park, PA 15101 

(412) 492-5409 

Lloyd H. Stone 

Corporate HSE Manager 

The Westlake Group 

2801 Post Oak Blvd. 

Houston, TX 77056 

(713) 585-2816 

John Pastuck 

Environmental & Safety Manager 

Formosa Plastics Corporation, U.S.A. 

9 Peach Tree Hill Road 

Livingston, NJ 07039 

(973) 716-7387 

RobertS. Schuster 

Director - HES Compliance Assurance 

Occidental Chemical Corporation 

P.O. Box 809050 

Dallas, Texas 75380 

(972) 404-2434 

2 
VI. Testing Facility -

WIL Research 

1407 George Road 

Ashland, Ohio 44805 

Study Director: Melissa J. Beck, Ph.D. 

Assistant Director, Neurosciences 

( 419) 289-8700 

VII. Professional Qualifications 

Curricula vitae providing the training and experience of each professional involved in 

this testing are attached as Appendix A to this study plan. 

VIII. Identity of Test Substance 

1,2-Dichloroethane (CAS No. 107-06-2) 

CzH4Ch 

MW=98.96 

Clear, colorless, oily, highly flammable liquid 

BP = 83.5° C 

Soluble at 8490-9000 mg/L in water (20° C) 

LD50 orally in rats= 770-967 mg/kg 

An analysis of the product used for the study is attached as Appendix B. 

IX. Study Protocols 

The protocol for the extended one-generation drinking water reproductive toxicity study 

of ethylene dichloride in rats is attached as Appendix C. The outline of an analytical 

validation and stability study of 1,2-dichloroethane in aqueous formulations is also 

attached in Appendix C. 

X. Test Schedule 

Experimental Start Date: 

Experimental Termination Date: 

Final Report: 

3738332 3 

August 14, 2012 

TBD 

June 28, 2013 (unless second 

generation study is triggered) 
ATTACHMENT A 
Formal Education: 

2001 

1995 

Professional Experience: 

08/06/05 - Present 

12/04/10- Present 

MELISSA J. BECK 

Neurosciences 

University of Michigan 

Ann Arbor, MI 

PhD Toxicology 

Ashland University 

BS Toxicology 

WIL Research Laboratories, LLC 

Ashland, OH 

Position: Assistant Director 

Neurosciences 

CURRICULUM VITAE 

February21, 2012 

Responsible for assisting with the direction of all administrative 

and scientific functions within the discipline of Neurosciences at 

WIL Research Laboratories, LLC. Support various 

pharmaceutical, agrochemical and veterinary medical product 

development programs. Assists in selecting, installing and 

validating new test systems into the Neurosciences Department. 

Assists in training personnel in the Neurosciences Department. 

Assists in the marketing of studies into the Neurosciences 

Department. Study Director, simultaneously oversee between 30-

40 developmental, reproductive and neurotoxicity studies. 

Responsible for the planning, conducting and successful reporting 

of assigned studies in accordance with FDA, EPA and various 

international regulatory requirements. 

Position: Head of Juvenile Toxicology 

In addition to the above responsibilities, added the responsibility 

for assisting with the direction of all administrative and scientific 

functions involving nonclinical juvenile toxicity safety assessment 

at WIL Research Laboratories, LLC. Assists in the marketing of 

nonclinical juenile toxicity studies. 

Professional Experience (Continued): 
Formal Education: 

2001 

1995 

Professional Experience: 

08/06/05 - Present 

12/0411 0 - Present 

10/01/01 - 08/05/05 

MELISSA J. BECK 

Neurosciences 

University of Michigan 

Ann Arbor, MI 

PhD Toxicology 

Ashland University 

BS Toxicology 

WIL Research Laboratories, LLC 

Ashland, OH 

Position: Assistant Director 

Neurosciences 

CURRICULUM VITAE 

February21, 2012 

Responsible for assisting with the direction of all administrative 

and scientific functions within the discipline of Neurosciences at 

WIL Research Laboratories, LLC. Support various 

pharmaceutical, agrochemical and veterinary medical product 

development programs. Assists in selecting, installing and 

validating new test systems into the Neurosciences Department. 

Assists in training personnel in the Neurosciences Department. 

Assists in the marketing of studies into the Neurosciences 

Department. Study Director, simultaneously oversee between 30-

40 developmental, reproductive and neurotoxicity studies. 

Responsible for the planning, conducting and successful reporting 

of assigned studies in accordance with FDA, EPA and various 

international regulatory requirements. 

Position: Head of Juvenile Toxicology 

In addition to the above responsibilities, added the responsibility 

for assisting with the direction of all administrative and scientific 

functions involving nonclinical juvenile toxicity safety assessment 

at WIL Research Laboratories, LLC. Assists in the marketing of 

nonclinical juenile toxicity studies. 

Position: Staff Toxicologist 

Developmental, Reproductive and Neurotoxicology 
Melissa J. Beck 

Continuing Education: 

CURRICULUM VITAE 

Study Director for developmental, reproductive and neurotoxicity 

studies. Responsible for the planning, conduct and successful 

reporting of assigned studies. Participate in report review and 

generation process. Develop new capabilities for WIL Research 

Laboratories, Inc. Communicate with Sponsors as required. 

Mechanisms, Biomarkers, Data Interpretation: An Integrative Risk Assessment Using 

Developmental Neurotoxicity as a Model. Teratology Society, 2002. 

The Effects of Non-Reproductive Hormones on the Reproductive System and the Implications for 

Toxicology. Society of Toxicology, 2003. 

Signaling Pathways and Tissue Interactions in Organ-System Development. Teratology Society, 

2004. 

Risk Assessment in Pharmaceutical Development. Society of Toxicology, 2005. 

Neuropathology for the Toxicologist. Society of Toxicology, 2006. 

Reproductive Toxicity Testing: Study Designs, Evaluation, Interpretation and Risk Assessment. 

Society of Toxicology, 2006. 

Professional Activities: 

Toxicological Highlight Manuscript for Toxicological Sciences, Volume 62, Number 1, 2001 

Society Affiliations: 

2003 - Present 

2003 - Present 

2009 - Present 

Melissa J. Beck 

Member, Teratology Society 

Member, Neurobehavioral Teratology Society 

Member, Society for Neurosciences 

Date 
Formal Education: 

1999 

1990 

1985 

Professional Experience: 

10/23/10 - Present 

12/20/08- 10/22/10 

DONALD G. STUMP 

Developmental and Reproductive Toxicology 

Diplomate American Board of Toxicology 

Vanderbilt University 

Nashville, TN 

PhD Biochemistry 

Philadelphia College of Pharmacy and Science 

Philadelphia, P A 

BS Toxicology 

WIL Research Laboratories, LLC 

Ashland, OH 

CURRICULUM VITAE 

January 4, 2012 

Position: Senior Director, Developmental and Reproductive 

Toxicology 

Responsibilities include directing the activities of Study 

Directors and managerial and technical personnel in the 

Developmental and Reproductive Toxicology, Juvenile 

Toxicology and Neuroscience Departments. Serves in a lead 

role in the conceptual design of studies for various product 

development programs, and serves as Study Director for 

developmental and developmental toxicology, adult 

neurotoxicology, and juvenile and developmental 

neurotoxicology studies. Provides consultation to clients on 

scientific issues, data interpretation and program development. 

Position: Director, Developmental and Reproductive 

Toxicology 

Provide senior guidance in the scientific and operational domains 

of the discipline. Directs the activities of Study Directors, 

managerial and technical personnel in a GLP-environment. 

Provide lead role in the conceptual design and internal ration of 

studies for various product development programs and litigation 
Donald G. Stump CURRICULUM VITAE 

Professional Experience (Continued): 

11/10/00- 12/19/08 

11/6/99 -11110/00 

11108/97- 11/5/99 

support. Responsible for the development of new business 

within the field. Study Director for various reproductive and 

developmental toxicology and adult neurotoxicology, juvenile 

and developmental neurotoxicology studies. 

Position: Associate Director, Developmental and Reproductive 

Toxicology 

Responsible for managing junior level study directors. Study 

Director for developmental and reproductive toxicology studies. 

Responsible for the planning, conducting and successful 

reporting of assigned studies in accordance with FDA, EPA and 

various international regulatory requirements. Responsible for 

assisting with the direction of all fmancial and scientific 

functions within the discipline of Developmental and 

Reproductive Toxicology at WIL Research Laboratories, LLC. 

Position: Associate Director, Developmental, Reproductive 

and Neurotoxicology 

Responsible for managing a technical staff of approximately 30 

individuals. Study Director for developmental, reproductive and 

neurotoxicology studies. Responsible for the planning, 

conducting and successful reporting of assigned studies in 

accordance with FDA, EPA and various international regulatory 

requirements. Responsible for assisting with the direction of all 

financial and scientific functions within the discipline of 

Developmental, Reproductive and Neurotoxicology at WIL 

Research Laboratories, Inc. 

Position: Assistant Director, Developmental, Reproductive, 

and Neurotoxicology 

Responsible for assisting with the direction of all financial and 

scientific functions within the discipline of Developmental, 

Reproductive and Neurotoxicology at WIL Research 

Laboratories, Inc. Support various pharmaceutical, 

agrochemical and veterinary medical product development 

programs. Study Director, simultaneously oversee between 30-

40 developmental, reproductive and neurotoxicity studies. 

Responsible for the planning, conducting and successful 

reporting of assigned studies in accordance with FDA, EPA 

and various international regulatory requirements. 
Donald G. Stump CURRICULUM VITAE 

Professional Experience (Continued): 

1985- 1990 

1/84-6/84 

Vanderbilt University School of Medicine 

Nashville, TN 

Position: Graduate Student 

Performed graduate study for a Ph.D. in Biochemistry. 

Participated in laboratory instruction of graduate and medical 

students. In Dr. F. Chytil's laboratory, his thesis research focused 

on the characterization of the binding activity of rat cellular 

retinol-binding protein. He made extensive use of site-directed 

mutagenesis, bacterial protein expression and protein purification 

techniques in his research. Thesis title: Rat Cellular RetinolBinding Protein: Site-Directed Mutagenesis and Characterization 

of the Genomic Sequence. 

Lederle Laboratories 

Pearl River, NY 

Position: Industrial Traineeship, Department of Toxicology 

Served as a study director for several acute studies following 

Good Laboratory Practices and internal standard operating 

procedures. He also assisted in the conduct of safety assessment 

studies. 
Donald G. Stump CURRICULUM VITAE 

Continuing Education: 

Preclinical Developmental Toxicity Studies, Teratology Society Meeting, 2011. 

Postmarketing Surveillance and Communication of Risk, Teratology Society Meeting, 2011. 

Postnatal Drug Disposition and Teratogenesis. Teratology Society Meeting, 2010. 

ICH Initiatives for Conducting Pharmaceutical Preclinical Safety Studies: New and Revised 

Guidelines and Challenges. Society of Toxicology, 2010. 

Epigenetic Mechanisms. Teratology Society Meeting, 2009. 

Bio-Imaging: New Approaches for Safety Assessment and Toxicology. American College of 

Toxicology Meeting, 2008. 

CNS Functional Teratology. Teratology Society Meeting, 2008. 

Nanotoxicology: The Science of Developing a Safe Technology. Society of Toxicology 

Meeting, 2008. 

Principles in Teratology: Basic Concepts and Research Applications. Teratology Society 

Meeting, 2007. 

The Biology and Toxicology of Peri- and Post-Natal Development. Society of Toxicology 

Meeting, 2007. 

US FDA Critical Path Initiative: Building a New Toolkit to Transform Drug Development. 

Meeting of the American College ofToxicology, 2006. 

Biomarkers as Predictive and Mechanistic Tools in Toxicology. The EUROTOX 2006/6 

CTDC Congress, 2006. 

Vertebrate Embryology. Teratology Society Meeting, 2006. 

Non-traditional Animal Models in Toxicology. Meeting of the American College of 

Toxicology, 2005. 

Understanding Stem Cells: Current Research and Potential Clinical Applications. Teratology 

Society Meeting, 2005. 

Signaling Pathways and Tissue Interactions m Organ-System Development. Teratology 

Society Meeting, 2004. 

Understanding Lifespan Changes in Form and Function of the Female Reproductive System to 

Assess and Interpret Toxicity. Society of Toxicology Meeting, 2004. 
Donald G. Stump CURRICULUM VITAE 

Continuing Education (Continued): 

Integration of Human and Animal Data in Understanding Mechanisms and Risk for Human 

Birth Defects. Teratology Society Meeting, 2003. 

Mechanisms, Biomarkers, Data Interpretation: An Integrative Risk Assessment usmg 

Developmental Neurotoxicity as a Model. Teratology Society Meeting, 2002. 

Incorporation of Pharmacokinetic and Pharmacodynamic Data into Risk Assessments. 

Society of Toxicology Meeting, 2002. 

Principles of Teratology: Application of New Technologies Toward Understanding 

Abnormal Development. Teratology Society Meeting, 2001. 

Improving Risk Assessment for Human Developmental Defects: The Promise of Recent 

Advances in Developmental Biology and Genomics. Society of Toxicology Meeting, 2001. 

Signal Transduction: Pathways to Growth, Differentiation, and Morphogenesis. Teratology 

Society Meeting, 2000. 

Advanced Neurotoxicity: Biomarkers and Mechanisms of Oxidative Stress-Induced 

Neurotoxicity. Society ofToxicology Meeting, 2000. 

Evaluation of Male Reproductive Toxicity: Sperm Markers and Epididymal Mechanisms of 

Toxicity. Society of Toxicology Meeting, 1999. 

Experimental Approaches for Studying Mechanisms of Abnormal Development. Teratology 

Society Meeting, 1997. 

Endocrine Control of Reproductive Development - Normal and Abnormal Aspects. Society 

of Toxicology Meeting, 1997. 

Risk Assessment in Developmental Toxicology. Teratology Society Meeting, 1996. 

Principles in Teratology II. Teratology Society Meeting, 1995. 

New Endpoints in Developmental and Reproductive Regulatory Studies: Methods for 

Success. Society of Toxicology Meeting, 1995. 
Donald G. Stump CURRICULUM VITAE 

Working Group Participation: 

International Life Sciences Institute, Risk Science Institute Project (2005-2007). Improving the 

Use of Toxicity Data in Constructing Statistically Based SAR Models for Developmental 

Toxicity. 

Journal Editorial Boards: 

Reproductive Toxicology (2008 - ) 

Congenital Anomalies (2009 - ) 

Teaching Experience: 

Adjunct professor in the Department of Biology, Ashland University, Ashland, OH. 

Professional Affiliations: 

Middle Atlantic Reproduction and Teratology Association 

Society of Toxicology 

Teratology Society 

Midwest Chapter of the Society of Toxicology 

Japanese Society ofToxicology 

Donald G. Stump Date 
Formal Education: 

2006 

2003 

1993 

1985 

1982 

GARY D. COLEMAN 

Anatomic Pathology 

U.S. Army War College 

Carlisle Barracks, P A 

MS of Strategic Studies 

CURRICULUM VITAE 

February I, 2012 

Johns Hopkins Bloomberg School of Public Health 

Baltimore, MD 

PhD Molecular Microbiology & Immunology 

Boston University 

Boston, MA 

MS Education 

Auburn University College ofVeterinary Medicine 

Aubum,AL 

DVM 

Morehead State University 

Morehead, KY 

BS Biology 

Board Certifications and Licensure: 

Diplomate, American College ofVeterinary Pathologists, 1998 

Diplomate, American College ofVeterinary Preventive Medicine, 1994 

Licensed to practice veterinary medicine in Alabama 
Gary D. Coleman 

Professional Experience: 

06/13/11 -Present 

2008-2011 

WIL Research Laboratories, LLC 

Ashland, OH 

Position: Senior Pathologist 

Anatomic Pathology 

CURRICULUM VITAE 

Performs microscopic examination of material obtained from 

various in-house studies utilizing different species. These will 

include acute, subacute and chronic studies. Records the 

microscopic findings in the on-line computer system utilizing 

the pathology master list of terms or microscopic lesions 

established for the given species or organ. Correlates the clinical 

pathology data with the microscopic observations. Writes the 

pathology summary, which includes organ weight, gross, 

microscopic and clinical pathology interpretations on various 

studies. Provides consultation to the necropsy group during the 

examination of individual animals sacrificed or which died on 

study. Provides direction and professional consultation for the 

department during the absence of the Director. Scientific and 

technical adviser for necropsy and histology labs. Maintains 

professional level of expertise by attending scientific meetings, 

reading appropriate professional journals, and participating in 

professional organizations. Special interests in neuropathology 

include brain morphometry in developmental toxicity studies. 

Charles River Laboratories 

Frederick, MD 

Position: Veterinary Pathologist 

Responsible for performing gross and microscopic evaluation 

of tissues, recording results, preparing reports delineating 

pathological findings, and assisting with necropsy supervision. 

May provide guidance to less experienced staff members. 

May assist with necropsy supervision and may conduct 

necropsies, including gross pathology interpretation, sample 

collection and fixation. Enter pathology findings in data 

capturing system(s). Write comprehensive report narratives 

detailing all test article effects. As needed, work with study 

directors to ensure data is accurately integrated into study 

reports. May serve as a study director or principal investigator. 

Provide scientific expertise for experimental design and other 

aspects of research studies as needed. 
Gary D. Coleman CURRICULUM VITAE 

Professional Experience (Continued): 

2009-2011 

2008-2011 

Office of the Surgeon General of the Army 

Falls Church, VA 

Position: Deputy Director 

Department of Defense Veterinary Service Activity 

Assist the Director in his role as principal veterinary staff 

officer to The Surgeon General, principal veterinary advisor to 

Health Affairs and the Chief, US Army Veterinary Corps on 

veterinary medical issues. Provide strategic planning, 

advocacy, oversight and policy guidance, maintain situational 

awareness and coordinate Veterinary Service with Department 

of Defense, Federal and state agencies and civilian entities. 

Oversee accomplishment of mission priorities of food safety, 

animal medicine and veterinary medical research throughout 

Department of Defense. Integrate the activities of the Army 

Veterinary Service with Department of Defense and Federal 

Agencies. Develop and disseminate veterinary regulatory and 

doctrinal publications for Department of Defense food safety, 

veterinary public health, animal medicine and zoonotic 

diseases. Represent the Director in his absence to Office of the 

Surgeon General, Department of the Army and Department of 

Defense. 

U.S. Army 

Position: Consultant to the Surgeon General 

Veterinary Pathology 

Manage personnel assignments for U.S. Army veterinary 

pathologists supporting Department of Defense research 

institutions and the Armed Forces Institute of Pathology. 

Oversee career development for veterinary pathologists within 

the Army Veterinary Corps. Advise the Surgeon General and 

the Veterinary Corps Chief on the development and selection 

of veterinary officers for continuing education. Develop 

strategic policy for pathology support to the Department of 

Defense. Represent the veterinary pathology specialty to the 

larger military and civilian veterinary community and recruit 

incoming residents to the specialty. 
Gary D. Coleman CURRICULUM VITAE 

Professional Experience (Continued): 

2008-2009 

2004-2008 

Defense Threat Reduction Agency 

Fort Belvoir, VA 

Position: Deputy Chief 

Chemical-Biological Medical Science & 

Technology Division 

Oversee research program development and execution 

addressing chemical, biological and radiologic medical 

countermeasures. Within a joint military and intergovernmental environment, guide a team of scientists and 

acquisition professionals with a $140 Million annual budget 

working to fill gaps in warfighter protection against chemical, 

biological and radiologic warfare and terrorism threats. Lead 

and coordinate technology transition efforts with the Joint 

Program Executive Office Advanced Developer for Medical 

Countermeasures. Assist Division Chief in daily operations 

and provide administrative oversight of, and support to, 

Science and Technology Managers. Represent the Medical 

Chem-Bio Division and Directorate at national and 

international chem-bio defense conferences and meetings. 

Provide responsive support to the Medical Chem-Bio 

Directorate, the Research and Development Enterprise and the 

Defense Threat Reduction Agency. 

Walter Reed Army Institute of Research/ 

Naval Medical Research Command 

Silver Spring, MD 

Position: Chair, Institutional Animal Care and Use 

Committee 

As the Institutional Official's representative, oversee the 

administrative support of all animal research protocols within 

the two institutes to include animal use, approval, monitoring, 

and personnel qualifications in accordance with the Animal 

Welfare Regulations and Department of Defense policy. 

Responsible for maintaining Association of Assessment and 

Accreditation for Laboratory Animal Care (AAALAC) 

accreditation. 
Gary D. Coleman CURRICULUM VITAE 

Professional Experience (Continued): 

2004-2008 

2002-2004 

1997- 1999 

Walter Reed Army Institute of Research 

Silver Spring, MD 

Position: Director 

Division of Pathology 

Lead the Division of Pathology at the largest medical research 

institute within the Department of Defense. Supervise 3 

departments composed of approximately 69 military and 

civilian pathologists, Ph.D. scientists and technical and 

administrative staff, with a combined annual budget of 

approximately $1.3 Million. As the senior pathologist at 

WRAIR and the Naval Medical Research Center, direct 

medical research within the division and pathology services to 

the two institutes to support the development and testing of 

medical countermeasures against infectious agents and their 

toxins and other military-relevant diseases, health hazards and 

injuries. Responsible for resource planning and allocation to 

achieve efficient and effective utilization of fiscal, personnel 

and material resources in the division. 

Position: Chief 

Diagnostic Pathology 

Guides and develops a professional staff for two laboratories 

providing Immunohistochemical and ultrastructural support to 

researchers at the Walter Reed Army Institute of Research, Walter 

Reed Army Medical Center and the Naval Medical Research 

Center. Develops and integrates advanced molecular pathology 

techniques for research and extramural collaborations. Provides 

comprehensive gross, histological and Immunohistochemical 

support of the pathology mission at the WRAIR, including 

professional consultation on the use of laboratory animals, animal 

models of human disease, and research techniques. Serves as 

Associate Investigator on research protocols evaluating 

prophylactic and therapeutic candidates against agents of military 

significance. 

Position: Chief 

Joint Immunohistochemistry Laboratory 

Coordinates the joint laboratory effort between the Armed 

Forces Institute of Pathology and the Walter Reed Army 

Institute of Research. Conceives and develops protocols to 
Gary D. Coleman CURRICULUM VITAE 

Professional Experience (Continued): 

2004-2008 

2002-2004 

1997- 1999 

Walter Reed Army Institute of Research 

Silver Spring, MD 

Position: Director 

Division of Pathology 

Lead the Division of Pathology at the largest medical research 

institute within the Department of Defense. Supervise 3 

departments composed of approximately 69 military and 

civilian pathologists, Ph.D. scientists and technical and 

administrative staff, with a combined annual budget of 

approximately $1.3 Million. As the senior pathologist at 

WRAIR and the Naval Medical Research Center, direct 

medical research within the division and pathology services to 

the two institutes to support the development and testing of 

medical countermeasures against infectious agents and their 

toxins and other military-relevant diseases, health hazards and 

injuries. Responsible for resource planning and allocation to 

achieve efficient and effective utilization of fiscal, personnel 

and material resources in the division. 

Position: Chief 

Diagnostic Pathology 

Guides and develops a professional staff for two laboratories 

providing Immunohistochemical and ultrastructural support to 

researchers at the Walter Reed Army Institute of Research, Walter 

Reed Army Medical Center and the Naval Medical Research 

Center. Develops and integrates advanced molecular pathology 

techniques for research and extramural collaborations. Provides 

comprehensive gross, histological and Immunohistochemical 

support of the pathology mission at the WRAIR, including 

professional consultation on the use of laboratory animals, animal 

models of human disease, and research techniques. Serves as 

Associate Investigator on research protocols evaluating 

prophylactic and therapeutic candidates against agents of military 

significance. 

Position: Chief 

Joint Immunohistochemistry Laboratory 

Coordinates the joint laboratory effort between the Armed 

Forces Institute of Pathology and the Walter Reed Army 

Institute of Research. Conceives and develops protocols to 
Gary D. Coleman 

Professional Affiliations: 

CURRICULUM VITAE 

support research and diagnostic efforts. Performs gross, 

histological, and Immunohistochemical evaluation and 

interpretation of laboratory tissues in support of the research 

mission. Provides comprehensive necropsy and biopsy reports 

to research scientists. Provides professional consultation and 

advice to scientists and investigators on the use of laboratory 

animals, animal models of human disease, and techniques to 

improve the quality and efficiency of Department of Defensedirected biochemical research. Periodically serves as Acting 

Chief of the Department of Diagnostic Pathology. 

Chair, Government Policy Committee, American College of Veterinary Pathologists, 2009 -

Present. 

Member, Public Visibility Committee, American College of Veterinary Pathologists, 2009 -

Present. 

Member, Role Delineation Task Force, American College of Veterinary Pathologists, 2008-

2009. 

Member, Role Delineation Focus Group - Anatomic Pathology, American College of 

Veterinary Pathologists, 2008. 

Member, Board of Trustees, Association for the Assessment and Accreditation of Laboratory 

Animal Care, International, Inc. (AAALAC), representing the American College of 

Veterinary Pathologists, 2002 - 2011. 

Representative, U.S. Army Medical Research and Materiel Command, to the 

Armed Forces Institute of Pathology Board of Governors Strategy Panel, Falls Church, 

Virginia, 2005. 

Government Liaison, National Prion Research Program Scientific Merit Peer Review 

Meeting, Fairfax, Virginia, 2002. 

Member, American Society for Veterinary Clinical Pathology, 1998- Present. 

Member, American Veterinary Medical Association, 1985- Present. 

Gary D. Coleman Date 
GEORGE ALLEN PARKER 

Anatomic Pathology 

Formal Education: 

CURRICULUM VITAE 

November 22, 2010 

1999 University of Medicine & Dentistry ofNew Jersey 

Newark, NJ 

1977 

1976 

1972 

1968 

PhD 

Armed Forces Institute of Pathology 

Washington, DC 

Residency Program 

National Institute of Health Graduate School 

Bethesda, MD 

Postgraduate Courses 

Auburn University 

School ofVeterinary Medicine 

Aubum,AL 

DVM 

University of Kentucky 

Lexington, KY 

Pre-Veterinary Curriculum 

Board Certifications and Licensure: 

1977 - Diplomate, American College of Veterinary Pathologists 

2000- Diplomate, American Board of Toxicology 

North Carolina Veterinary Medical Board (active) 

Ohio Veterinary Medical Board (inactive) 

Kentucky Veterinary Medical Board (inactive) 

Virginia Veterinary Medical Board (inactive) 
George A. Parker 

Professional Experience: 

10/23110 - Present 

10/01/05 - 10/22/10 

2009 - Present 

10/01/2005- Present 

1986- 1987 

WIL Research Laboratories, LLC 

Ashland, OH 

Position: Vice President, Pathology 

WIL Research Laboratories, LLC 

CURRICULUM VITAE 

Directs and coordinates pathology-related services of WIL 

Research Laboratories LLC. Responsible for administration of 

pathology department divisions in Ashland, OH and 

Hillsborough, NC, which includes 14 pathologists and 

approximately 70 technical and clerical personnel. Services 

include histology, clinical pathology, histopathology, flow 

cytometry, immunopathology, immunohistochemistry, 

morphometry and stereological analysis. Provides consultation 

to corporate management, sponsors, study directors and 

pathologists regarding issues related to pathology. Evaluates 

new methodology and technology, and develops new services 

when appropriate to meet research objectives. Interfaces with 

management of various scientific and support departments to 

insure the proper integration of pathology services. Participates 

in business development activities as required to meet corporate 

objectives. 

Position: Director, Pathology 

Auburn University College ofVeterinary Medicine 

Auburn, AL 

Position: Adjunct Professor, Pathology 

Position: Laboratory Director 

Serves as the local management of the histology and 

histopathology laboratory (Biotechnics LLC) located in 

Hillsborough, NC. 

Virginia-Maryland Regional College ofVeterinary Medicine 

Blacksburg, VA 

Position: Adjunct Professor 

Department of Large Animal Clinical Sciences 
George A. Parker CURRICULUM VITAE 

Professional Experience (Continued): 

1980-2005 

1978- 1980 

1978 

1977- 1980 

1974- 1977 

1975- 1976 

1973- 1974 

Biotechnics, LLC 

Hillsborough, NC 

Position: President and Laboratory Director 

Department of Veterinary Pathology 

Armed Forces Institute of Pathology 

Washington, DC 

Position: Adjunct Staff Member 

Uniformed Services University of the Health Sciences 

Bethesda, MD 

Position: Histology Instructor 

Armed Forces Radiobiology Research Institute 

National Naval Medical Center 

Bethesda, MD 

Position: Head, Comparative Pathology Division 

Armed Forces Institute of Pathology 

Washington, DC 

Position: Residency in Veterinary Pathology 

National Zoological Park 

Smithsonian Institution 

Washington, DC 

Position: Prosecutor, Office ofPathology 

Ubon Royal Thai Air Force Base 

Ubon, Thailand 

Position: Base Veterinarian 
George A. Parker 

Professional Experience (Continued): 

1972- 1973 

Professional Affiliations: 

Macdill Air Force Base 

Tampa, FL 

Position: Assistant Base Veterinarian 

American College of Veterinary Pathologists 

Society of Toxicologic Pathologists 

Society of Toxicology 

American College of Toxicology 

European Society of Toxicologic Pathology 

Japanese Society of Toxicologic Pathology 

American Society for Veterinary Clinical Pathology 

CURRICULUM VITAE 
Formal Education: 

1992 

1988 

1984 

1980 

CATHERINE A. PICUT 

Biotechnics 

Yale Law School 

New Haven, CT 

JD 

Cornell University 

School of Veterinary Medicine 

Ithaca, NY 

Diplomate, ACVP 

University of Pennsylvania 

School ofVeterinary Medicine 

Philadelphia, P A 

VMD 

University of Rochester 

Rochester, NY 

BABiology 

Board Certifications and Licensure: 

2004 Board Certification by American Board of Toxicology 

CURRICULUM VITAE 

January 27, 2011 

2000 Registered Quality Assurance Professional, Good Laboratory Practices (RQAPGLP) 

2000 North Carolina Veterinary Medical Board, License Number 4759 

2000 Quality Assurance Professional, Society of Quality Assurance 

1993 Patent Attorney Reg. No. 37,419 

1993 District of Columbia Bar (inactive status) 

1992 Virginia State Bar (#34785) (inactive status) 

1992 New Jersey State Bar (active status) 

1988 Board Certification by the American College of Veterinary Pathologists 

1984 Pennsylvania State Veterinary Medical License (inactive status) 

1984 New York State Veterinary Medical License (inactive status) 
CatherineA. Picut 

Professional Experience: 

10/01/05- Present 

01/95 - 09/05 

01/99 - 09/05 

11/93 - 12/95 

WIL Research Laboratories, LLC/Biotechnics 

Hillsborough, NC 

Position: Senior Pathologist 

CURRICULUM VITAE 

Provides GLP-compliant research pathology services to client 

laboratories engaged in the safety and efficacy evaluation of new 

drugs, biologics, medical devices and environmental chemicals. 

Specializes in on-site services at client facilities to ensure rapid 

and efficient completion of high profile projects. Interface 

extensively with study directors, histology supervisors, and 

quality assurance personnel to harmonize and expedite pathology 

support services. 

Biotechnics, Inc. 

Hillsborough, NC 

Position: Vice President and Pathologist 

Provides GLP-compliant research pathology services to client 

laboratories engaged in the safety and efficacy evaluation of new 

drugs, biologics, medical devices and environmental chemicals. 

Specializes in on-site services at client facilities to ensure rapid 

and efficient completion of high profile projects. Interface 

extensively with study directors, histology supervisors, and 

quality assurance personnel to harmonize and expedite pathology 

support services. 

Position: Quality Assurance Advisor/Legal Counsel 

Hoffman-La Roche 

Nutley, NJ 

Position: Patent Attorney 

50% Prosecution, 30% Collaborative Agreements, 20% Legal 

Opinions 
CatherineA. Picut 

Pro(essional Experience (Continued): 

06/91 - 1 0/93 

King & Spalding 

Washington, DC 

Position: Attorney 

Food and Drug Law 

Veterinary Labs of America/Biotechnics 

Sterling, VA 

05190 - 09/90 Position: Diagnostic Pathologist 

Veritas Laboratories, Inc. 

Burlington, NC 

09/88 - 09/89 Position: Senior Toxicologist Pathologist 

Pro(essional Affiliations: 

New Jersey Intellectual Property Law Association 

American College of Veterinary Pathologists 

Society of Toxicologic Pathologists 

North Carolina Society of Quality Assurance 

Society of Quality Assurance 

Catherine A. Picut Date 

CURRICULUM VITAE 
Formal Education: 

1988 

1973 

1971 

ANN E. RADOVSKY 

Anatomic Pathology 

Purdue University 

West Lafayette, IN 

PhD in Veterinary Pathology 

Drexel University 

Graduate School 

Philadelphia, P A 

MS in Biomedical Engineering 

Purdue University 

College ofVeterinary Medicine 

West Lafayette, IN 

Doctor of Veterinary Medicine 

CURRICULUM VITAE 

January I 0, 20 II 

Board Certifications and Licensure: 

Diplomate, American College ofVeterinary Pathologists, 1991 

Diplomate, American Board of Toxicology, 1996; Recertified for 2002-2011 

Licensed to practice veterinary medicine in Indiana 

Professional Experience: 

03/23/02 - Present 

WIL Research Laboratories, LLC 

Ashland, OH 

Position: Senior Pathologist 

Anatomic Pathology 

Performs microscopic examination of material obtained from 

various in-house studies utilizing different species. These will 

include acute, subacute and chronic studies. Records the 

microscopic findings in the on-line computer system utilizing 

the pathology master list of terms or microscopic lesions 

established for the given species or organ. Correlates the 

clinical pathology data with the microscopic observations. 
Ann E. Radovsky CURRICULUM VITAE 

Professional Experience (Continued): 

05/04/98 - 03/22/02 

Writes the pathology summary, which includes organ weight, 

gross, microscopic and clinical pathology interpretations on 

various studies. Provides consultation to the necropsy group 

during the examination of individual animals sacrificed or 

which died on study. Provides direction and professional 

consultation for the department during the absence of the 

Director. Scientific and technical adviser for necropsy and 

histology labs. Maintains professional level of expertise by 

attending scientific meetings, reading appropriate professional 

journals, and participating in professional organizations. 

Special interests in neuropathology include brain morphometry 

in developmental toxicity studies. 

Position: Staff Pathologist 

Anatomic Pathology 

Performs microscopic examination of material obtained from 

various in-house studies utilizing different species, including 

acute, subacute and chronic type (duration) studies. Records the 

microscopic findings in the on-line VAX system utilizing the 

pathology master list of terms or microscopic lesions established 

for the given species or organ. Correlates the clinical pathology 

data with the microscopic observations. Writes the pathology 

summary, which includes gross, microscopic and clinical 

pathology on various studies in a format acceptable to report 

writing and the study director. Provides consultation to the 

necropsy group during the examination of individual animals 

sacrificed or which died on study. Provides direction and 

professional consultation for the department during the absence 

of the Director. Maintains professional level of expertise by 

attending meetings and reading appropriate professional journals. 

Special interests in neuropathology include brain morphometry 

in developmental toxicity studies. Assists with the clinical care 

of study animals. 
Ann E. Radovsky CURRICULUM VITAE 

Professional Experience (Continued): 

1993 - 2005 

1993- 1998 

1988- 1993 

Safar Center for Resuscitation Research 

University of Pittsburgh 

Position: Neuropathology Consultant 

Describes, evaluates and semi-quantitates histopathologic brain 

damage in experimental animals (rats and dogs) after transient 

cardiac arrest, shock or trauma in tests of therapeutic 

interventions. 

Laboratory of Experimental Pathology National Institute of 

Environmental Health Sciences 

Position: Veterinary Pathologist 

Responsible for toxicologic pathologist evaluations, interpreting 

and defending results of chronic rodent bioassays for toxicity and 

carcinogenicity of administered environmental compounds and 

conducting research investigating characteristics of reactions to 

foreign substances. Evaluated data for risk assessment. 

Consulted regularly in peer review on histopathology and data 

assessment. Conducted training sessions on basic histology and 

histopathology for biological scientists and technicians. 

Conducted reviews of slides and written histopathology data and 

determine target organs and toxic reactions. Generated reports 

and presentations interpreting pathology fmdings, dose response 

and toxicokinetics. 

Safar Center for Resuscitation Research 

University of Pittsburgh 

Position: Assistant Professor 

Conducted comparative and experimental neuropathology 

studies, evaluated the severity of brain damage in rats and dogs, 

semi-quantitative/computer-assisted methods. Supervised histotechnicians and trained research fellows. 
Ann E. Radovsky CURRICULUM VITAE 

Professional Experience (Continued): 

1983-1988 

1982- 1983 

1981 - 1982 

1979- 1981 

Purdue University 

West Lafayette, IN 

Position: Graduate Instructor 

Combined Ph.D. - Residency in Veterinary Pathology. 

Residency training in diagnostic pathology of all domestic 

species; necropsy and biopsy service, extensive experience in 

general and toxicologic pathology, electron microscopy and 

statistics. Ph.D. thesis correlated heart pathology with pacing 

threshold and electrical impedance. 

Federated Medical Resources 

Honeybrook, P A 

Position: Clinical Veterinarian for Laboratory Animal Facility 

Clinical medicine for breeding colonies of dogs, African Green 

Monkeys, sheep, pigs and rodents. 

University of Pennsylvania 

Philadelphia, P A 

Position: Research Associate 

Developed pre-clinical safety protocol for external 

electromagnetic bone stimulator using beagle dogs. 

Quonset Animal Hospital 

East Greenwich, RI 

Position: Clinical Veterinarian in small animal practice. 
Ann E. Radovsky CURRICULUM VITAE 

Professional Experience (Continued): 

1976- 1979 

1974- 1979 

1971 - 1973 

University of Rhode Island 

Kingston, RI 

Position: Research Associate and Lecturer 

Developed and taught a graduate course in rodent and rabbit 

experimental techniques. Assisted with acute surgical 

experiments in dogs (e.g. flowmeters around vessels). 

Support from National Institute of Dental Research, University 

of Pennsylvania 

Philadelphia, P A 

Position: Post-Doctoral Fellow 

Assisted with the study of the clinical effects of electrical 

stimulation of dentin in dogs. 

Support from National Heart and Lung Institute, Drexel 

University, P A 

Position: Post-Doctoral Fellow 

Evaluated the gross, clinicopathologic and histopathologic 

effects of electrically-active biogalvanic power cells and 

correlated subcutaneous histopathology with measured electrical 

performance. 
Ann E. Radovsky CURRICULUM VITAE 

Professional Affiliations: 

1991 - Present 

1995 -Present 

1996 - Present 

American College of Veterinary Pathologists (Diplomate) 

Member, Society of Toxicologic Pathologists 

American Board of Toxicology (Diplomate) 

Continuing Education: 

Neuropathology. STPIIFSTP 2010 Joint Symposium, 2010. 

Experimental Biology Annual Meeting, 2007. 

Second RTP Rodent Pathology Course, 2004. 

Second Annual Workshop on the Pathology of Mouse Models for Human Disease 2003, 

Cornell University, 2003. 

Workshop on Evaluating Toxicity to the Male Reproductive Tract, 2002. 

Annual Symposium of the Society of Toxicologic Pathologists, 1999- 2002, 2005, 2006, 2009 

Annual Meeting of the American College of Veterinary Pathologists, 1992-1997. 

Systemic Pathology of Laboratory Animals. International Life Sciences Institute, 1992-1995. 

Mid-America Toxicology Course, 1996. 

Concepts in Molecular Biology, 1996. 

Midwest Association of Veterinary Pathologists: Attended and made oral presentations at 

annual summer meetings 1999, 2000, 2001 

Ann E. Radovsky Date 
ATTACHMENT 8 
Attn: Peter Voytek 

Date: 6/19/2012 

Sample: Composite of 4 - 4 Liter bottles 

of 1 ,2-Dichloroethane 

EDC Purity 

Richard Rhodes 

1,2- Dichloroethane 

DOW RESTRICTED - For internal use only 

The Dow Chemical Company 

2301 Brazosport Blvd 

Freeport, Texas 77541-3257 

ANALYZED 

PPM/WT 

99.97 
ATTACHMENT C 
WILR s RCt-1 

Page I of 53 

PROTOCOL 

An Extended One-Generation Drinking Water 

Reproductive Toxicity Study of Ethylene Dichloride in Rats 

[OECD Guideline 443] 

WIL Study Number: WIL-417005 

Submitted To: 

HAP Task Force 

c/o Peter Voytek, PhD 

P.O. Box 331 

Millwood, VA 22646 

WIL Research 

1407 George Road 

Ashland, OH 44805-8946 

ATTACHMENT 

PROTOCOL 

Wll-417005 

June 25. 2012 
Page 2 of 53 

1 OBJECTIVE: 

WIL-417005 

June 25, 2012 

To evaluate the potential effects of the test substance on reproduction in an extended 

one-generation study covering life stages that are not covered by other types of 

toxicity studies. This will include determining the effects of the test substance on 

male and female reproductive processes including gonadal function, estrous cyclicity, 

mating behavior, conception, gestation, parturition, lactation, and weaning, and on 

survival, growth, and development of the offspring. A minimum of one litter will be 

produced. 

As a part of this assessment, offspring will also be evaluated for effects on the 

nervous system, alterations in endocrine function (including thyroid perturbations), 

and effects on other systemic toxicity parameters. 

The extended one-generation reproductive toxicity study will serve as a substitute for 

the oral subchronic neurotoxicity and multigeneration reproductive studies described 

in Tier 2 testing for ethylene dichloride (EDC) under an enforceable consent 

agreement (U.S. EPA, 2003, U.S. EPA, 2009) between the HAP Task Force and the 

U.S. EPA. 

This study will be conducted in accordance with the Organisation for Economic 

Cooperation and Development (OECD) Guideline for the Testing of Chemicals 

Guideline 443, Extended One-Generation Reproductive Toxicity Study, July 2011. 

This study will be conducted in compliance with the U.S. EPA Toxic Substances 

Control Act (TSCA; 40 CFR Part 792), the OECD Series on Principles of Good 

Laboratory Practice and Compliance Monitoring, Number 1 OECD 

ENV/MC/CHEM(98)17 Principles of Good Laboratory Practice, and the European 

Community (EC) Europ~an Parliament and Council Directive 2004/1 0/EC (O.J. No. 

L 50/44, 02 February 2004). 

2 PERSONNEL INVOLVED IN THE STUDY: 

2.1 Sponsor Representative: 

Peter E. Voytek, PhD 

PO Box 331 

1680 Bishop Meade Highway 

Millwood, VA 22646 

Tel: (540) 837-1602 

Email: pvoytekRSI@aol.com 
Page 3 of 53 

2.2 WIL Studv Director: 

Melissa J. Beck, PhD 

Associate Director, Neurosciences 

and Head of Juvenile Toxicology 

Tel: (419) 289-8700 

Fax: (419) 289-3650 

Email: melissa. beck@wilresearch.com 

2.3 WIL Study Pathologist: 

To be assigned at Study Initiation. 

2.4 Contributing Scientist, Ovarian Primordial Follicle Evaluation: 

Catherine A. Picut, VMD, DACVP, DABT 

Senior Pathologist 

WIL Research 

31 0 Millstone Dr. 

Hillsborough, NC 27278 

Tel: (919) 245-3114 x106 

Fax: (919) 245-3115 

Email: catherine. picut@wilresearch.com 

2.5 Principal Investigator (Ovarian Histology): 

Sirena Hudgins, HT (ASCP) 

Supervisor, Histology 

WIL Research 

310 Millstone Dr. 

Hillsborough, NC 27278 

Tel: (919) 245-3114 x114 

Fax: (919) 245-3115 

Email: sirena.hudgins@wilresearch.com 

WIL-417005 

June 25,2012 
Page 4 of 53 

2.6 Test Site Quality Assurance: 

For histology conducted at WIL Research at Hillsborough: 

Janice Jackson-Freeman 

Compliance Specialist, Quality Assurance 

WIL Research 

310 Millstone Dr. 

Hillsborough, NC 27278 

Tel: (919)245-3114xl19 

Fax: (919) 245-3115 

Email: janice. freeman@wilresearch. com 

2.7 WIL Departmental Responsibilities: 

Pragati Sawhney Coder, PhD, DABT 

Senior Toxicologist, Developmental and 

Reproductive Toxicology 

Emergency Contact 

Email: pragati.sawhney.coder@wilresearch.com 

Steven D. Barkyoumb, DVM, PhD, DACVP 

Senior Vice President, 

Global Operations- Safety Assessment 

Donald G. Stump, PhD, DABT 

Senior Director, Developmental 

and Reproductive Toxicology 

Eddie D. Sloter, PhD 

Assistant Director, Developmental and 

Reproductive Toxicology 

Bennett J. Varsho, MPH, DABT 

Director, Operations 

Gwendalyn M. Maginnis, DVM 

Clinical Veterinarian 

CarolS. Wally, BA, SRS, RLATG 

Group Manager, Sample Processing Laboratory 

Carol A. Kopp, BS, LAT 

Manager, Gross Pathology and 

Developmental Toxicology Laboratory 

WIL-417005 

June 25, 2012 
Pa e 5 of 53 

George A. Parker, DVM, PhD, DACVP, DABT 

Vice President, Pathology 

Michael J. Schlosser, PhD, DABT 

Vice President, Analytical, 

Metabolism and In Vitro Toxicology 

Ronald E. Wilson, BS 

Director, Global Scientific Application Architecture 

Heather L. Johnson, BS RQAP-GLP 

Manager, Quality Assurance 

Robert A. Wally, BS 

Operations Manager, Reporting & 

Technical Support Services 

3 STUDY SCHEDULE: 

Proposed Experimental Starting 

(Animal Receipt) Date: 

Proposed Experimental Start 

(First Day of Dosing) Date: 

Proposed Experimental 

Completion/Termination Date: 

Proposed Audited Report Date: 

(without Optional Second Generation) 

July 31,2012 

August 14, 20 12 

To be determined 

May 14,2013 

WIL-417005 

June 25,2012 
4 TEST SUBSTANCE DATA: 

4.1 Test Substance Shipment: 

Page 11 of 53 

WIL-417005 

June 25, 2012 

Test substance and applicable documentation, including a Certificate of 

Analysis, will be shipped under Sponsor's responsibility to: 

Formulations Laboratory 

Attn: Terri Rafeld, Manager 

(WIL-417005; Melissa Beck) 

WIL Research 

1407 George Road 

Ashland, Ohio 44805-8946 

Tel: (419) 289-8700 

Fax: ( 419) 289-3650 

Email: terri.rafeld@wilresearch.com 

4.2 Identification: 

Ethylene dichloride (EDC; 1 ,2-Dichloroethane) 

4.3 CAS Number 

107-06-2 

4.4 Lot Number: 

Not Applicable; there is no lot number assigned as the manufacturer has a 

continuous running plant. 

4.5 Expiration/Retest Date: 

Not Applicable. 

4.6 Purity: 

99.97% for the material provided. 

4.7 Storage Conditions: 

In a flammables cabinet in a climate controlled atmosphere. 

4.8 Stability: 

The test article is considered to be stable under the storage conditions provided 

by the Sponsor. 
Page 7 of 53 

4.9 Physical Description: 

To be documented by WIL Research. 

4.10 Reserve Samples: 

WIL-417005 

June 25,2012 

Reserve samples of the test substance will be taken in accordance with 

WIL Standard Operating Procedures (SOPs) and stored in the WIL Research 

Archives indefmitely, unless otherwise specified. 

4.11 Personnel Safety Data: 

To be provided by the Sponsor. It is the responsibility of the Sponsor to notifY 

the testing facility of any special handling requirements for the test substance. 

A Material Safety Data Sheet (MSDS) should accompany the test substance 

upon arrival at the laboratory. 

4.12 Test Article Disposition: 

With the exception of the reserve sample for each batch of test substance, all 

neat test substance remaining at study completion will be returned to the 

Sponsor (storage conditions, contact, address, fax number and telephone to be 

provided). Alternatively, the test substance can be retained for subsequent 

studies. 

5 TEST SYSTEM: 

5.1 Species: 

Rat 

5.2 Breed/Strain: 

Sprague Dawley Crl:CD(SD) 

5.3 Source: 

Charles River Laboratories, Inc. 

(Facility to be documented in the study records) 

5.4 Number of Animals: 

108 Males and 108 Females (maximum of 120 males and 120 females 

purchased; males and females will be ordered from separate barrier rooms to 

ensure avoidance of sibling mating). Animals not assigned to the study will be 
Page X of 53 

WIL-417005 

June 25, 2012 

transferred to the stock animal colony or will be euthanized by carbon dioxide 

inhalation and discarded. 

The number of animals is based on the OECD Guideline for the Testing of 

Chemicals: Guideline 443, Extended One-Generation Reproductive Toxicity 

Study, July 2011, which recommends including a sufficient number of mating 

pairs to yield at least 20 pregnant females per dose group. Given the possibility 

of non-gravid animals, unexpected deaths, total litter losses, or treatment-related 

moribundity and/or mortality in each generation of the study, this is an 

appropriate number of animals to achieve the desired number of animals in each 

generation. 

5.5 Body Weight Range: 

At randomization: 

5.6 Approximate Age: 

At initiation of dosing: 

5.7 Identification System: 

Males 300-450 g. 

Females 175-300 g. 

Males and Females: 90 days 

The F0 animals will be uniquely identified by a Monel® metal ear tag displaying 

the animal number. Individual cage cards will be affixed to each cage and will 

display the animal number, group number, study number, dosage level and sex 

of the animal. Offspring will be identified by tattoo markings applied to the 

digits after parturition and by ear tag after weaning. 

5.8 Justification for Selection: 

This species and strain of animal is recognized as appropriate for reproduction 

and neurotoxicity studies. WIL Research has reproductive and neurotoxicity 

historical control data in the Crl:CD(SD) rat. This animal model has been 

proven to be susceptible to the effects of reproductive and neurological 

toxicants. 

6 SPECIFIC MAINTENANCE SCHEDULE: 

6.1 Animal Housing: 

Fa animals will be housed individually in clean suspended wire mesh cages in 

an environmentally controlled room during acclimation and continuing until 

euthanasia (for males) or until mating (females). The cages will be elevated 

above cage board or other suitable material, which will be changed at least three 

times each week. 
Page 9 of 53 

WIL-417005 

June 25,2012 

During cohabitation, females will be paired ( 1: 1) with a male from the same 

treatment group in a suspended wire mesh cage (home cage of the male). 

following successful mating the females will be housed individually in a plastic 

maternity cage containing nesting material (Aspen Bed TM 1) and will remain in 

these cages until weaning of the offspring. Analyses for possible contamination 

are conducted on representative lots of bedding and documented in the raw data. 

The F0 females will then be individually housed in wire-mesh cages until 

euthanasia. Aspen bedding has been shown to be a suitable bedding for 

reproduction studies; it does not affect pup survival (Burkhart, 1978) or 

significantly induce liver enzymes like soft-pine bedding, including heat-treated 

pine wood bedding (Weichbrod, 1988). Hardwood bedding, such as Aspen 

BedT"1, does not need to be heat treated because it does not release oils and 

resins like soft-pine bedding. Therefore, the use of Aspen bedding (non-heattreated) in the current study is not expected to impact the outcome ofthe study. 

All F 1 offspring selected to remain on study after weaning will be housed (by 

sex within litter) in plastic cages with nesting material until postnatal day 

(PND) 28. F1 offspring not selected to continue on study will be necropsied on 

PND 21. Subsequently, the F1 males and females will be individually housed in 

clean suspended wire-mesh cages in an environmentally controlled room and 

will remain in these cages until euthanasia. 

Nylabone® (or a similar enrichment device) will be provided to each animal for 

environmental enrichment and to aid in maintaining the animal's oral health, 

beginning during acclimation and continuing throughout the course of the study. 

The cages will be subjected to routine cleaning at a frequency consistent with 

maintaining good animal health and WIL Standard Operating Procedures. The 

facilities at WIL Research are accredited by the Association for Assessment and 

Accreditation of Laboratory Animal Care International (AAALAC 

International). The cage sizes and housing conditions employed in this study 

will be in compliance with the Guide for the Care and Use of Laboratory 

Animals (1996). 

6.2 Environmental Conditions: 

Controls will be set to maintain temperature at 71 ± 5°F (22 ± 3°C) and relative 

humidity at 50 ± 20%. Temperature and relative humidity will be monitored 

continuously. Data for these two parameters will be scheduled for automatic 

collection on an hourly basis. Fluorescent lighting controlled by light timers 

will provide illumination for a 12-hour light/dark photoperiod (on at 6 a.m. and 

off at 6:00p.m). The ventilation rate will be set at a minimum of 10 room air 

changes per hour, 100% fresh air. 
Pa e I 0 of 53 

6.3 Drinking Water: 

WIL-417005 

June 25,2012 

Cage banks will not be connected to an automatic watering system. Municipal 

water (with test substance added during the treatment period for the animals 

assigned to Groups 2-4) will be available ad libitum to all animals and will be 

provided from standard plastic water bottles (approximately 250 mL). Bottles 

will be checked daily for spillage and supplemented as necessary. The 

occurrence of spillage will be documented. Filters servicing the water supply 

will be changed regularly according to WIL SOPs. The municipal water 

supplying the laboratory is analyzed according to WIL SOPs on a routine basis 

to ensure that contaminants are not present in concentrations that would be 

expected to affect the outcome of the study. 

6.4 Basal Diet: 

Harlan Laboratories 2016CM Teklad Global 16% Protein Rodent Diet will be 

offered ad libitum during the study. The batch of feed used on this study must 

have a genistein-equivalent content (aglycone) of less than 300 ppm (Owens 

eta/., 2003). Periodic analyses of the certified feed are performed by the 

manufacturer to ensure that heavy metals and pesticides are not present at 

concentrations that would be expected to affect the outcome of the study. 

Results of the analyses are provided to WIL Research by the manufacturer. 

Feeders will be changed and sanitized once per week. Samples (approximately 

100 g each) of each batch of the diet used during the study will be retained 

frozen (approximately -20°C) until issuance of the final report, in case the study 

results necessitate further analysis of diet ingredients. 

7 EXPERIMENTALDESIGN: 

7.1 Animal Receipt and Quarantine: 

Each animal will be inspected by a veterinarian or qualified technician under the 

direct supervision of a laboratory veterinarian upon receipt. Rats judged to be 

in good health and suitable as test animals will be immediately placed in 

quarantine for a minimum of 10 days. During this time, the animals will also be 

acclimated to the water bottles that will be used during the study. All rats will 

be initially weighed, permanently identified with a metal ear tag and receive a 

clinical observation. During the quarantine period, each rat will be observed 

twice daily for changes in general appearance and behavior. Prior to the start of 

the in-life phase, those animals judged to be suitable test subjects will be 

identified. 
Pa e 11 of 53 

7.2 Randomization: 

WIL-417005 

June 25, 2012 

At the conclusion of the quarantine period, animals judged to be suitable test 

subjects and meeting acceptable body weight requirements will be assigned at 

random using a computer program. At that time, the animal numbers and 

corresponding body weights will be entered into the WIL Toxicology Data 

Management System (WTDMS TM). A printout containing the animal numbers 

and individual group assignments will be generated based on body weight 

stratification into a block design. Animals will then be arranged into the groups 

according to the printout. Body weights at randomization will be within ±20% of 

the mean for each sex. The control group and three test substance groups will 

consist of27 males and 27 females. 

Any animal assigned to the study that is found dead, euthanized in extremis or 

exhibits abnormal clinical signs, reduced food consumption or body weight 

losses prior to the start of dosing may be replaced by an animal of appropriate 

age when possible. Replacement animals will be arbitrarily assigned (not 

computer randomized) to the study based on comparable body weights (if 

possible) with respect to the animal that was replaced. 

7.3 Route and Rationale of Test Substance Administration: 

The route of administration will be oral via the drinking water as this route of 

exposure may be used to estimate equivalent inhalation exposures (Sweeney 

and Parker, 2010; see Section 7.4.1) and since this route of administration is a 

reliable and accurate method of delivering test substance in an extended onegeneration reproductive toxicity study. Historically, this route has been used for 

studies ofthis nature. 

7.4 Organization of Test Groups, Dosage Levels and Treatment Regimen: 

7.4.1 Rationale for Dose Selection: 

The dosage levels were determined from results of previous studies and 

were provided by the Sponsor Representative. 

The primary exposure route of concern for EDC is inhalation. Based on 

a pharmacokinetic (PK) study (Saghir et al., 2006) and a physiologically 

based PK (PBPK) model describing disposition in rats following oral or 

inhalation routes of exposure (Sweeney and Parker, 2010), oral doses of 

50, 150, and 300 mg/kg/day were selected. The model will then be used 

to estimate equivalent inhalation exposures. A 300 mg/kg/day exposure 

to EDC (Z% in drinking water) would be expected to yield blood EDC 

peak and cumulative exposures that would produce some maternal 

toxicity. This selection is based on the assumption that the narcotic-type 
Page 12 of 53 

WIL-417005 

June 25, 2012 

effects (central nervous system depression) may be attributed to elevated 

levels ofEDC rather than metabolite levels or GSH depletion. The low 

dose of 50 mg/kg/day (X%) is predicted to identifY a clear no-observedadverse-cfiect level (NOAEL). If treatment-related effects are seen in 

the high-dose group, the mid dose of 150 mg/kg/day (Y%) is expected to 

provide dose-response data. 

7.4.2 Organization of Test Groups: 

The following table presents the study group arrangement. 

Drinking Approximate Number of 

Group Test Substance Water Dosage Level Animals 

Number Concentration 

(%) (mg/kg/day)" Male Female 

1 Control (Drinking_ Water) 0 0 27 27 

2 Ethylene Dichloride X 50 27 27 

3 Ethylene Dichloride y 150 27 27 

4 Ethylene Dichloride z 300 27 27 

a - Approxtmate dosage level based on htstoncal water consumptton values. Achteved 

dosage levels will be calculated at the conclusion of the study based on water 

concentration, recorded body weight, and water consumption values for each 

animal. 

7.4.3 Vehicle (Drinking Water): 

Municipal water. 

7.4.4 Treatment Regimen: 

Untreated or test substance-treated drinking water formulations will be 

offered ad libitum via standard plastic water bottles (approximately 

250 mL) with metal sipper tubes, and supplied for the duration of the 

study, as noted in Sections 7.4.4.1 and 7.4.4.2. Fresh vehicle and test 

substance drinking water formulations will be provided approximately 

weekly. 

7.4.4.1 F0 Generation: 

Males: Following randomization, F0 male animals will be 

offered the control or test substance-treated drinking water for 

approximately 28 days prior to mating, throughout mating (up to 

14 days), and for approximately 49 additional days until 

euthanasia. 

Females: Following randomization, F0 female animals will be 

offered the control or test substance-treated drinking water for 

approximately 28 days prior to mating and continuing throughout 
Page 13 of 53 

WIL-417005 

June 25, 2012 

mating (up to 14 days), gestation (approximately 21 days), and 

lactation (approximately 21 days) until euthanasia on lactation 

day(LD) 22 .. 

7.4.4.2 F1 Generation (Fo Offspring): 

The F 1 generation will potentially be exposed to the test 

substance in utero, through nursing during lactation and via the 

drinking water from the time of weaning through the scheduled 

euthanasia (as appropriate for the relevant Cohort). 

7 .4.5 Adjustment of Doses: 

Drinking water concentration adjustments are designed to provide a 

relatively constant mglkg body weight/day dose throughout all study 

phases. 

The proposed drinking water adjustments during lactation and postweaning are presented below. During lactation, drinking water 

concentrations will be adjusted using historical control water 

consumption data for lactating females to account for the large and rapid 

increase in food consumption (2-3 fold) typical for rats in mid- to late 

lactation. Dams awaiting necropsy will receive drinking water 

containing the same concentration of EDC that was provided during 

breeding (i.e., during LD 21-22). 

To account for the large amount of water consumed per kg of body 

weight in weanling pups, from PND 21-35, male and female weanlings 

will receive drinking water containing the same concentration of EDC 

that was provided to the Fo females during the second week of lactation 

(half of the adult concentration). On PND 35, F1 male and female 

offspring will receive adult water concentrations until termination, 

unless an Fz generation is required. If a second generation is produced, 

similar drinking water adjustments will be employed for the F1 dams 

during lactation. 

Exposure Unadjusted Adjustment Adjusted 

TMI Increase• Concentration Concentration Period (%) Factor (%) 

LD 7-14 3.1X X,Y,Z 2 X, YzY, YzZ 

LD 14-21 3.8X X,Y,Z 3 X,YzY,YzZ 

PND 21-28b 2.4X X,Y,Z 2 X, YzY, YzZ 

PND 28-35c l.9X X,Y,Z 2 X,YzY,YzZ a Test Matenal1ntake relative to nonpregnant adult females 

b Assumes water intake mirrors food intake in young animals (Marty et a!., 2003) 

which is true at other time points included in this table. 

c After PND 35, pups return to unadjusted drinking water concentrations. 
Pa e 14 of 53 

WIL-417005 

June 25, 2012 

7.5 Preparation and Analysis of Test Substance-Treated Drinking Water: 

7.5.1 Preparation of Test Substance-Treated Drinking Water: 

The drinking water solutions will be prepared as a fixed percent EDC in 

water. Based on the physical characteristics of the test substance, 

appropriate methods will be used to ensure the best possible mixing of 

the test substance in the drinking water. Test substance formulations 

will be prepared approximately weekly. The test substance will be 

added to the drinking water and mixed to obtain the appropriate drinking 

water concentrations, and the formulations will be mixed periodically 

throughout the study based on stability data. 

Any special procedures required for formulation will be documented 

according to Good Laboratory Practice Standards and presented in the 

final report of this study. 

7.5.2 Analysis of Test Substance-Treated Drinking Water: 

Drinking water solutions will be analyzed at WIL Research using an 

analytical method that will be developed and validated under a 

concurrent study (see Appendix B). 

7.5.3 Stability of Test Substance-Treated Drinking Water: 

Stability of the test substance in the drinking water preparations will be 

established prior to the start of dosing, if possible. Representative 

batches of the drinking water at each of the selected dosage levels will 

be mixed, and duplicate sets of samples will be taken from the middle of 

the control formulation and each test formulation. One set of samples 

will be stored under laboratory conditions for up to 15 days after 

preparation. These samples will be analyzed for stability after eight and 

15 days. The other set of samples will be stored frozen at approximately 

-20°C as backup samples. 

7.5.4 Homogeneity and Concentration of Test Substance in the Drinking 

Water: 

Prior to the first exposures, duplicate sets of samples of drinking water 

will be collected from the top, middle, and bottom strata of each dosage 

formulation, and duplicate sets of samples from the middle of the 

controls. One set of samples from all dosage levels, including controls, 

will be analyzed for test substance concentration. If stability permits, 

analytical verification will be performed prior to introduction of the test 

substance-treated water to the animals. The other set of samples will be 

stored frozen at approximately -20°C and discarded at the end of the in-
Page 15 of 53 

WIL-417005 

June 25, 2012 

life phase following acceptance of the analytical results by the Study 

Director. 

Samples will be taken from the middle of each dosing formulation 

prepared during the first 4 weeks of the dosing period and monthly 

thereafter for the remainder of the study. Any backup samples kept at 

WIL Research will be discarded following acceptance of the analytical 

results by the Study Director. 

7.5.5 Reserve Samples: 

Samples (from each day of preparation will be collected from each dose 

level (including controls). These samples will be stored frozen at 

approximately -20°C in sealed vials in the WIL Research Archives and 

will be discarded following acceptance of the study results. 

7.6 .Eo Determination of Estrous Cycles: 

Daily vaginal lavages will be performed to determine the stage of estrus 

beginning 14 days after the initiation of test substance administration. Estrous 

cycles will be evaluated for 2 weeks prior to mating, and during cohabitation 

until evidence of copulation (vaginal copulatory plug or evidence of sperm in a 

vaginal lavage) is observed. Vaginal lavages will continue for those females 

with no evidence of copulation until termination of the 14-day mating period. 

On the day of necropsy, a vaginal lavage will also be performed to determine 

stage of estrus. An analysis of the female cycle pattern will be reported. 

7.7 Fo Breeding Procedures: 

After a minimum of 28 days of treatment, one female will be cohabited with 

one male rat of the same treatment group, avoiding sibling mating (Charles 

River Laboratories will supply non-litter mates). Detection of mating will be 

confirmed by the appearance of a vaginal copulatory plug or by evidence of 

sperm in a vaginal lavage. After confirmation of mating, the female will be 

placed in an individual suspended plastic cage containing nesting materiaL and 

the day will be designated as day 0 of gestation. If a breeding male dies or is 

removed from study, a second male (from the same dose group) that has already 

completed mating will be provided, if available. 

A maximum of 14 days will be allowed for mating. If no evidence of 

copulation is obtained after 14 days, the animals will be separated without 

further opportunity for mating, and the female will be placed in a plastic cage 

containing nesting material. 
Pa e \6 of 53 

7.8 F0 Parturition and Lactation and Ft Litters: 

WIL-417005 

June 25,20\2 

The day parturition is initiated will be designated as day 0 of lactation. Any 

difficulties at the time of parturition will be recorded. When parturition is 

judged complete, litters will be sexed, examined for gross malformations and 

the number of stillbirths and live pups will be recorded. Any changes or 

abnormalities in nesting and nursing behavior will be recorded. The dam and 

litter will remain together until weaning on lactation day 21. 

7.9 Standardization of F1 Litter Size and Identification: 

Following completion of delivery, all pups will be individually identified by 

tattoo markings applied to the digits. To reduce variability among the litters, on 

PND 4, 10 pups of equal sex distribution (if possible) from each litter will be 

randomly selected. For litters consisting of fewer than 10 pups, adjustments for 

litter size will not be performed. 

Following selection and blood/thyroid collection for PND 4 thyroid hormone 

analysis and possible histopathology (See Section 7.10), any remaining culled 

pups will be euthanized by an intraperitoneal injection of sodium pentobarbital, 

examined macroscopically, and discarded. 

7.10 Thyroid Hormone Analysis (PND 4 Culled Pups): 

On PND 4, culled pups will be anesthetized by isoflurane inhalation and as 

much blood as possible will be collected via cardiac puncture and pooled by 

litter until a total of 15 samples/dose level (minimum of 1.5 mL each) has been 

obtained. Blood may be pooled by litter to ensure a sufficient volume for 

hormone analysis. To the extent possible, samples from the first 15 litters at 

each dose level with sufficient numbers of culled pups will be used. Additional 

litters may be used if there is an insufficient number of pups to obtain the 

desired blood volume. The following parameters will be evaluated: 

Thyroxine (T4; Cobas e411) 

Thyroid stimulating hormone (TSH; Radioimmunoassay) 

Blood will be collected into tubes without anticoagulant and immediately placed 

on ice. Serum will be obtained as soon as possible following blood collection, 

and stored frozen (approximately -70°C) until analyzed. If the volume of serum 

samples in insufficient, additional samples may be collected and documented in 

the study records. 

While under deep anesthesia, culled pups will be examined macroscopically, 

blood will be collected (as described above), and the thyroid glands will be 

harvested for thyroid hormone analyses. The trachea, with the thyroid gland 

attached, will be removed, placed into cassettes labeled to match the pooled 
Page 17 of 53 

WIL-417005 

June 25,2012 

blood samples, and placed in 10% neutral-buffered formalin for possible future 

histopathological examination. Following thyroid gland removal, the animals 

will be euthanized by decapitation. If no histopathologic examination 1s 

required, these tissues will be discarded after acceptance of the fmal report. 

7.11 Assignment ofF1 Animals to Cohorts: 

Prior to PND 21, 3 F 1 pups/sex/litter from all available litters (2:20 litters/group) 

will be selected to remain on study for further examinations. Pups will be 

selected randomly; only pups not expected to survive due to notable physical 

limitations and/or obvious runts (animals with body weight more than 

2 standard deviations lower than the respective mean litter weight) will not be 

available for selection. A detailed evaluation of each pup excluded from 

selection will be recorded. 

The selected pups will be randomly assigned to one of three cohorts. Cohorts 

1A and 1B (containing a minimum of 20 pups/sex/group/Cohort) will be 

assigned to reproductive/developmental toxicity testing, and Cohort 2 (2A and 

2B; containing a minimum of 10 pups/sex/group/Cohort and representing a 

minimum of20 litters) will be assigned to developmental neurotoxicity testing. 

The cohorts will be subdivided as follows: 

Cohort Number of Animals Primary Assessments 

lA l /sexilitter/group Primary reproductive/developmental 

(>20/sexigroup) toxicity testing 

lB l /sex/litter/group Follow-up reproductive assessment, if 

(>20/sex/group) required 

2A l pup/litter (2: l 0/sexigroup, Neurobehavioral testing 

representing 2':20 litters) 

2B l pup/litter (2':1 0/sexigroup, PND 22 Neuropathology 

representing 2':20 litters) 

Assignment of same-sex littem1ates to a particular Cohort will be avoided 

whenever possible. If there are insufficient numbers of pups to fill all of the 

designated pup assignments, the following prioritization plan will be used 

(highest to lowest priority): 

Cohort lA, Cohort 1B, Cohort 2A, Cohort 2B 

Animals assigned to Cohort lB may be maintained on study with exposure to 

the test substance continuing beyond PND 90. If necessary, these animals may 

be bred to produce an F2 generation (See Section 7.16). 
Page IK of 53 

7.12 Examination ofNonselected Pups: 

7.12.1 Macroscopic Examination 

WIL-417005 

June 25,2012 

Nonselected Ft pups will be weighed and anesthetized via isoflurane 

inhalation on PND 21. Blood will be collected from a subset of 

weanlings as described in Section 7.12.2, and all animals will be 

euthanized via decapitation and necropsied as described in Section 8.1 

with an emphasis on evaluation of developmental morphology and 

organs of the reproductive system. The animals will not be fasted prior 

to euthanasia. All gross lesions will be collected and placed in 

10% neutral-buffered formalin. 

7.12.2 Blood Collection and Thyroid Hormone Analysis: 

Blood will be collected via cardiac puncture from a subset of 

nonselected Ft pups (15/sex/group) for thyroid hormone analysis. The 

site of collection, including right or left, will be documented for each 

animal. The following parameters will be evaluated: 

Thyroid stimulating hormone (TSH; RIA) 

Thyroxine (T4; Cobas e411) 

Blood will be collected into tubes without anticoagulant and 

immediately placed on ice. Serum will be obtained as soon as possible 

following blood collection, and stored frozen (approximately -70°C) 

until analyzed. If the volume of serum samples in insufficient, 

additional samples may be collected and documented in the study 

records. Following blood collection, all animals will be euthanized by 

decapitation. 

7.12.3 Organ Weights: 

The following organs will be weighed and placed in 10% neutralbuffered formalin for possible future histopathologic examination. The 

brains, spleens, and thymuses of 10 randomly selected pups/sex/group 

(representing 20 litters/group, if possible) will be weighed, while the 

thyroids will be obtained from the same 15 pups/sex/group used for 

blood collection. Organ-to-fmal-body-weight ratios will be calculated. 

Brain 

Spleen 

Thymus 

Thyroids+ 

+ From the same 15 pups/sex/group used for blood collection. Tissue will be weighed 

after fixation in 10% neutral-buffered formalin. 
Page 19 of 53 

WIL-417005 

June 25, 2012 

Mammary tissue and any gross lesions will also be collected from all 

pups and placed in 10% neutral-buffered formalin for possible future 

histopathologic examination. 

7.13 Euthanasia of F0 Generation: 

Following completion of weaning of the F1 offspring, all Fo rats will be fasted 

overnight, and urine will be collected from the males as described in 

Section 7.18. On the following day, all animals will receive a detailed physical 

examination; all females will receive a vaginal lavage to determine the stage of 

estrus. The Fo animals will then be euthanized by carbon dioxide inhalation, 

blood will be collected as described in Section 7 .18, the animals will be 

subjected to a gross necropsy, and tissues will be preserved as described in 

Section 8.1. Detailed evaluation of the process of spermatogenesis will be 

performed on all Fo males as described in Section 7.20. 

Any F0 rats not expected to survive to the next observation period (moribund) 

will be euthanized by carbon dioxide inhalation, subjected to a gross necropsy 

and tissues saved as described in Section 8. For females found dead or 

euthanized in extremis during gestation, the number of corpora lutea and 

implantation sites will be recorded. Viable fetuses will be euthanized by a 

subcutaneous injection of sodium pentobarbital in the scapular region. 

Recognizable fetuses will be examined externally and discarded. For females 

found dead or euthanized in extremis during lactation, all pups will be examined 

externally and examined according to Section 7 .14. 7. 

7.14 General Observations During The Experimental Period: 

7.14.1 Parental Appearance and Behavior: 

The parental rats will be observed twice daily for moribundity, 

mortality, and the availability of feed and water at least once in the 

morning and once in the afternoon from the day of animal receipt until 

euthanasia. This twice-daily observation will include cage-side 

examinations, while the animals are in their cages, to detect any 

significant clinical abnormalities that would be visible upon a limited 

examination, and to monitor the general health of the animals; only the 

presence of adverse clinical signs will be recorded. 

A detailed physical examination will be conducted weekly, beginning 

one week prior to the initiation of test substance administration, on all 

parental animals. Detailed physical examinations will also be conducted 

on gestation days 0, 7, 14, and 20 for all mated (sperm- or plug-positive) 

females and on lactation days 0 (if possible), 1, 4, 7, 14 and 21. 

Mortality and all signs of overt toxicity will be recorded on the day 
Page ~o of 53 

WIL-417005 

June 25,2012 

observed. The observations shall include, but are not limited to, 

evaluation for changes in appearance of skin and fur, eyes, mucous 

membranes, respiratory, circulatory, autonomic and central nervous 

system function, somatomotor activity and behavior patterns. Special 

attention will be paid to the degree of salivation and lacrimation, 

presence or absence ofurination and defecation (including polyuria and 

diarrhea), pupil size, degree of palpebral closure, presence of 

convulsions, tremors or abnormal movements, presence of posture and 

gait abnormalities, the presence of any unusual or abnormal behaviors 

and any repetitive actions (stereotypies). During the period of expected 

parturition, the dams will be observed twice daily for dystocia, 

prolonged labor, delayed labor or other difficulties at parturition 

(beginning on or around gestation day 20). Any abnormalities in 

nursing behavior will be recorded. All animals will also be observed on 

the day of necropsy and any findings will be recorded. 

7.14.2 Detailed Clinical Observations: 

All Fo animals will receive a Detailed Clinical Observation prior to the 

initiation of test substance administration, and weekly during the 

exposure period. The Detailed Clinical Observations utilized at WIL 

Research, are based on previously developed protocols (Moser et a!., 

1991; Irwin, 1968; Gad, 1982; Moser et a!., 1988; Haggerty, 1989; 

O'Donoghue, 1989). Testing will be performed at approximately the 

same time each day by the same biologists, whenever possible, who will 

not know the animals' group assignments. Animals will be observed for 

the following: 

Ease of removal from cage 

Lacrimationlchromodacryorrhea 

Piloerection 

Palpebral closure 

Red/Crusty deposits 

Eye prominence 

Mobility 

Convulsions/tremors 

Grooming 

Bizarre/Stereotypic behavior 

Pupillary response 

General body posture 

Ease of handling animal in hand 

Salivation 

Fur appearance 

Respiratory rate/character 

Mucous membranes/eye/skin color 

Muscle tone 

Gait 

Arousal 

Urination/ defecation 

Backing 
Pa e 21 of 53 

7.14.3 Parental Body Weights: 

WIL-417005 

June 25,2012 

All animals will have a final body weight recorded on the day on 

euthanasia. 

7.14.3.1 Males: 

Recorded individually on a weekly basis, beginning 1 week prior 

to the initiation of test substance administration, until euthanasia. 

7.14.3.2 Females: 

Recorded individually on a weekly basis, beginning 1 week prior 

to the initiation of test substance administration, until evidence of 

copulation is observed, and on gestation days 0, 7, 14, and 20 and 

lactation days 1, 4, 7, 14, and 21. 

Females with no evidence of mating or that fail to deliver will 

continue to be weighed on a weekly basis until the scheduled 

euthanasia. 

7.14.4 Parental Food Consumption: 

Individual food consumption measurement will be suspended during 

periods of mating as the animals will be cohabited. 

7.14.4.1 Males: 

Recorded individually on a weekly basis on the days of body 

weight collection, beginning 1 week prior to the initiation of test 

substance administration, until euthanasia. 

7.14.4.2 Females: 

Recorded individually on a weekly basis on the days of body 

weight collection, beginning 1 week prior to the initiation oftest 

substance administration, until the start of the mating period and 

on gestation days 0, 7, 14, and 20 and lactation days 1, 4, 7, 14, 

and 21. 

For females with no evidence of mating or that fail to deliver, 

individual food consumption will continue to be weighed on a 

weekly basis until the scheduled euthanasia. 
Pa e 22 of 53 

7.14.5 Parental Water Consumption: 

WIL-417005 

June 25, 2012 

Individual water consumption measurement will be suspended during 

periods of mating as the animals will be cohabited. 

7.14.5.1 Males: 

Recorded (by weight) individually on a weekly basis on the days 

of body weight collection, beginning 1 week prior to the 

initiation oftest substance administration, until euthanasia. 

7.14.5.2 Females: 

Recorded (by weight) individually on a weekly basis on the days 

of body weight collection, beginning 1 week prior to the 

initiation of test substance administration, until the start of the 

mating period and on gestation days 0, 7, 14, and 20 and lactation 

days 1, 4, 7, 14, and 21. 

For females with no evidence of mating or that fail to deliver, 

individual water consumption will continue to be recorded on a 

weekly basis until scheduled euthanasia. 

7.14.6 Examination of Offspring: 

7.14.6.1 Appearance and Behavior: 

All pups will be observed daily for general appearance, behavior 

and survival during lactation. A detailed physical examination 

will be recorded for each pup on postnatal days 1, 4, 7, 14 and 21 

of lactation. Any abnormalities in nesting and nursing behavior 

will be recorded. The pups will be sexed on lactation days 0, 1, 4 

(before culling), 7, 14 and 21. 

Following weaning, cage-side examinations (as described in 

Section 7 .14.1) will continue to be conducted at least twice daily, 

and detailed physical examinations will be conducted weekly 

until euthanasia. 

7.14.6.2 Body Weights: 

Each pup will be weighed on PND 1, 4 (before culling), 7, 14, 

and 21, on a weekly basis following weaning, and on the day of 

euthanasia. 
Page 23 of 53 

7.14.6.3 Food Consumption: 

WIL-417005 

June 25, 2012 

Cage food consumption will be recorded on PND 21 and 28. 

Individual food consumption will be recorded on a weekly basis, 

beginning on PND 28 (when the animals are individually 

housed) until the day prior to euthanasia. 

7.14.6.4 Water Consumption: 

Cage water consumption (by weight) will be recorded on PND 

21 and 28. Individual water consumption (by weight) will be 

recorded on a weekly basis, beginning on PND 28 (when the 

animals are individually housed) until the day prior to euthanasia. 

7.14.6.5 Preweaning Developmental Landmarks: 

7.14.6.5.1 Anogenital Distance: 

The anogenital distance of all F 1 pups will be 

measured on PND 1 (Gallavan et al., 1999). 

Absolute and relative (to the cube root of body 

weight) will be reported. 

7.14.6.5.2 Assessment of Areolae/Nipple Anlagen: 

On PND 12, all F1 offspring (male and female) will 

be evaluated for the presence of nipples/areolae in 

accordance with the methods described by Grey 

eta!. (1990). A finding of 'yes' and the number of 

nipples will be recorded if nipples are present, and a 

finding of 'no' will be recorded if nipples are 

absent. Testing will be performed by biologists 

who will not know the animals' group assignments. 

If there is a statistically or biologically significant, 

treatment-related increase in retained 

nipples/areolae in males on PND 12, males assigned 

to Cohort 1A will also be examined at necropsy for 

nipple/areolae retention (upon written authorization 

by the Study Director). This examination will be 

similar to the PND 12 examination, with the 

additional requirement that the adult males will be 

shaved on the ventral surface to allow the accurate 

assessment of nipple/areola number. 
Page 24 of 53 

WIL-417005 

June 25,2012 

7.14.6.6 Pup Detailed Clinical Observations: 

Beginning on PND 21 (after weaning), all offspring assigned to 

Cohort 2A will receive a weekly Detailed Clinical Observation 

(DCO). The DCOs will be conducted at approximately the same 

time each examination day. The DCO list utilized at WIL 

Research is based on previously developed protocols (Moser 

eta!., 1991; Irwin, 1968; Gad, 1982; Moser et al., 1988; 

Haggerty, 1989; O'Donoghue, 1989). Testing will be performed 

by the same biologists, whenever possible, who will not know 

the animals' group assignments. Animals will be observed for 

endpoints listed below. 

Arousal 

Backing 

B izarre/Stereotypic behavior 

Convulsions/tremors 

Ease of handling animal in 

hand 

Ease of removal from cage 

Eye color 

Eye prominence 

Forelimb grip strength 

Fur appearance 

Gait 

General body posture 

Grooming 

Hind limb grip strength 

Lacrimation/ 

chromodacryorrhea 

Mobility 

Mucous membranes and 

skin color 

Muscle tone 

Palpebral closure 

Red/Crusty deposits 

Piloerection 

Pupillary response 

Respiratory rate/character 

Salivation 

Urination/ defecation 

7.14.6.7 Postweaning Developmental Landmarks: 

All offspring will be evaluated for attainment of the following 

landmarks of sexual maturity: 

7.14.6.7.1 Balanopreputial Separation: 

Each male pup will be observed for balanopreputial 

separation beginning on PND 35 (Korenbrot et a/, 

1977). Examination of the males will continue 

daily until balanopreputial separation is present. 

Any abnonnalities of genital organs (e.g., persistent 

preputial threads) will be noted. The body weight 

of each male will be recorded on the day of 

attainment ofbalanopreputial separation. 
Pa e 25 of 53 

WIL-417005 

June 25, 2012 

7.14.6.7.2 Vaginal Patency: 

Each female pup will be observed for vaginal 

patency on PND 25 (Adams et al, 1985). 

Examination of the females will continue daily until 

vaginal patency is present. Any abnormalities of 

genital organs (e.g., persistent vaginal threads) will 

be noted. The body weight of each female will be 

recorded on the day of attainment of vaginal 

patency. 

7.14.6.8 Evaluation for First Estrus and Determination of Estrous 

Cycles (F 1 Cohort 1A): 

Beginning on the day of vaginal patency, daily vaginal lavages 

will be performed for all female offspring assigned to Cohort lA, 

and the vaginal lavages will be examined daily until the first sign 

of estrus (cornified cells) is observed. 

Daily vaginal lavages will also be performed for all female 

offspring assigned to Cohort 1A to determine the stage of estrus 

for 2 weeks from PND 75 through PND 88. On the day of 

necropsy, a vaginal lavage will also be performed to determine 

stage of estrus. 

7.14.7 Pup Deaths: 

7.14.7.1 Pups 0 to 4 Days of Age: 

Moribund pups will be euthanized by an intraperitoneal injection 

of sodium pentobarbital. Stillborn pups, pups found dead 

between birth and PND 4, and any pups that are euthanized 

in extremis will be sexed, examined for the presence of milk in 

the stomach (PND 0 or 1 only), and dissected (including the 

heart, and the brain examined by a mid coronal slice) by a 

technique described by Stuckhardt and Poppe (Stuckhardt and 

Poppe, 1984). If a skeletal anomaly is suspected, the pups will 

be eviscerated, cleared and stained with Alizarin Red S as 

described by Dawson (Dawson, 1926) and examined. The cause 

of death will be determined, if possible. Representative 

specimens with malformations may be preserved in 10% neutralbuffered formalin at the discretion of the study director. 
Pa e 26 of 53 

7.14.7.2 Pups 5 Days of Age to Weaning: 

WIL-417005 

June 25, 2012 

Moribund pups will be sexed and euthanized by an 

intraperitoneal injection of sodium pentobarbital. A gross 

necropsy will be performed on pups found dead or euthanized in 

extremis, the sex will be determined, and the carcass of all pups 

with gross lesions will be preserved in 10% neutral-buffered 

formalin for possible future histopathological examination. The 

cause of death will be determined, if possible. 

7.15 Developmental Neurotoxicity (Ft Cohort 2A): 

F1 animals assigned to Cohort 2A will be assigned to the following tests: 

auditory startle response, functional observational battery, locomotor activity, 

and neuropathological and brain weight evaluations on PND 78. 

Behavioral testing will be balanced across time of day such that all groups are 

represented at each testing session. Body weights will be recorded on the days 

of behavioral testing; these data will be maintained in the study records. 

7.15.1 Auditory Startle Response: 

Auditory startle response will be assessed on PND 24 using the 

StartleMonitor System (Kinder Scientific Company, LLC, Poway, CA) 

as described by WIL standard operating procedures. Auditory startle 

response testing will be performed in a sound-attenuated room equipped 

with a white noise generation system set to operate at 70 ±10 dB. 

The test sessions will consist of a five-minute acclimation period with a 

65 ± 5-dB broadband background white noise. The startle stimulus for 

each trial will be a 115 ± 5-dB mixed frequency noise burst stimulus 

(approximately 20 ms in duration). Responses will be recorded during 

the first 100 ms following onset ofthe startle stimulus for each trial. 

The test session will consist of 50 trials with an eight-second intertrial 

interval. Startle response data will be analyzed in five blocks of 10 trials 

each. Startle response measures obtained are maximum response 

amplitude (MAX), average response amplitude (AVE) and latency to 

MAX (TMAx). 

7.15.2 Locomotor Activity: 

Locomotor activity will be monitored on PND 65. Locomotor activity 

will be measured using the Kinder Scientific Motor Monitor System 

(Kinder Scientific Company, LLC, Poway, CA) as described by WIL 

standard operating procedures. Locomotor activity sessions will be 
Pa e 27 of 53 

WIL-417005 

June 25,2012 

performed in a room equipped with a white noise generator set to 

operate at 70 ± 10 dB. 

The test session will be of60 minutes duration and will consist of twelve 

5-minute intervals. The 12 collection intervals will be presented in six 

1 0-minute intervals for reporting purposes. Locomotor activity 

measures evaluated are total and ambulatory activity counts obtained for 

the 60-minute test session. 

7.15.3 Functional Observational Battery (FOB): 

A functional observational battery will be conducted on all animals 

assigned to Cohort 2A on PND 65, prior to the locomotor activity 

assessment. If possible, at least 10 males and 10 females (from 20 

litters) will be evaluated. The functional observational battery (FOB) 

utilized at WIL Research is based on previously developed protocols 

(Moser et al., 1991; Irwin, 1968; Gad, 1982; Moser et al., 1988; 

Haggerty, 1989; O'Donoghue, 1989). Testing will be performed in a 

sound-attenuated room equipped with a white noise generation system 

set to operate at 70 ±10 db. Testing will be performed by the same 

technicians, whenever possible, who will not know the animal's group 

assignment. Animals will be observed for the following as described 

below (see WIL SOP No. T1-112, as revised, for a description of 

scoring criteria): 

7.15.3.1 Home Cage Observations: 

Posture 

Convulsions/tremors 

Feces consistency 

7.15.3.2 Handling Observations: 

Ease of removal from cage 

Lacrimation/ 

chro modacryorrhea 

Piloerection 

Palpebral closure 

Red/crusty deposits 

Eye prominence 

Ease of handling animal in hand 

Biting 

Palpebral (eyelid) closure 

Salivation 

Fur appearance 

Respiratory rate/character 

Mucous membranes/eye/skin 

color 

Muscle tone 
Page 2S of 53 

7.15.3.3 Open Field Observations: 

WIL-417005 

June 25, 2012 

Evaluated over a 2-minute observation period 

Mobility 

Rearing 

Convulsions/tremors 

Grooming 

Bizarre/stereotypic behavior 

Time to first step (seconds) 

7.15.3.4 Sensory Observations: 

Approach response 

Startle response 

Pupil response 

Forelimb extension 

Air righting reflex 

7 .15.3.5 Neuromuscular Observations: 

Hindlimb extensor strength 

Hindlimb foot splay 

7.15.3.6 Physiological Observations: 

Catalepsy 

Body temperature 

7.16 Production of a Second Generation: 

Gait 

Arousal 

Urination/ defecation 

Gait score 

Backing 

Touch response 

Tail pinch response 

Eyeblink response 

Hindlimb extension 

Olfactory orientation 

Grip strength - hind and 

forelimb 

Rotarod performance 

Body weight 

If warranted, the F1 animals may be mated to produce a second generation (with 

Sponsor approval, at additional cost). Reproductive and systemic toxicity data 

from the Fo generation will be used to assess whether a second generation will 

be needed. Males and females of the same dose group would be cohabited on or 

after PND 90, but not later than PND 120. Subsequent procedures will be 

similar to those outlined for the F0 generation/F1 litters unless otherwise 

specified in a protocol amendment. For example, based on a weight of 

evidence, it may be sufficient to terminate the F2 litters on PND 4. 
Page 29 of 53 

7.17 Euthanasia ofF 1 Reproductive/Developmental Toxicity Animals 

(Cohorts 1A and 1B): 

WJL-417005 

June 25, 2012 

On PND 91, all F1 males and females assigned to Cohort lA will be fasted 

overnight prior to euthanasia, and urine will be collected as described in Section 

7.18. On the following day, all F1 Cohort lA animals will be weighed and 

receive a detailed physical examination. If a second mating is not required, F1 

male and female rats assigned to Cohort lB will be weighed and receive a 

detailed physical examination on PND 120. All F, females in Cohorts lA and 

1B will receive a vaginal lavage to determine the stage of estrus prior to being 

transported to the Necropsy Department. The rats will be euthanized by carbon 

dioxide inhalation, and blood will be collected from Cohort 1A animals as 

described in Section 7.18. All Cohort 1 rats will be subjected to a gross 

necropsy, and tissues will be preserved as described in Section 8.1. Detailed 

evaluation of the process of spermatogenesis (Cohort lA only) will be 

performed as described in Section 7.20. 

Any F1 rats not expected to survive to the next observation period (moribund) 

will be euthanized by carbon dioxide inhalation and subjected to a gross 

necropsy and tissues saved as described in Section 8. 

7.18 Clinical Pathology Evaluations (F0 and F1 Cohort 1A): 

Urine will be collected from the first 10 F0 males that are available on study day 

91 and from the first 10 F1 Cohort 1A animals/sex/group that are available on 

PND 91. Urine will be collected overnight using metabolism cages. Water will 

be available during this procedure (animals will be fasted during the collection 

period). Urine will not be collected from F0 females. 

Prior to the scheduled necropsy (LD 21 for the Fo females, study day 91 for the 

Fo males, and PND 90 for the Cohort lA males and females), all Fo animals and 

F1 animals assigned to Cohort 1A will be fasted overnight. Blood samples will 

be collected from the first 10 Fo and F1 Cohort lA animals/sex/group sent to the 

Necropsy Department. Blood samples for hematology and serum chemistry 

will be collected from the retro-orbital sinus from animals anesthetized with 

isoflurane. Blood samples for coagulation parameters will be collected by 

necropsy personnel from the inferior vena cava at the time of euthanasia from 

animals euthanized via C02 inhalation. In addition, blood smears will be 

prepared, stained with Wright-Giemsa stain, cover-slipped, and retained for 

possible future evaluation. If needed, additional samples may be collected to 

ensure the target sample size (n=lO/sex/group) is achieved. Any additional 

samples will be documented in the study file. Blood will not be obtained from 

animals that die or are euthanized in extremis prior to their scheduled necropsy. 
Page 30 of 53 

WTL-417005 

June 25,2012 

Anticoagulants used will be potassium EDT A for the hematology samples and 

sodium citrate for the coagulation test. Samples for serum chemistry will be 

collected without anticoagulants. 

Any requirement for additional testing of serum or plasma samples must be 

determined prior to 90 days after the specimen has been tested. Consequently, 

any remaining samples may be discarded 90 days after testing is completed, 

unless directed otherwise by protocol. 

7.18.1 Urinalysis: 

Bilirubin 

Color and clarity 

Glucose 

Ketones 

Leukocytes 

Microscopy of sediment 

7 .18.2 Hematology: 

Differential leukocyte counta 

Erythrocyte count 

Total hemoglobin 

Hematocrit 

Mean corpuscular hemoglobin 

Mean corpuscular volume 

Occult blood 

pH 

Protein 

Speci fie gravity 

Urobilinogen 

Volume 

Mean corpuscular hemoglobin 

concentration 

Platelet count 

Reticulocyte count 

Total leukocyte count 

a- If performed manually, results will include platelet estimates and RBC morphology. 

7.18.3Serum Chemistry: 

Alanine aminotransferase 

Albumin 

Aspartate aminotransferase 

Alkaline phosphatase 

Albumin/globulin ratio 

(calculated) 

Calcium 

Chloride 

Creatinine 

Gamma glutamyltransferase 

Globulin (calculated) 

Glucose 

Phosphorus 

Potassium 

Sodium 

Total bile acids 

Total bilirubin 

Total cholesterol 

Total protein 

Triglycerides 

Urea nitrogen 

Appearanceb 

b -will include degree of hemolysis, lipemia, and icterus (individual tables only) 
Page 31 of 53 

7.18.4Coagulation Parameters: 

Prothrombin time 

WIL-417005 

June 25, 2012 

7.19 Thvroid Hormone Analysis (F0 Males and F1 Cohort lA Males and 

Females): 

Blood (approximately 1.5 mL) will be collected via the jugular vein from F0 

males (15/group) on study day 85 and F1 males and females assigned to Cohort 

1 A ( 15/sex/group) on PND 85 under isoflurane anesthesia to avoid the effects of 

fasting on thyroid hormone levels. The site of collection, including right or left, 

will be documented for each. The following parameters will be evaluated: 

Thyroid stimulating hormone (TSH; RIA) 

Thyroxine (T4; Cobas e411) 

Blood will be collected into tubes without anticoagulant and immediately placed 

on ice. Serum will be obtained as soon as possible following blood collection, 

separated into two tubes containing approximately equal aliquots, and stored 

frozen (approximately -70°C) until analyzed. If the volume of serum samples is 

insufficient, additional samples may be collected and documented in the study 

records. 

Blood for thyroid hormone analysis will not be collected from Fo females as 

these animals are lactating and must provide care to the F 1 offspring until 

overnight fasting and termination on LD/PND 22. 

7.20 Spermatogenesis Evaluation (F0 Males and F1 Cohort lA Males): 

The following quantitative assessment ofthe process of spermatogenesis will be 

performed on all F0 males and all F 1 males assigned to Cohort IA that survive 

to the scheduled euthanasia. 

If the left testis or epididymis is noted with a gross lesion, these organs will not 

be homogenized. Instead, the right testis and epididymis will be used for 

enumeration of epididymal and testicular sperm and sperm production rate and 

the left testis and epididymis will be used for motility/viability and morphology 

assessments as well as for preservation in modified Davidson's solution for 

subsequent microscopic examination. 

7.20.1 MotilityNiability Assessment: 

Immediately after euthanasia, the reproductive tract of the male will be 

exposed via a ventral mid-line incision. The right testis and epididymis 

will be excised and weighed separately. An incision will be made in the 

distal region of the right cauda epididymis, and the cauda will be placed 
Page 32 of 53 

WIL-417005 

June 25, 2012 

in a beaker containing Dulbecco's phosphate-buffered saline (maintained 

at approximately 37°C) with 10 mg/ml Bovine Serum Albumin (BSA). 

A small sample will be loaded into a 100-f.lm chamber for determination 

of motility. Because sperm motility can be affected by temperature 

shock, all slides and diluents will be warmed prior to use in an 

incubator, and motility determinations will be performed under constant 

temperature (approximately 3 7°C) using the Hamilton-Thome 

HTM-IVOS Version 12.3 computer-assisted sperm analysis (CASA) 

system. Analysis of at least 200 motile and nonmotile spermatozoa per 

animal, if possible, from all dose groups will be performed by the 

analyzer. The motility score will be reported as the total percentage of 

motile sperm (showing any motion) and the total percentage of 

progressively motile sperm (showing net forward motion). The right 

testis and epididymis will then be preserved in accordance with 

Section 8. 

7.20.2 Morphology Assessment: 

For all F0 males in the control and high dose groups and all F1 males 

assigned to Cohort lA in the control and high dose groups, sperm 

morphology will be evaluated by a modification of the wet-mount 

evaluation technique (Linder et al, 1992) by trained biologists who do 

not know the animals' group assignments. Abnormal forms of sperm 

(double heads, double tails, microcephalic or megacephalic, etc.) from a 

differential count of 200 spermatozoa (if possible) per animal in all 

groups will be recorded. If a treatment-related effect is observed, males 

from the lower dose groups will be evaluated (with Sponsor approvaL at 

additional cost). 

7.20.3 Enumeration of Epididymal and Testicular Sperm and Sperm 

Production Rate (SPR): 

For all F0 males in the control and high dose groups and all F1 males 

assigned to Cohort lAin the control and high dose groups, the left testis 

and epididymis will be weighed, homogenized and evaluated for sperm 

numbers and the sperm production rate will be calculated (Blazak eta!, 

1985) utilizing the Hamilton-Thome CASA system. The homogenized 

sample will be mixed with a DNA-specific fluorescent dye to allow 

identification of the sperm under fluorescent illumination. If a 

treatment-related effect is observed, males from the lower dose groups 

will be evaluated (with Sponsor approval, at additional cost). 
Page 33 of 53 

8 ANATOMIC PATHOLOGY: 

WIL-417005 

June 25, 2012 

8.1 Macroscopic Examination (Fo~1 Cohorts lA and lB, and Unscheduled 

Deaths): 

A complete necropsy will be conducted on all Fo and F 1 post-weaning animals 

dying spontaneously, euthanized in extremis (including animals with dystocia), 

or at the scheduled euthanasia for F0 animals and F 1 animals assigned to 

Cohorts lA and 1 B, This will include examination of the external surface, all 

orifices, the cranial cavity, the external surface of the brain and spinal cord, and 

the thoracic, abdominal, and pelvic cavities, including viscera. Special attention 

will be paid to the organs ofthe reproductive system. In addition, the eyes will 

be examined in situ by application of a moistened microscope slide to each 

cornea. For F0 females that were mated, the number of former implantation 

sites and corpora lutea will be recorded. 
Pa e 34 of 53 

WIL-417005 

June 25,2012 

At the time of necropsy, the following tissues and organs will be collected and 

placed in 10% neutral-buffered formalin (except as noted): 

Adrenals (2) 

Aorta (1) 

Bone with marrow 

(sternebrae) 

Brain (cerebrum, brainstem, 

cerebellum) (3) 

Coagulating glands (2t 

Eyes with optic nerve (2)b 

Gastrointestinal tract 

Esophagus 

Stomach 

Duodenum 

Jejunum 

Ileum 

Cecum 

Colon 

Rectum 

Heart 

Kidneys (2) 

Lacrimal/Harderian 

glands (2) 

Larynx (1) 

Liver (sections oftwo 

lobes)(3) 

Lungs (including bronchi, 

fixed by inflation with 

fixative) (5) 

Lymph nodes 

Mediastinal ( 1) 

Mesenteric ( 1) 

Mediastinal tissues (1) 

Mesenteric tissues ( 1) 

Nasal tissues/pharynx (2) 

Oral tissues (2) 

Ovariesc,d and oviduct (2t 

Pancreas (1 t 

Peripheral nerve (tibial) 

Pituitary (l)a 

Prostate (1 t 

Mandibular salivary 

glands (1) 

Seminal vesicles (2t 

Skeletal muscle (rectus 

femoris)( 1) 

Skin with mammary 

gland and subcutis( 1 t 

Spinal cord (cervical, 

thoracic, lumbar) (3) 

Spleen (1) 

Testes with epididymides 

and vas deferens (1 t.f,g 

Tongue (0) 

Thymus 

Thyroids with Tracheah 

[with parathyroids if 

present ( 1) t 

Urinary bladder 

Uterus with cervix and 

vagina (4t 

All gross lesions 

(all groupst 

The munber in parentheses is the minimum number of sections to be examined if the tissue is chosen for 

histopathologic examination. All tissues will be collected from animals that are found dead or 

euthanized in extremis. 

a- May also be examined for F1 Cohort 18 animals if Cohort lA results are equivocal or if reproductive or 

endocrine toxicity is suspected. 

b- Eyes with optic nerves will be fixed in Davidson's solution. 

c- Ovaries will be fixed in 10% neutral-buffered formalin for approximately 48 hours following which all 

ovaries will be transferred to 70% Ethanol. 

d - An additional section will be taken from Fo females for qualitative evaluation; additional sections (See 

Section 8.3) will be taken from F1 Cohort lA females for quantitative evaluation of primordial follicles. 

e - For females; a corresponding section of skin will be taken from the same anatomic area for males. 

f- Testis and epididymis (right only) will be fixed in modified Davidson's solution. Both testes and 

epididymides from males found dead or euthanized in extremis will be fixed in modified Davidson's 

solution. Care will be taken to ensure separation between the left and right organs. 

g- If the left testis or epididymis is noted with a gross lesion, the left testis and epididymis will be fixed in 

modified Davidson's solution and the right testis and epididymis will be homogenized according to 

section 7.20.3. 

h - Thyroids and parathyroids (if present) will be removed from the trachea following fixation to ensure 

tissue structure is maintained during prosection. 
Page 35 of 53 

8.2 Organ Weights: 

WJL-417005 

June 25,2012 

The following organs will be weighed from Fo animals and F1 animals assigned 

to Cohorts lA and lB at the scheduled termination as noted below. Organ-tofinal-body-weight ratios will be calculated. 

Adrenalsa 

Brain a 

Epididymides* 

(total and cauda)b 

Heart a 

Kidneysa 

Livera 

Ovariesb 

Pituitary+b 

Prostate"b 

Seminal vesicles with coagulating glands 

(with accessory fluids)b 

Spleena 

Testes*b 

Thyroids with parathyroidsa+ 

Thymusa 

Uterus (with oviducts and cervix)b 

a- Collected from F0 and F1 Cohort IA animals only 

b - Collected from F0 and F 1 Cohort 1 A and 1 B animals 

* - These paired organs will be weighed separately. 

+-Tissue will be weighed after fixation in 10% neutral-buffered formalin 

" - If there is a treatment-related effect on prostate weights, the dorsolateral and 

ventral segments will be dissected (after fixation) and weighed separately. 

8.3 Microscopic Examination (F0 and Ft Cohorts lA and lB): 

Microscopic examination will be performed on the tissues listed in Section 8.1 

from Fo animals and F1 animals assigned to Cohort lAin the control and highdose groups. These tissues will also be examined from all parental animals 

dying spontaneously or euthanized in extremis. For F1 animals assigned to 

Cohort lB, these indicated tissues will be processed to the block stage (in 

paraffin) and will be examined (upon Sponsor approvaL at additional cost) if 

results from Cohort lA are equivalent or in cases of suspected reproductive or 

endocrine toxicity. 

Tissues and organs fixed in 10% neutral-buffered formalin, or other fixatives (as 

noted in Section 8.1) will be embedded in paraffin, sectioned, and stained with 

hematoxylin and eosin (H&E) for microscopic evaluation. Processing of the 

testes, epididymides, and ovaries will be performed as noted below. 

Sections of 2-4 microns of the testis (transverse) and epididymis (longitudinal) 

will be stained with Periodic Acid-Schiffs stain (PAS) and hematoxylin in 

addition to routine hematoxylin and eosin (H&E) staining. Testes from males 

that are found dead or euthanized in extremis will be stained with H&E only. 

The following regions of the epididymis will be paraffin embedded: caput, 

corpus, and cauda; the vas deferens will be examined when possible. 

Histopathological examination of the testis will include a qualitative assessment 

of the stages of spermatogenesis. For males that survive to the scheduled 
Page 36 of 53 

WIL-417005 

June 25, 2012 

euthanasia, microscopic evaluation will include a qualitative assessment of the 

relationships between spermatogonia, spermatocytes, spermatids, and 

spennatozoa seen in cross-sections of the seminiferous tubules. The 

progression of these cellular associations defines the cycle of spermatogenesis. 

In addition, sections of both testes will be examined for the presence of 

degenerative changes (e.g., vacuolation of the germinal epithelium, a 

preponderance of Sertoli cells, sperm stasis, inflammatory changes, 

mineralization, and fibrosis). When possible, sections of the rete testis will be 

examined in the F1 Cohort lA males. 

Five (5) sections will be taken approximately 100 J..lm apart from the inner third 

of each ovary from 10 F1 Cohort IA females in the control and high-dose 

groups at scheduled termination and for any F0 females suspected of reduced 

fertility. In addition, a single section will be taken from l 0 F0 and 10 F 1 females 

per dose level for a qualitative bilateral evaluation of each ovary. For all 

F 1 females in the control and high-dose groups at scheduled termination, and for 

any females suspected of reduced fertility, a quantitative histopathologic 

evaluation of multiple sections will be conducted. This examination will 

include enumeration ofthe total number ofprimordial follicles according to the 

methods of Bolon eta/. (1997), Bucci eta/. (1997) and Picut eta/. (2008). 

For the F1 Cohort 1A females, a tiered approach will be used for the evaluation 

of ovarian follicle counts. A quantitative evaluation of primordial and growing 

follicles in at least 1% of the ovary will be performed as a screening measure 

for potential adverse effects. If the results are within 10% of controls, no 

further action will be required. If results are less than 90% of controls, a 

quantitative evaluation of primordial and growing follicles in at least 5% of the 

ovary will be performed as a definitive measure for potential adverse effects. If 

the results are equivocal, a quantitative evaluation of primordial and growing 

follicles in at least 5% of the ovary may be conducted on F1 females assigned to 

Cohort lB. 

If treatment-related effects are seen, qualitative (Fo) or quantitative (F 1; 

following mating to produce a second generation [see Section 7 .16]) ovarian 

assessments will be performed on animals from the low- and mid-dose groups. 

Uterine and ovarian histopathology will be considered in light of the terminal 

estrous stage. 

For females found dead or euthanized in extremis, a single section from each 

ovary will be qualitatively evaluated. 

If a target organ is identified in the high-dose group, that organ will be 

examined from all animals in the low and mid-dose groups (after the approval 

of the Sponsor and at additional cost). In addition, reproductive organs of all 

animals suspected of reduced fertility, e.g., those that failed to mate, conceive, 
Page 37 of 53 

WIL-417005 

June 25,2012 

sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, 

motility or morphology were affected, will be subjected to histopathological 

evaluation. 

Ovarian tissues will be shipped to the attention of the Principal Investigator 

(Ovarian Histology) at the address provided in Section 2. This is a WILdesignated test site. Histology slide preparation will be conducted following the 

Standard Operating Procedures of WIL Research at Hillsborough, and in 

accordance with the Protocol and applicable GLPs. The Principal Investigator 

(Ovarian Histology) will be responsible for all delegated-phase activities. Any 

deviations from the GLPs or Protocol and any study specific SOP deviations 

will be reported to the Study Director. 

8.4 F1 Offspring Scheduled for Neuropathology/Brain Weight Measurements 

(Cohorts 2A and 2B): 

8.4.1 PND 21 Neuropathology and Brain Weights (Cohort 2B) 

8.4.1.1 Neuropathology- Macroscopic Examination: 

On PND 21, F1 animals assigned to Cohort 2B (2:10/sex/group) 

will be macroscopically examined for neuropathology. The pups 

will be deeply anesthetized by intraperitoneal injection of sodium 

pentobarbital and perfused in situ as described in Section 8.4.2.1. 

The brains will be removed (including olfactory bulbs), weighed 

and the size (length [excluding olfactory bulbs] and width) will 

be recorded. Any abnormal coloration or lesions of the brain and 

spinal cord will be recorded. The brain, head, and any relevant 

gross lesions from all animals will be placed in 10% neutralbuffered formalin for possible future histopathological 

examination. 

8.4.1.2 Neuropathology- Microscopic Examination: 

F, pups assigned to Cohort 2B will be prepared for microscopic 

neuropathologic examination as described in Section 8.4.2.2. 

Brains for all groups will be embedded in paraffin; however, only 

pups in the control and high-dose groups will be initially 

evaluated. The brains will be prepared for a qualitative 

histopathological examination by embedding in paraffin, 

sectioning, and staining with hematoxylin and eosin. Sections 

from all major brain regions (olfactory bulbs, cerebral cortex, 

hippocampus, basal ganglia, thalamus, hypothalamus, midbrain, 

brainstem and cerebellum) will be examined. In addition, 
Pa e38of53 

WIL-417005 

June 25, 2012 

sections will be prepared from the trigeminal ganglion and nerve, 

and the pituitary gland. 

Based on the results of the qualitative examination and the 

professional judgment of the pathologist, selected sites and 

cellular components may be further evaluated by the use of 

additional methods (at additional cost). If no neuropathological 

alterations are observed in samples from the high-dose group, 

subsequent analysis will not be performed. If a lesion is 

observed in the high-dose group, then animals from the low- and 

mid-dose groups will be examined (at additional cost) by the 

pathologist without knowledge of the animals' group 

assignments. 

8.4.2 PND 78 Neuropathology and Brain Weights (Cohort 2A): 

8.4.2.1 Neuropathology- Macroscopic Examination: 

F 1 offspring assigned to Cohort 2A will be fasted overnight and 

weighed on PND 78 prior to being transported to the Necropsy 

Department. The F1 Cohort 2A animals will be deeply 

anesthetized by intraperitoneal injection of sodium pentobarbital 

and perfused in situ as described in WIL Standard Operating 

Procedure T3-034, as revised. The central and peripheral 

nervous system tissues will be dissected and preserved as 

described in WIL Standard Operating Procedure T3-035, as 

revised. The whole brains shall be removed (including olfactory 

bulbs), weighed, and the size (length [excluding olfactory bulbs] 

and width) recorded. Any abnormal coloration or lesions of the 

brain or spinal cord will be recorded. Nervous system tissues 

will be retained from all animals. 

8.4.2.2 Neuropathology - Microscopic Examination: 

The offspring perfused in situ on PND 78 will be processed for 

neuropathological evaluation. Brains for all groups will be 

embedded in paraffm, however, only rats in the control and highdose groups will be initially evaluated. 
Page 39 of 53 

WIL417005 

June 25, 2012 

The following tissues will be examined microscopically: 

Brain - olfactory bulbs, cerebral cortex, hippocampus, basal 

ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, 

and medulla oblongata (including nucleus gracilis/cuneatus) 

Spinal cord - at cervical swellings C3-C7 and at lumbar swellings 

Tl3-L4 

Trigeminal ganglia/nervesa 

Lumbar dorsal root ganglia at Tl3-L4• 

Lumbar dorsal root fibers at T 13-L4 • 

Lumbar ventral root fibers at Tl3-L4• 

Cervical dorsal root ganglia at C3-C7' 

Cervical dorsal root fibers at C3-C7' 

Cervical ventral root fibers at C3-C7• 

Pituitary gland 

Caudal nerves <

2

l+ 

Sciatic nerves<2)+ 

Sural nerves<2)+ 

Tibial nerves<2)+ 

Peroneal nerves<2l+ 

Nasal tissue with olfactory epithelium 

Optic nervesa 

Eyes (retinat 

Skeletal muscle (anterior tibial and gastrocnemius) 

a 

* 

+ 

Both processed and evaluated microscopically, 

4-6 tissues will be collected at necropsy; 2 will be evaluated microscopically, 

One processed for microscopic examination, 

(2) Indicates that two sections (one cross and one longitudinal) of the tissue will be 

evaluated from the right hind leg. The tissues from the left hind leg and both 

forelimbs will be collected and preserved for possible future evaluation. 

The central nervous system tissues listed above will be embedded 

in paraffin. The peripheral nervous system tissues will be 

embedded in plastic. Tissues will be prepared for a qualitative 

histopathological evaluation by sectioning and staining with 

hematoxylin and eosin. The histopathological examination will 

include a simple morphometric analysis. 

Based on the results of the qualitative examination and the 

professional judgment of the pathologist, selected sites and 

cellular components may be further evaluated by the use of 

additional methods (at additional cost). If no neuropathological 

alterations are observed, subsequent analysis will not be 

performed. 
Pa e 40 of 53 

8.4.3 Subjective Diagnosis: 

WIL-417005 

June 25, 2012 

If any evidence of neuropathological alterations is found in the 

qualitative examination of pups euthanized on PND 22 (Cohort 2B) or 

PND 78 (Cohort 2A), then a subjective diagnosis will be performed for 

the purpose of evaluating possible dose-response relationships. All 

regions of the nervous system exhibiting any evidence of 

neuropathological changes shall be included in this evaluation. Sections 

from all dose groups from each region will be examined in random 

order, without knowledge of the group assignment. The type and 

severity of each lesion will be recorded. Each type of dose related lesion 

(if present) and normal areas for comparison will be photographed. 

9 DURATION OF STUDY: 

The two generations to be studied (parental animals and first generation offspring) 

will be termed F0 and F 1, respectively. The conduct of this study will require 

approximately six months for acclimation, mating, gestation, and lactation of the 

Fo generation, and the growth and/or behavioral testing of the Ft generation until 

euthanasia. 

10 STATISTICAL METHODS: 

All analyses will be two tailed for minimum significance levels of 5% and 1%. All 

means will be presented with standard deviations and/or standard errors ofthe mean. 

Coefficients of variation will also be presented for brain weights and morphometric 

measurements. All statistical tests will be performed by a computer with appropriate 

programming as referenced below. Following completion of the mating period, data 

from nongravid females, females with resorptions only, and/or females found to be 

gravid following staining of the uteri will be excluded from calculation of means and 

from comparative statistics. The litter, rather than the pup, will be considered as the 

experimental unit. 

10.1 In Life Data: 

Continuous data variables (mean body weights, body weight gains, and food, 

water, and test substance consumption at each interval), pre-coital intervals, 

mean estrous cycle length~ gestation length, former implantation sites, 

unaccounted-for sites, mean days of attainment of developmental landmarks 

(balanopreputial separation and vaginal patency) and the body weight on the 

day of attainment, anogenital distance (absolute and relative to the cube root of 

body weight), numbers of nipples/areola, and time from vaginal patency to first 

estrus, will be subjected to a parametric one-way analysis of variance 

(ANOV A) (Snedecor and Cochran, 1980) to determine intergroup difference. If 

the results of the ANOV A are significant (p<0.05), Dunnett's test (Dunnett, 
Page 41 of 53 

WIL-417005 

June 25,2012 

1964) will be applied to the data to compare the treated groups to the control 

group. 

Fo male and female mating, fertility, copulation, and conception indices, and 

F1 nipple retention of the treated groups will be compared to the control group 

using the Chi-square test with Yates' correction factor (Hollander and Wolfe, 

1999). 

10.2 Litter Data: 

The mean litter proportions (% per litter) of pup viability during the postnatal 

period and sex ratio at birth will be subjected to the Kruskal-Wallis 

nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine 

intergroup difference. Ifthe results of the ANOVA are significant (p<0.05), the 

Dunn's Test (Dunn, 1964) will be applied to compare the treated and control 

groups. Mean litter weights will be subjected to the parametric ANOVA test 

(Snedecor and Cochran, 1980) and Dunnett's test (1964) as described above 

with the litter representing the experimental unit. 

10.3 Histopathology, Clinical Patholoey, Thyroid Hormone, and Orean Weight 

Data: 

Histopathologic fmdings of the treated groups will be compared to those of the 

control group by the Fisher's Exact Test (Steel and Torrie, 1980). The mean 

number of ovarian primordial follicles, clinical pathology and thyroid hormone 

(T4 and TSH) values, organ weights (absolute and relative to body weights) and 

mean brain lengths and widths will be subjected to a parametric ANOV A test 

(Snedecor and Cochran, 1980) and Dunnett's test (1964) as described above. 

10.4 Spermatogenic Endpoint Evaluations: 

The percentage of motile (showing any motion) and progressively motile 

(showing net forward movement) spermatozoa and percentage of sperm with 

normal morphology will be subjected to the Kruskal-Wallis nonparametric 

ANOVA (Kruskal and Wallis, 1952) followed by the Dunn's test (1964) as 

described above. Slides containing less than 200 sperm will be excluded from 

analysis. Epididymal and testicular sperm counts and sperm production rates 

will be analyzed by a parametric ANOVA test (Snedecor and Cochran, 1980) 

and Dunnett's test ( 1964) as described above. 
Page 42 of 53 

10.5 Detailed Clinical Observation, Functional Observational Battery, 

Locomotor Activity, and Auditory Startle Data: 

WIL-417005 

June 25, 2012 

Repeated Measures Analysis will be conducted by BioSTAT Consultants, Inc. 

10.5.1 Detailed Clinical Observation and Functional Observational Battery 

Data: 

Continuous Detailed Clinical Observation (DCO) and Functional 

Observational Battery (FOB) data (including the average of 3 grip 

strength trials and 3 landing foot splay trials) will be subjected to a 

parametric one way ANOVA (Snedecor and Cochran, 1980) to 

determine intergroup differences. If significant differences are indicated 

by the ANOVA, Dunnett's test (1964) will be used to compare the 

control and treated groups. FOB and DCO parameters which yield 

scalar and descriptive data will be analyzed by the Fisher's Exact Test 

(Steel and Torrie, 1980). 

10.5.2 Locomotor Activity Data: 

Total and ambulatory activity counts will be analyzed, by sex and 

session, with a repeated measure analysis of variance (RMANOVA). 

Factors in the model will include treatment group (TR T), time interval 

(TIME), and the interaction of time interval and treatment group 

(TRT*TIME). The SAS® procedure PROC MIXED will be used for 

analysis with the random effect of animal included as the repeated 

measurement. The covariance structure across time will be selected by 

comparing Ak:aikc's Information Criterion (AIC). 

The monotonic dose response relationship will be evaluated using 

sequential linear trend tests based on ordinal spacing of dose levels. The 

linear dose by time interaction (LinTrt*Time) will be evaluated and, if 

significant at the 0.05 level, trend tests on treatment means will be 

performed at the 0.05 level for each time interval. If the linear dose by 

time interaction is not significant, the trend test will be conducted across 

the pooled time intervals of the entire session only. 

Nonmonotonic dose responses will be evaluated whenever no significant 

linear trends arc detected but TRT and/or TRT*TIME interaction is 

significant at the 0.01 level. Within the framework ofthe RMANOVA, 

pairwise comparisons will be made for each individual treated group 

with the control group through linear contrasts. If TRT*TIME is 

significant, the comparisons will be conducted for each time interval. If 

only the TRT effect is significant, the comparisons will be conducted 

across the pooled time intervals of the entire session. These 
Page 43 of 53 

WJL-417005 

June 25,2012 

nonmonotonic dose response compansons will be conducted at the 

0.01 significance level. 

All statistical analyses will be conducted using SAS version 9.2 

(SAS Institute, Inc., 2002-2008), or higher, software. 

10.5.3 Auditory Startle Response Data: 

MAX and Tmax will be analyzed, by sex and session with a RMANOV A. 

Factors in the model will include TRT, trial block (TRIAL), and the 

interaction of trial block and treatment group (TRT*TRIAL). The SAS® 

procedure PROC MIXED will be used for analysis with the random 

effect of animal included as the repeated measurement. The covariance 

structure across time will be selected by comparing AIC. 

The monotonic dose response relationship will be evaluated using 

sequential linear trend tests based on ordinal spacing of dose levels. The 

linear dose by trial interaction (LinTrt*Trial) will be evaluated and, if 

significant at the 0.05 level, trend tests on treatment means will be 

performed at the 0.05 level for each block of trials. If the linear dose by 

trial interaction is not significant, the trend test will be conducted across 

the pooled trials of the entire session only. 

Nonmonotonic dose responses will be evaluated whenever no significant 

linear trends are detected but TRT and/or TRT*TRIAL interaction is 

significant at the 0.01 level. Within the framework ofthe RMANOVA, 

pairwise comparisons will be made for each individual treated group 

with the control group through linear contrasts. If TRT*TRIAL is 

significant, the comparisons will be conducted for each block of trials. 

If only the TRT effect is significant, the comparisons will be conducted 

across the pooled trials ofthe entire session. These nonmonotonic dose 

response comparisons will be conducted at the 0.01 significance level. 

All statistical analyses will be conducted using SAS version 9.1 

(SAS Institute, Inc., 2002-2003), or higher, software. 

Average response will be subjected to a parametric one-way ANOV A 

(Snedecor and Cochran, 1980) to determine intergroup difference. If 

significant differences are indicated by the ANOVA, Dunnett's test (1964) 

will be used to compare the control and treated groups. 

10.6 Brain Morphometric Data {Cohort 2A): 

Brain morphometric data will be analyzed by the parametric one-way ANOV A 

(Snedecor and Cochran, 1980) to determine intergroup differences. If 
Pa c44of53 

WIL-417005 

June 25,2012 

significant differences are indicated by the ANOVA, Dunnett's test (1964) will 

be used to compare the control and treated groups. 

11 QUALITY ASSURANCE: 

The study will be audited by the WIL Quality Assurance Department while in 

progress to assure compliance with Good Laboratory Practice Regulations, adherence 

to the protocol and to WIL Standard Operating Procedures. The protocol, protocol 

amendments, raw data, draft report and final report will be audited by the WIL 

Quality Assurance Department prior to submission to the Sponsor to assure that the 

fmal report accurately describes the conduct and the findings of the study. This study 

will be conducted in compliance with the EPA TSCA ( 40 CFR Part 792), OECD 

Series on Principles of Good Laboratory Practice and Compliance Monitoring, 

Number I OECD ENV/MC/CHEM(98)17 Principles of Good Laboratory Practice, 

and the European Community (EC) European Parliament and Council Directive 

2004/10/EC (O.J. No. L 50/44, 02 February 2004) and OECD 

ENV/MC/CHEM(98)17 Principles of Good Laboratory Practice. 

Histology slide preparation for ovarian primordial follicle evaluation will be 

conducted following the Standard Operating Procedures of WIL Research at 

Hillsborough, and in accordance with the Protocol and GLPs. Quality Assurance 

monitoring of these analyses for Protocol, SOP and GLP compliance is the 

responsibility ofWIL Research at Hillsborough. Inspection reports will be supplied to 

the Study Director. Upon completion of the prescribed activities, WIL Research at 

Hillsborough will provide a signed Quality Assurance Statement to the Study Director 

which will be included in the fmal report. 

Data requmng RMANOV A statistical analysis will be analyzed by 

BioSTAT Consultants, Inc. following the current procedural guidelines of 

BioSTAT Consultants, Inc. BioST AT Consultants, Inc. will provide a statistical 

analysis report, which will be included as an appendix to the fmal report. Quality 

Assurance auditing of the statistical report (for internal consistency and consistency 

with the study report) will be conducted under the direction of the Quality Assurance 

Department of WIL Research. 

This study will be included on the WIL master list of regulated studies. 

12 RECORDS TO BE MAINTAINED: 

All original raw data records, as defined by WIL SOPs and the applicable GLPs, will 

be stored as described in Section 13 in the Archives at WIL Research. 

13 WORK PRODUCT: 

The Sponsor will have title to all documentation records, raw data, specimens and 

other work product generated during the performance of the study. Any remaining 
Pa e 45 of 53 

WJL-417005 

June 25,2012 

samples (e.g. formulation or blood/plasma/serum) will be discarded after issuance of 

the Final Report. All work product generated at WIL Research Hillsborough will be 

returned to WIL Research Ashland. All work product, including raw paper data, 

pertinent electronic storage media and specimens, will be retained at no charge for six 

months following issuance of the final report in the WIL Research Archives. 

Thereafter, WIL Research will charge a monthly archiving fee for retention of all 

work product. All work product will be stored in compliance with regulatory 

requirements. 

Any work product, including documents, specimens, and samples, that are required 

by this protocol, its amendments, or other written instructions of the Sponsor, to be 

shipped by WIL Research to another location will be appropriately packaged and 

labeled as defined by WIL's SOPs and delivered to a common carrier for shipment. 

WIL Research will not be responsible for shipment following delivery to the common 

carrier. 

Appropriate supporting documentation for statistical analyses conducted and reported 

by BioSTAT Consultants, Inc. will be maintained in the WIL Research Archives. 

14 REPORTS AND STUDY PROGRESS UPDATES: 

The Study Director will provide periodic updates of study progress to the Sponsor. 

Draft summary tables of unaudited electronically recorded data may accompany these 

updates. 

The final report will contain a summary, test substance information, methods and 

procedures, appropriate individual animal and summary data tables, WIL Historical 

Control Data, supporting sub-reports (e.g. analytical chemistry report, etc.), a copy of 

the protocol and amendment(s), if any, and an interpretation and discussion of the 

study results. 

In addition, the final report will also include a graphical depiction* of mean data for: 

a. Maternal body weights during gestation 

b. Maternal body weights during lactation 

c. Litter body weights during lactation 

d. Male and female body weights ofpostweaning animals 

c. Locomotor activity interval data at each age 

f. Auditory startle peak and latency to peak response data by trial block at each age 

g. Forelimb and hindlimb grip strength values across each age tested 

* Graphs should depict the smnmary data presented after statistical analysis (i.e. data should be 

combined unless statistically analyzed separately by sex) 
Pa e46of53 

WIL-417005 

June 25,2012 

The final report will be comprehensive and shall defme Ievel(s) inducing toxic effects 

as well as no-effect Icvcl(s) under the conditions of this investigation. The report will 

contain all information necessary to conform with current regulatory specifications. 

WIL Research will submit an electronic copy (PDF and MS Word copy of the report 

text for editing and comments) of the audited draft report in a timely manner upon 

completion of data collection prior to issuance of the final report. It is expected that 

the Sponsor will review the draft report and provide comments to WIL Research 

within a two-month time frame following submission. WIL Research shall provide a 

revised draft report that incorporates the Sponsor's reasonable revisions and 

suggestions. One revision will be permitted as part ofthe cost of the study; additional 

changes or revisions may be made, at extra cost. WIL Research shall submit the final 

report within two weeks of receiving authorization to finalize the report from the 

Sponsor. Ifthe Sponsor's comments and/or authorization to finalize the report have 

not been received at WIL Research within one year following submission of the draft 

report, WIL Research may elect to finalize the report following appropriate written 

notification to the Sponsor. An electronic copy (bookrnarked and hyperlinked PDF) 

of the final report will be provided; requests for paper copies of the fmal report may 

result in additional charges. 

15 ANIMAL WELFARE ACT COMPLIANCE: 

This study will comply with all applicable sections of the Final Rules of the Animal 

Welfare Act (A W A) regulations (9 CFR). The protocol will be reviewed by the WIL 

Institutional Animal Care and Use Committee (IACUC) prior to commencement of 

study specific activities. The Sponsor should make particular note of the following: 

• The Sponsor Representative's signature on this protocol documents for the Study 

Director the Sponsor's assurance that the study described in this protocol does not 

unnecessarily duplicate previous experiments. 

• Whenever possible, procedures used in this study have been designed to avoid or 

minimize discomfort, distress or pain to animals. All methods are described in this 

study protocol or in written laboratory Standard Operating Procedures. 

• 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 

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. 

• Methods of euthanasia used during this study are in conformance with the 

above-referenced regulation. 

• The Sponsor/Study Director has considered alternatives to procedures that may 

cause more than momentary or slight pain or distress to the animals and has 
Page 47 of 53 

WIL-417005 

June 25, 2012 

provided a written nanative description (A W A covered species) of the methods and 

sources used to determine that alternatives arc not available. 

16 PROTOCOL MODIFICATION: 

Modification of the protocol may be accomplished during the course of this 

investigation. However, no changes will be made in the study design without the 

verbal or written permission of the Sponsor. In the event that the Sponsor verbally 

requests or approves a change in the protocol, such changes will be made by 

appropriate documentation in the form of a protocol amendment. All alterations of 

the protocol and reasons for the modification(s) will be signed by the Study Director 

and the Sponsor Representative. 

17 REFERENCES: 

Adams, J.; Buelke-Sam, J.; Kimmel, C.A.; Nelson, C.J.; Reiter, L.W.; Sobotka, T.J.; 

Tilson, H.A.; Nelson, B.K. Collaborative behavioral teratology study: protocol design 

and testing procedure. Neurobehavioral Toxicology and Teratology 1985, 7, 579-586. 

Biel, W.C. Early age differences in maze performance in the albino rat. Journal of 

Genetic Psychology 1940, 56, 439-453. 

Blazak, W.F.; Ernst, T.L.; Stewart, B.E. Potential indicators of reproductive toxicity: 

testicular sperm production and epididymal sperm number, transit time and motility in 

Fischer 344 rats. Fundamental and Applied Toxicology 1985, 5 (6 Pt. I), 1097-1103. 

Bolon, B., Bucci, T.J., Warbritton, A.R., Chen, J.J., Mattison D.R. and Heindel, J.J. 

Differential follicle counts as a screen for chemically induced ovarian toxicity in mice: 

Results from continuous breeding bioassays. Fundam. Appl. Toxicol. 1997,39, 1-10. 

Bucci, T.J., Bolon, B., Warbritton, A.R., Chen, J.J., and Heinde~ J.J. Influence of 

Sampling on the reproducibility of ovarian follicle counts in mouse toxicity studies. 

Reprod Toxicol. 1997, II (5), 689-696. 

Burkhart, C.A. and Robinson, J.L. High rat pup mortality attributed to the use of 

cedar-wood shavings as bedding. Laboratory Animals 1978, I2, 221-222. 

Dawson, A.B. A note on the staining of the skeleton of cleared specimens with Alizarin 

RedS. Stain Technology 1926, I, 123-124. 

Dunn, O.J. Multiple comparisons using rank sums. Technometrics 1964, 6(3), 241-252. 

Dunnett, C.W. New tables for multiple comparisons with a control Biometrics 1964, 

20, pp. 482-491. 
Page 48 of 53 

WIL-417005 

June 25, 2012 

Gad, S. A neuromuscular screen for use in industrial toxicology. Journal of Toxicology 

and Environmental Health 1982, 9:691-704. 

Gallavan, R.H., Jr., Holson, J.F., Stump, D.G., Knapp, J.F., and Reynolds, V.L. 

Interpreting the toxicological significance of alterations in anogenital distance: potential 

for confounding effects of progeny body weights. Reprod. Toxicol. 1999, 13(5), 

383-390. 

Gray Jr., L. E., Ostby, J., Monosson, E., Kelce, W. R. Environmental antiandrogens: low 

doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicology 

and Industrial Health. (1990) 15, 48-64. 

Haggerty, G.C. Development of Tier 1 neurobehavioral testing capabilities for 

incorporation into pivotal rodent safety assessment studies. Journal of the American 

College Toxicology 1989, 8:53-69. 

Hollander, M.; Wolfe, D.A. Nonparametric Statistical Methods, 2nd ed.; Hollander, M., 

Wolfe, D.A., Eds.; John Wiley and Sons, Inc.: New York, NY, 1999; p 468. 

!twin, S. Comprehensive observational assessment: Ia. A systematic quantitative 

procedure for assessing the behavioral physiological state of the mouse. 

Psychopharmacologia 1968, 13:222-256. 

Korenbrot, C.C.; Huhtaniemi, LT.; Weiner, R.W. Preputial separation as an external 

sign of pubertal development in the male rat. Biology of Reproduction 1977, 17, 

298-303. 

Kruskal, W.H.; Wallis, W.A. Use ofranks in one-criterion variance analysis. Journal 

~/the American Statistical Association 1952,47, 583-621. 

Linder, R.E.; Strader, L.F.; Slott, V.L.; Suarez, J.D. Endpoints ofspermatotoxicity in the 

rat after short duration exposures to fourteen reproductive toxicants. Reproductive 

Toxicology 1992, 6, 491-505. 

Marty, M.S., Johnson, KA., Carney, E.W. Effect of feed restriction on Hershberger and 

pubertal male assay endpoints. Birth Defects Research, Part B: Developmental and 

Reproductive Toxicology, 2003, 68, 363-374. 

Moser, V.C. McCormick, J.P.; Creason, J.P. and MacPhail, R.C. Comparison of 

Chloridimeform and Carbaryl Using a Functional Observational Battery. Fundamental 

and Applied Toxicology 1988, 11, 189-206. 

Moser, V.C.; McDaniel, K.L.; Phillips, P.M. Rat strain and stock comparisons using a 

functional observational battery: Baseline values and effects of Amitraz. Toxicology 

and Applied Pharmacology 1991, 108, 267-283. 
Page 49 of 53 

WIL-417005 

June 25, 2012 

O'Donoghue, J.L. Screening for neurotoxicity using a neurologically based examination 

and neuropathology. Journal of the American College Toxicology 1989, 8:97-116. 

Owens, W.; Ashby, J.; Odum, J. and Onyon, L. The OECD program to validate the rat 

uterotrophic bioassay. Phase 2: dietary phytoestrogen analyses. Environmental Health 

Perspectives 2003, 111 (12), 1159-67. 

Picut, C.A.; Swanson, C.L.; Scully, K.L.; Roseman, V.C.; Parker, RF.; Remick, A.K. 

Ovarian Follicle Counts Using Proliferating Cell Nuclear Antigen (PCNA) and 

Semi-Automated Image Analysis in Rats. Toxicol Pathol. 2008 May 8. 

Saghir, S.A., Clark, A.J., Gilles, M.M. et a!. 1,2-Dichloroethane (EDC): Limited 

pharmacokinetics and metabolism study in Fischer 344 rats. The Dow Chemical 

Company, Midland, MI, 2006. 

SAS® Proprietary Software, Version 9.2; SAS Institute, Inc.: Cary, NC, 2002-2008. 

Snedecor, G.W.; Cochran, W.G. One Way Classifications; Analysis of Variance. In 

Statistical Methods, 7th ed.; The Iowa State University Press: Ames, IA, 1980; 

pp. 215-237. 

Steel, RG.D.; Torrie, J.H. Principles and Procedures of Statistics, A Biometrical 

Approach, 2nd ed.; McGraw-Hill Book Company: New York, NY, 1980; pp. 504-506. 

Stuckhardt, J.L.; Poppe, S.M. Fresh visceral examination of rat and rabbit fetuses used 

in teratogenicity testing. Teratogenesis, Carcinogenesis and Mutagenesis 1984, 4, 

181-188. 

Sweeney, L. M., and Parker, A. Internal dosimetry considerations for dose selection for 

an extended one-generation reproductive toxicity study of 1 ,2-dichloroethane in the rat. 

Report ofToxicology Excellence for Risk Assessment, Cincinnati, Ohio, 2010. 

U.S. EPA. 1,2-Ethylene Dichloride: Final Enforceable Consent Agreement and Testing 

Consent Order, Fed. Reg. 2003,68: 33125-33129. 

U.S. EPA. 1,2-Ethylene Dichloride: Completion of EPA Program Review, Fed Reg. 

2009, 74: 36196-36198. 

Weichbrod, RH.; Cisar, C.F.; Miller, J.G.; Simmonds, RC.; Alvares, A.P.; Ueng, T.H. 

Effects of Cage Beddings on Microsomal Oxidative Enzymes in Rat Liver. Laboratory 

Animal Science 1988; 38(3), 296-298. 
II • II 

a II 

••••• I II II 

II • ••••• 

••••• 

6/25/2012 7:4S AH FRml: 5032419078 TO: 14192893650 PAGE: 001 OF 002 

W!l.-·117WJ~ 

·-·· ~----· ----- Jli)t!L' -~-~~-~~----- .----···--··-------·--~-~-··· ·-·•·•~·----~~-~~~~~~~-~-~--

lH PROTOCOLAPPHOVAL: 

Sponsor <~pprov~ll reeei\·ed via_£1Cl'~L __________ on_..!:!_/cUi/JO I'd, 

IIi\ P Task Force 

__ lthUu_ ~-e~j_wal_ Peter E. VoyJt.- PhD 

Sponsor Representative 

WIL Rc:~carch 

___ :):x_L<-l~Qadi_ir_d.==---------- 1vlelissa J. Beck, PhD 

Study Director 

Dmc 

,}fi_~.,, d.Or ~~~. Date 
27 F0 Males/Group 

All F1 Pups 

Anogenital Distance on PND 1 

Nipple/ Areola Retention on PND 12 

F1 Culled Pups (PND 4} 

Thyroid Hormone Assessment 

(Minimum of 15 Samples/Group) 

Tissues for Possible Thyroid 

Histopathology 

Pa e 51 of 53 

APPENDIX A 

STUDY DESIGN 

F0 GENERATION 

Exposure to Test Substance for 28 Days 

Prior to Mating 

Weekly Detailed Clinical Observations 

beginning Prior to Initiation of Test 

Substance Administration 

Determination of Estrous Cycles 

Beginning 2 weeks after Initiation of 

Test Substance Administration 

MATE TO PRODUCE F1 LITTERS 

· 1 Male x 1 Female 

Mate 14 days 

Exposure to Test Substance Throughout 

Gestation and Lactation Periods (Fo 

Males and Females) 

Urinalysis Conducted on F0 Males; 

Hematology and Serum Chemistry 

Conducted on all F0 Animals prior to the 

Scheduled Necropsy 

F0 adults euthanized after F1 weaning 

WIL-417005 

June 25,2012 

27 F0 Females/Group 

.E2 Males 

Thyroid Hormone Assessment I 

Week Prior to Necropsy 
Page 52 of 53 

WIL-417005 

June 25, 2012 

F0 Histopathology 

Fo Gross Pathology 

Spermatogenesis Evaluation 

Thyroid Hormone Assessment 

(15 Males/Group) 

27 rats/sex (Control & High) 

PND 22 Nonselected F1 Weanlings 

Thyroid Hormone Assessment 

15/sex/group 

F1 WEANLINGS 

At Weaning, F1 Pups from 

2:20 Litters/Group Assigned to Cohorts 

(2/Sex/Li tter) 

Weekly Detailed Clinical Observations 

Conducted Weekly beginning on 

PND21 

I 

I 

Cohort 1 

(Reproductive and Developmental Toxicology) I 

Cohort 2 

(Developmental N eurotoxicology) 

Cohort IA 

2:20/sex/group 

(1/sex/litter) 

Sexual Developmental 

Landmarks 

Day of First Estms 

Daily Estrous Cycle 

(PND 75-88) 

Thyroid Hormone 

Assessment 

(-PND 85) 

Spermatogenesis 

Evaluations 

Clinical Pathology 

Assessments 1 Week 

Prior to Necropsy 

Euthanasia on 

-PND 91 

Histopathology 

Cohort IB 

2:20/sex/group 

(!/sex/litter) 

Sexual Developmental 

Landmarks 

Maintained for Potential 

F 1 Mating and/or 

Additional 

Histopathology 

Euthanasia on -PND 120 

Cohort2A 

10/sex/group 

(representing 20 litters, 

if possible) 

Sexual Developmental 

Landmarks 

Neurobehavioral 

Assessments 

Euthanasia on -PND 78 

Neuropathology 

Cohort 2B 

1 0/sex/group 

(representing 20 litters, 

if possible) 

Sexual Developmental 

Landmarks 

Euthanasia on PND 21 

Neuropathology 
Page 53 of 53 

APPENDIXB 

WIL-417005 

June 25, 2012 

Analytical Validation and Stability Study of 1,2-Dichloroethane in Aqueous Formulations 

P99e 1 of 1 

Analytical Validation and Stability Study of 1.2-Dichloroethan<! in Aqueous Formulations 

CompiJanc.e· F::ll>.' GLP 

OeVIIIopm&nl and validation ol a msthod lor the detarmrnation of analyle concentration in aqueous 

formulations: 

MeL"od del'eiopmeM usua!y incU:es tbu! rs r-ot limiied to) the following 

activitiea: (1 i ;,-,,-estigation ol po;en!ia! so'ubility 'imrtations; (2) lhl analysis 

of slzndaros prepared in an approprillle solwn! to es!ablisb 

cllromalogt~~phy, inc""'ding re:errtion times, resolution, ;and to check 

propcrtbr.-lily of respon!l!; (3) the 11nlliysis a' the anlllyte prepared in the 

m11trix io confirm the presence or ab..<erce of interferences, to ellliiUi!l!e 

potential •tabilrty lim~atior:s, and to evaiua:e response proportionafrty. 

Method development will be bile<! a! a rate of $25JI'hour and will not 

exceed !he amO!Jm propcsed 'Nill'o!1 sponsor llill-"'lCVaL 

Validabon wiil be conducted using the currem Wtl SOP guidelines for the 

auenment of system suitabil!y. method speeficltylselectivity, in1ra- 11nd 

mler·eesSIOn method oalibratior acoepteb<lrty, irnr&· and inter-session 

melhod accuracy are precision, ruggednes.s, and processed sample 

stabilty. A mnmum of three V1!1ida!ioo sessions win be conducted. All 

laboratory wort; associated with vartdatioos will be conducted in 

accordaoce wilh applicable GLP regu\aOOns. 

Unl1orm11y and stablltty u .. nrMnt of analyte in aqu.ous formulations: 

Quallly Assuranea: 

CONFIDENTIAL 

Testing itlcludu the assessment of tes! article uniformity in formulations 

spam~ the range of concentration arlioipaled an t<..rture studoes. In 

adddion, statlilrty will be IISM!SMd tallowtng a single stora~ auratrorr 

Additional stabiwty lime-colnts can be added for an acdilional fee. Afl 

laboratory work. eS50Ciated with sample analysis viii be conducted in 

aooc«la!"Ce with applicable GLP regu:ations. 

The study will be cond~cted ir co!"1pfiaN:I! with Good LaooratD!j' Practice 

(GlPl standards ar.d wi'l be morr.cred by the Qu;a!ily Assurance Unit 

We have listening down to a science. 
ATTACHMENT 

ECA 

Federal Register/Val. 68, No. 106/Tuesday, June 3, 2003/Notices 33125 

Region VII, 901 N. 5th Street, Kansas 

City, Kansas 66101. 

SUPPLEMENTARY INFORMATION: This 

proposed settlement is intended to 

resolve the liability of Saveway 

Petroleum at the Great Lakes Container 

Corporation Superfund Site in St. Louis, 

Missouri. 

Great Lakes Container Corporation is 

a former drum reclamation company 

who operated at the Site from 1976 to 

1985. The same business was operated 

as Northwestern Cooperage from the 

1950's to 1976 and then operated as 

Great Lakes Container Corporation. EPA 

conducted a time-critical removal 

completed in 1998 that consisted 

primarily of soil and drum removal. The 

EPA incurred costs of approximately 

$9,127,244.30. The hazardous 

substances at this Site consisted 

primarily of lead and polychlorinated 

biphenyls. Liability is based on the 

theory that de minimis parties arranged 

for disposal of hazardous substances at 

the Site by shipping drums for 

reclamation coated with paint 

containing lead. The de minimis parties 

either admitted that they sent drums for 

reclamation to the Site or EPA had 

separate evidence to prove that de 

minimis parties sent drums for 

reclamation to the Site. 

This settlement is being offered to 

Saveway because it is liable for no more 

than one quarter a percent (.25%) of 

EPA's past costs at the Site. The 

majority of de minimis parties are each 

required to pay $4,839.44 or $5,133.72 

depending on whether the party was 

required to pay prejudgment interest. 

Other settlements made for six de 

minimis parties varied from $3,794.19 to 

$22,856.56 because more volumespecific information was available for 

them allowing EPA to refine the 

calculation. The amount and toxicity of 

hazardous substances contributed by 

Saveway was minimal as compared to 

other parties' shares of hazardous 

substances. The EPA determined this 

amount to be Saveway's fair share of 

liability based on the amount of 

hazardous substances generated and 

disposed of at the Site and the volume 

of waste contributed. However, because 

Saveway has demonstrated an inability 

to pay, it will not be required to pay any 

of EPA's past costs at the Site. As a 

result, Saveway has agreed to provide 

access to EPA and maintain records for 

five (5) years. 

The settlement also includes 

contribution protection from lawsuits by 

other potentially responsible parties as 

provided for under section 122(g)(5) of 

CERCLA, 42 U.S.C. 9622(g)(5). The de 

minimis settlement provides that EPA 

covenants not to sue Saveway for 

response costs at the Site or for 

injunctive relief pursuant to sections 

106 and 107 of CERCLA and section 

7003 of the Resource Conservation and 

Recovery Act of 1976, as amended 

(RCRA), 42 U.S.C. 6973. The settlement 

contains a reopener clause which 

nullifies the covenant not to sue if any 

information becomes known to EPA that 

indicates that Saveway no longer meets 

the criteria for a de minimis settlement 

set forth in section 122(g)(l)(A) of 

CERCLA, 42 U.S.C. 9622(g)(l)(A). The 

United States maintains the ability to 

bring an action in the event that the 

financial information provided by 

Saveway was false. The covenant not to 

sue does not apply to the following 

matters: 

(a) Claims based on the future 

arrangement for disposal or treatment of 

any hazardous substance, pollutant, or 

contaminant at the Site after the 

effective date of the de minimis 

settlement; 

(b) Criminal liability; or 

(c) Liability for damages cir injury to, 

destruction of, or loss of the natural 

resources and for the costs of any 

natural resource damage assessments. 

The de minimis settlement will 

become effective upon the date which 

the EPA issues a written notice to 

Saveway that the statutory public 

comment period has closed and that 

comments received, if any, do not 

require modification, of or EPA 

withdrawal from the settlement. 

Dated: May 22, 2003. 

James B. Gulliford, 

Regional Administrator, Region VII. 

[FR Doc. 03-13565 Filed 6-2-Q3; 8:45am] 

BILLING CODE 6560-511-P 

ENVIRONMENTAL PROTECTION 

AGENCY 

[OPPT -2003-001 0; FRL-7300--6] 

1 ,2-Ethylene Dichloride; Final 

Enforceable Consent Agreement and 

Testing Consent Order 

AGENCY: Environmental Protection 

Agency (EPA). 

ACTION: Notice. 

SUMMARY: Under section 4 of the Toxic 

. Substances Control Act (TSCA), EPA 

has issued a testing consent order 

(Order) that incorporates an enforceable 

consent agreement (ECA) with the Dow 

Chemical Company, Vulcan Materials 

Company; Occidental Chemical 

Corporation, Oxy Vinyls, LP, Georgia 

Gulf Corporation, Westlake Chemical 

Corporation, PPG Industries, Inc., and 

Formosa Plastics Corporation, U.S.A. 

The Companies are members of the 

Hazardous Air Pollutant (HAP) Task 

Force, which represents the 

manufacturers of 1,2-ethylene 

dichloride (EDC). The Companies have 

agreed to: Conduct toxicity testing, 

develop pharmacokinetics and 

mechanistic test data, and develop a 

computational dosimetry model for 

quantitative route-to-route 

extrapolations of dose-response for EDC 

for acute, subchronic, developmental, 

reproductive and neurotoxicity effects 

that were identified in a proposed test 

rule for hazardous air pollutants. This 

notice announces the ECA and Order for 

EDC and summarizes the terms of the 

ECA. 

DATES: The effective date of the ECA 

and Order is May 13, 2003. 

FOR FURTHER INFORMATION CONTACT: For 

general information contact: Barbara 

Cunningham, Acting Director, 

Environmental Assistance Division 

(7408M), Office of Pollution Prevention 

and Toxics, Environmental Protection 

Agency, 1200 Pennsylvania Ave., NW., 

Washington, DC 20460-0001; telephone 

number: (202) 554-1404; e-mail address: 

TSCA-Hotline@epa.gov. 

For technical information contact: 

Richard Leukroth or John Schaeffer, 

Chemical Control Division (7405M), 

Office of Pollution Prevention and 

Toxics, Environmental Protection 

Agency, 1200 Pennsylvania Ave., NW., 

Washington, DC 20460-0001; telephone 

number: (202) 564-8157; fax number: 

(202) 564-4765; e-mail address: 

ccd.citb@epa.gov. 

SUPPLEMENTARY INFORMATION: 

I. General Information 

A. Does this Action Apply to Me? 

This announcement is directed to the 

public in general. However, as described 

in Unit IV., this ECA and Order may 

affect others in that EPA has initiated 

rulemaking under TSCA section 12(b) 

(62 FR 67038, December 23, 1997) 

(FRL-5762-8). When finalized, that 

rulemaking will require all persons who 

export or intend to export EDC to 

comply with the export notification 

regulations at 40 CFR part 707, subpart 

D. Although others may be affected by 

subsequent actions related to this 

announcement, this ECA and Order 

only applies to those Companies that are 

specifically named in this ECA and 

Order. If you have any questions 

regarding the applicability of this action 

to a particular entity, consult the 

technical person listed under FOR 

FURTHER INFORMATION CONTACT. 
33126 Federal Register/Val. 68, No. 106/Tuesday, June 3, 2003/Notices 

B. How Can I Get Copies of this 

Documrmt and Other Related 

Information? 

1. Docket. EPA has established an 

official public docket for this action 

under docket identification (ID) number 

OPPT-2003-0010. The official public 

docket consists of the documents 

specifically referenced in this action, 

any public comments received, and 

other information related to this action. 

Although a part of the official docket, 

the public docket does not include 

Confidential Business Information (CBI) 

or other information whose disclosure is 

restricted by statute. The official public 

docket is the collection of materials that 

is available for public viewing at the 

EPA Docket Center, Rm. B102-Reading 

Room, EPA West, 1301 Constitution 

Ave., NW., Washington, DC. The EPA 

Docket Center is open from 8:30a.m. to 

4:30p.m., Monday through Friday, 

excluding legal holidays. The EPA 

Docket Center Reading Room telephone 

number is (202) 566-1744 and the 

telephone number for the OPPT Docket, 

which is located in the EPA Docket 

Center, is (202) 566-0280. 

2. Electronic access. You may access 

this Federal Register document 

electronically through the EPA Internet 

under the "Federal Register" listings at 

http:/ /www.epa.gov/fedrgstr/. 

An electronic version of the public 

docket is available through EPA's 

electronic public docket and comment 

system, EPA Dockets. You may use EPA 

Dockets at http://www.epa.gov/edocket/ 

to submit or view public comments, 

access the index listing of the contents 

of the official public docket, and to 

access those documents in the public 

docket that are available electronically. 

Although not all docket materials may 

be available electronically, you may still 

access any of the publicly available 

docket materials through the docket 

facility identified in Unit I.B.l. Once in 

the system, select "search," then key in 

the appropriate docket ID number. 

II. Background 

A. What is EDC? 

EDC is used as a chemical 

intermediate principally in the 

production of vinyl chloride, but also 

vinylidene chloride, 1,1,1-

trichloroethane, trichloroethylene, 

tetrachloroethylene, aziridines, and 

ethylene diamines. It is also used as a 

solvent. An estimated 77,111 workders 

are exposed to EDC (Ref. 1). The 

Chemical Abstract Service Registry 

Number (CAS No.) for EDC is 107-06-

2. 

B. Why Does EPA Need Health Effects 

Data on EDC? 

EPA proposed health effects testing 

under TSCA section 4(a) for a number 

of hazardous air pollutants ("HAPs" or 

"HAP chemicals"), including EDC (61 

FR 33178, June 26, 1996) (FRL-4869-1). 

as amended at 62 FR 67466, December 

24, 1997 (FRL-5742-2) and 63 FR 

19694, April21, 1998 (FRL-5780-6). In 

the original HAPs proposal, the Agency 

made preliminary findings for EDC (61 

FR 33178, 33190, 33191; and Ref. 1) 

that: 

1. EDC may present an unreasonable 

risk of injury to health. 

2. EDC is or will be produced in 

substantial quantities, and there is or 

may be substantial human exposures to 

the chemical. 

3. There are insufficient data to 

determine or predict the effects of 

activities on human health involving 

EDC. 

4. Testing is necessary to develop 

health effects data for EDC. 

The HAPs rule, as amended, proposed 

testing ofEDC for acute toxicity, 

subchronic toxicity, developmental 

toxicity, reproductive effects toxicity, 

and neurotoxicity (61 FR 33178, 33198, 

June 26, 1996; 62 FR 67466, 67483, 

December 24, 1997). 

III. ECA Development and Conclusion 

A. How is EPA Going to Obtain Health 

Effects Testing on EDC? 

In the proposed HAPs test rule, as 

amended, EPA invited the submission 

of proposals regarding the performance· 

of pharmacokinetics studies that would 

permit extrapolation from oral data to 

predict risk from inhalation exposure. 

Such proposals could provide the 

scientific basis for alternative testing to 

the testing proposed and form the basis 

for developing needed HAPs data via 

ECAs (61 FR 33178, 33189, June 26, 

1996; 62 FR 67466, 67474, December 24, 

1997). EPA uses ECAs to accomplish 

testing where a consensus exists among 

EPA, affected manufacturers and/or 

processors, and interested members of 

the public concerning the need for and 

scope of testing (40 CFR 790.1(c)). 

The procedures for ECA development 

are described at 40 CFR 790.22(b). 

In response to EPA's request for 

proposals for ECAs, the HAP Task Force 

submitted a proposal for alternative 

testing that included physiologicallybased pharmacokinetics (PBPK) studies 

and computational modeling to inform 

route-to-route extrapolations of doseresponse for EDC on November 22, 1996 

(Ref. 2). EPA responded to this proposal 

on June 26, 1997 (Ref. 3). indicating that 

the HAP Task Force alternative 

approach offered sufficient merit to 

proceed with discussions for developing 

an ECA for EDC. Consequently, EPA 

issued a document which was 

published in the Federal Register of 

December 19, 1997 (62 FR 66626) (FRL5763-1), soliciting interested parties to 

monitor or participate in these 

discussions. 

EPA held a public meeting to develop 

an ECA for EDC on January 12, 1998. 

Representatives .of the Companies and 

other interested parties attended this 

meeting. The participants reached 

consensus on the general scope of the 

testing to be required under the ECA. 

Following the public meeting, the HAP 

Task Force submitted (March 19, 1999) 

a revised proposal for a testing program 

(Ref. 4). On February 13, 2001, EPA 

responded to the HAP Task Force with 

comments on the revised proposal and 

by initiating a draft ECA for 

consideration by the HAP Task Force 

(Ref. 5). A final version of the ECA was 

later circulated to the HAP Task Force 

for signature, and returned to EPA for 

signature. On February 3, 2003, EPA 

received the ECA signed by the 

Companies. On May 13,2003, EPA 

signed the ECA and accompanying 

Order (Ref. 6). 

B. What Testing Does the EGA for EDC 

Require? 

The EDC ECA alternative testing 

program has four segments as follows: 

Tier I HAPs Testing, Tier I Program 

Review Testing, EPA Program Review, 

and Tier IT Testing. This is described in 

Table 1 in this unit and includes the 

following testing, reporting, and review 

activities: 

1. Tier I HAPs Testing. This testing 

consists of endpoint testing, conducted 

by inhalation exposure, that EPA 

deemed necessary to meet certain data 

needs identified in the proposed HAPs 

test rule, as amended, and includes 

acute toxicity with bronchoalveolar 

lavage (BAL) and histopathology, and 

acute neurotoxicity testing. These tests 

will be conducted under a combined 

protocol as described in Appendix D.1 

of the ECA. 

2. Tier I Program Review Testing. 

Under this segment of the EDC ECA 

alternative testing program, the 

Companies will conduct studies to 

extend the computational dosimetry 

model of D'Souza et al. (1987; 1988; 

Refs. 7 and 8) in order to apply the 

model to the specific health effects 

endpoints for EDC listed in the ECA, 

validate the model, and verify the 

model's ability to perform quantitative 

route-to-route extrapolations of doseresponse. In addition, the Companies 

will sponsor development of 
Federal Register/Val. 68, No. 106/Tuesday, June 3, 2003/Notices 33127 

pharmacokinetics and mechanistic (PK/ 

MECH) data to support the application 

of the model for the endpoints listed 

under Tier II of the EDC ECA. 

Specifically, the PK/MECH testing will 

develop data to inform: 

i. Oral-to-inhalation extrapolation of 

subchronic toxicity data reported by 

Daniel, et al. (1994; Ref. 9) relevant to 

corn oil gavage. 

ii. Oral-to-inhalation extrapolation of 

'subchronic neurotoxicity data relevant 

to drinking water exposure of a study to · 

be conducted under Tier II Testing. 

iii. Oral-to-inhalation extrapolation of 

reproductive effects testing conducted 

under Tier II Testing and each dosing 

paradigm of studies reported by Alumot 

et al. (1976; Ref. 10), Rao, et al. (1980; 

Ref. 11) and Lane et al. (1982; Ref. 12). 

In addition, the Companies will 

provide model simulations with point 

and uncertainty estimates of internal 

dose metrics (parent chemical peak and 

area under the curve (AUC) 

concentrations in blood and brain, and 

24-hour total glutathione-dependent 

metabolism) in rats and humans to 

inform quantitative route-to-route 

extrapolations of dose-response. These 

simulations will be used to evaluate the 

acceptability of: Subchronic 

neurotoxicity testing of oral exposure 

via drinking water in rats, extant oral 

subchronic toxicity data of Daniel et al. 

(1994; Ref. 9) in rats via corn oil gavage, 

and reproductive toxicity testing of oral 

e,?Posure via drinking water in rats. 

3l ~PA Program Review. Model 

· 'ffi:!velopment and data from Tier I 

Program Review Testing are subject to 

an EPA Program Review. It is essential 

to the success of the EDC ECA 

alternative testing program for EPA to 

ensure that the model and the PK/ 

MECH data used to support the routeto-route extrapolations of dose-response 

are of the highest quality. The purpose 

of the EPA Program Review will be to 

determine: 

i. Whether it is feasible and 

' appropriate to apply Tier I Program 

: Review Testing data and data from other 

studies acceptable to EPA to support 

' computational route-to-route 

extrapolations of dose-response for 

endpoints listed in the Tier II Testing 

segment of the ECA. 

ii. Whether the data from the Tier I 

Program Review Testing segment 

provide a sufficient basis for conducting 

the endpoint testing and/or the 

computational route-to-route 

extrapolations for the dose-responses 

specified in the Tier II Testing segment. 

iii. The nature and scope of any 

additional work that may be required 

before Tier II Testing and application of 

the EDC model for route-to-route 

extrapolation of dose-response reporting 

(e.g., development of additional PK/ 

MECH data, modification to the EDC 

model). 

4. Tier II Testing and/or Extrapolation 

Reporting. This segment of the EDC ECA 

alternative testing program consists of 

endpoint testing by drinking water 

exposure for subchronic neurotoxicity 

and reproductive toxicity. The 

reproductive effects toxicity testing is 

intended to confirm studies reported by 

Alumot et al. (1976; Ref. 10), Rao et al. 

(1980; Ref. 11), and Lane et al. (1982; 

Ref. 12), and provide data needed on 

fertility index, gestation index, gross 

necropsy, organ weight, histopathology, 

estrous cycle, sperm evaluation, vaginal 

opening, and preputial separation as 

described in the ECA. 

This segment will also include 

application of the EDC model for 

quantitative route-to-route extrapolation 

reporting (oral to inhalation) for Tier II 

endpoint testing (subchronic 

neurotoxicity and reproductive toxicity) 

and similar computational extrapolation 

reporting for extant subchronic toxicity 

reported by Daniel et al. (1994; Ref. 9). 

Testing conducted under this ECA 

will allow EPA to characterize certain 

potential health hazards resulting from 

inhalation exposure to EDC. The 

following Table 1 sets forth the required 

testing, test standard, and reporting 

requirements under the ECA for EDC. 

TABLE 1.-REQUIRED TESTING, TEST STANDARD, AND REPORTING REQUIREMENTS FOR EDC 

Testing Segment Required testing Test standard Deadline for final 

report1 (Months) 

'• 

Tier I HAPs Testing. Acute toxicity, with BAL and histopathology 40 CFR 799.9135 (as annotated in ECA 1& 

(inhalation). Appendix D.1). 

Acute neurotoxicity (inhalation). 40 CFR 799.9620 (as annotated in ECA 18 

Appendix D.1). 

Tier I Program Review PKIMECH data to support model validation and ECA Appendix C (1-4). 21 

Testing. verification of oral-to-inhalation extrapolation of 

dose-response for the following data needs in 

the F344 rat: 

a. Subchronic toxicity. 

b. Subchronic neurotoxicity. 

c. Reproductive toxicity. 

PBPK model simulations. ECA Appendix C (1-5). 21 

Tier II Testing and/or Ex- Subchronic toxicity route-to-route extrapolation of ECA Appendix C.2 and C.6. 36 

trapolation Reporting. dose-response (oral Tier II Testing to inhalation) 

of a study reported by Daniel eta/. (1994). 

Subchronic neurotoxicity (oral). 40 CFR 799.9620 (as annotated in ECA 42 

Appendix D.2). 

Subchronic neurotoxicity route-to-route extrapo- ECA Appendix C.3 and C.6. 52 

lation of dose-response (oral Tier II Testing to 

inhalation). 

Reproductive toxicity (oral). 40 CFR 799.9380 (as annotated in ECA 42 

Appendix D.3). 
33128 Federal Register/Val. 68, No. 106/Tuesday, June 3, 2003/Notices 

TABLE 1.-REQUIRED TESTING, TEST STANDARD, AND REPORTING REQUIREMENTS FOR EDC-Continued 

Testing Segment Required testing Test standard Deadline for final 

report' (Months) 

Reproductive toxicity route-to-route extrapolation EGA Appendix C.4 and C.6. 52 

of dose-response (oral data to inhalation, including Tier II Testing and extant studies reported 

by Alumot eta/. (1976), Rao et al. (1980), and 

Lane et a/. ( 1982)). 

' Number of months after the effective date of the Order that incorporates this EGA when the final report is due. In addition, every 6 months 

from the effective date of the Order until the end of the EGA testing program, interim reports describing the status of all testing to be performed 

under this EGA must be submitted by the Companies to EPA. 

C. What are the Uses for the Test Data 

forEDC? 

EPA would use the data obtained 

from testing to implement several 

provisions of section 112 of the Clean 

Air Act (CAA), including the 

determination of residual risk, the 

estimation of the risks associated with 

accidental releases of chemicals, and 

other HAP risk assessments. EPA will 

also use the data from this ECA to fulfill 

part of the Tier I Testing portion of the 

Voluntary Children's Chemical 

Evaluation Program (VCCEP). (For more 

information about VCCEP, see: http:// 

www.epa.gov/chemrtk/vceep/.) In 

addition, the data will be used by other 

Federal agencies (e.g., the Agency for 

Toxic Substances and Disease Registry 

(ATSDR), the National Institute for 

Occupational Safety and Health 

(NIOSH), the Occupational Safety and 

Health Administration (OSHA), and the 

Consumer Product Safety Commission 

(CPSC)) in assessing chemical risks and 

in taking appropriate actions within 

their programs (see the proposed HAPs 

test rule at 61 FR 33178, 33179, June 26, 

1996). 

D. Does the EGA for EDC Meet all the 

Testing Requirements for EDC that were 

Contained in the Proposed Test Rule? 

In the proposed HAPs test rule, as 

amended, EPA proposed testing of EDC 

for acute, subchronic, developmental, 

and reproductive effects and 

neurotoxocity by the inhalation route of 

exposure. The ECA alternative testing 

program for EDC requires inhalation 

testing for acute toxicity and acute 

neurotoxicity, and oral drinking water 

testing for subchronic neurotoxicity and 

reproductive effects toxicity. The ECA 

requires the development of PK/MECH 

data to support computational PBPK 

modeling to inform quantitative routeto-route extrapolations of dose-response 

(oral to inhalation) for the endpoints of 

subchronic toxicity, subchronic 

neurotoxicity, and reproductive effects 

toxicity as described in the ECA. 

During discussions to develop this 

ECA, EPA concluded that the 

developmental toxicity studies reported 

by Rao et al. (1980; Ref. 11), in rabbits, 

and Payan et al. (1995; Ref. 13), in rats, 

adequately fulfill the HAPs rulemaking 

testing requirement for developmental 

toxicity testing for EDC and, therefore, 

the ECA does not require, and the final 

HAPs test rule will not require this 

testing. In addition, the ECA does not 

require, and the final HAPs test rule will 

not require, macrophage function testing 

(a component of EPA's acute toxicity 

test gudeline 40 CFR 799,g135) because 

EPA considers existing data by 

Sherwood et al. (1987; Ref. 14) adequate 

to fulfill this aspect of the acute testing 

need. Furthermore, the Tier I HAPs 

Testing endpoints (acute toxicity and 

acute neurotoxicity) will not be 

included in the final HAPs test rule 

because the route of testing to be 

conducted under this ECA is identical 

to that specified in the HAPs 

rulemaking. Finally, depending on the 

outcome of EPA's Program Review, the 

Agency anticipates that the balance of 

the testing for EDC that was contained 

in the proposed HAPs test rule, as 

amended, will also not be included in 

the final HAPs test rule because the 

Companies will conduct equivalent 

testing as Tier II Testing and 

Extrapolation Reporting under this ECA 

alternative testing program for EDC. 

Therefore, EPA anticipates the fulfilling 

of all of the health effects testing 

requirements, identified in the HAPs 

proposed rule, as amended, by 

implementing the ECA and Order. 

The issuance of the ECA and Order 

constitutes final EPA action for 

purposes of 5 U.S.C. 704. 

E. What if EPA Should Require 

Additional Health Effects Testing on 

EDC? 

If EPA decides in the future that it 

requires additional health effects data 

on EDC, the Agency will initiate a 

separate action. 

IV. Other Impacts of the ECA for EDC 

The issuance of the ECA and Order 

under TSCA section 4 subjects the 

Companies that signed the ECA to 

export notification requirements under 

TSCA section 12(b)(1), as set forth at 40 

CFR part 707, subpart D, if they export 

or intend to export EDC. 

In the 12(b) proposal published in the 

Federal Register of December 23, 1997 

(62 FR 67038) (FRL-5762-8), EPA 

proposed to amend 40 CFR 799.5000 by 

adding EDC to the list of chemicals 

subject to testing consent orders. The 

listing of a chemical substance at 40 

CFR 799.5000 serves as notification to 

all persons who export or intend to 

export the chemical substance that: 

1. The chemical substance is the 

subject of an ECA and Order. 

2. EPA's export notification 

regulations at 40 CFR part 707, subpart 

D, apply to those exporters who have 

signed the ECA, as well as those 

exporters who have not signed the ECA 

(40 CFR 799.1g), 

When a final rule based on the 

proposed rule is published in the 

Federal Register, all persons who export 

or who intend to export EDC will be 

subject to export notification 

requirements. 

V. Paperwork Reduction Act 

The ECA and Order announced in this 

notice do not contain any information 

collection requirements that require 

additional approval by the Office of 

Management and Budget (OMB) under 

the Paperwork Reduction Act (PRA), 44 

U.S.C. 3501 et seq. The information 

collection requirements related to test 

rules and ECAs issued under TSCA 

section 4 have already been approved by 

OMB under OMB control number 2070-

0033 (EPA ICR No. 1139). The one-time 

public burden for this collection of 

information is estimated to be 

approximately 3,364 hours total (Ref. 

15). Under the PRA, "burden" means 

the total time, effort, or financial 

resources expended by persons to 

generate, maintain, retain, or disclose or 

provide information to or for a Federal 

agency. For this collection it includes 

the time needed to review instructions; 

complete and review the collection of 

information; and transmit or otherwise 

disclose the information. An agency 
Federal Register/Val. 68, No. 106/Tuesday, June 3, 2003/Notices 33129 

may not conduct or sponsor, and a 

person is not required to respond to a 

collection of information unless it 

displays a currently valid OMB control 

number. The OMB control number for 

EPA's regulations, after initial display in 

the final rule, are listed in 40 CFR part 

9. 

VI. References 

1. U.S. EPA, OPPT. I. Ethylene 

Dichloride (107-06-2). Pp 24-27 In: 

"TSCA Section 4 Findings for 21 

Hazardous Air Pollutants: A Supporting 

Document for Proposed Hazardous Air 

Pollutants (HAPs) Test Rule." (June 25, 

1996). 

2. The HAP Task Force. Letter from 

Peter E. Voytek to Charles M. Auer with 

attachment entitled: "Proposal for 

Pharmacokinetics Study of Ethylene 

Dichloride, November 22, 1996." 

(November 22, 1996). 

3. U.S. EPA. Letter from Charles M. 

Auer to Peter E. Voytek with attachment 

entitled: "Preliminary EPA Technical 

Analysis of Proposed Industry 

Pharmacokinetics (PK) Strategy for 

Ethylene Dichloride, June, 1997." (June 

26, 1997). 

4. The HAP Task Force. Letter from 

Peter E. Voytek to Charles M. Auer, U.S. 

EPA. (March 19, 1999). 

5. U.S. EPA. Letter from Charles M. 

Auer to Peter E. Voytek, HAP Task 

Force, Re: ECA Development of 

Ethylene Dichloride (EDC) (OPPTS 

42197C, with attachment: "EDC ECADRAFT, dated February, 2001." 

(February 13, 2001). 

6. Final Enforceable Consent 

Agreement for Ethylene Dichloride and 

Accompanying Testing Consent Order, 

signed by EPA on May 13, 2003. 

7. D'Souza, R.W., Francis, W.R., Bruce 

R.D., and Andersen, M.E. 

Physiologically based phamacokinetic 

model for ethylene dichloride and its 

application in risk assessment. Pp 286-

301, In: Pharmacokinetics in Risk 

Assessment. National Academy Press. 

Washington, D.C. (1987). 

8. D'Souza, R.W., Francis, W.R., and 

Andersen, M.E. Physiological model for 

tissue glutathione depletion and 

increased resynthesis after ethylene 

dichloride exposure. Journal of 

Pharmacology and Experimental 

Therapeutics 245(2):563-568. 1988. 

9. Daniel, F.B., Robinson, M., Olson, 

G.R., York, R.G., and Condie, L.W. Ten 

and ninety-day toxicity studies of 1,2-

dichloroethane in Sprague-Dawley rats. 

Drug and Chemical Toxicology 17: 463-

477. 1994. 

10. Alumot, E., Nachtomi, E., Mandel, 

E., Holstein, P., Bondi, A., and Herzberg, 

M. Tolerance and acceptable daily 

intake of chlorinated fumigants in the 

rat diet. Food, Cosmetics and 

Toxicology 14: 105-110. (1976). 

11. Rao, K.S., Murray, J.S., Deacon, 

M.M., John, J.A., Calhoun, L.L., and 

Young, J.T. Teratogenicity and 

reproduction studies in animals 

inhaling ethylene dichloride. Banbury 

Report 5: 149-166. (1980). 

12. Lane, R.W., Riddle, B.L., and 

Borzelleca, J.F. Effects of 1,2-

dichloroethane and 1,1,1-

trichloroethane in drinking water on 

reproduction and development in mice. 

Toxicology and Applied Pharmacology 

63: 409-421. 1982. 

13. Payan, J.P., Saillenfait, A.M., 

Bonnet, P., Fabry, J.P., Langonne, I., and 

Sabate J.P. Assessment of the 

developmental toxicity and placental 

transfer of the 1,2-dichloroethane in 

rats. Fundamental and Applied 

Toxicology 28: 187-198. 1995. 

14. Sherwood, R.L., O'Shea, W., 

Thomas, P.T., Ratajczak, H.V., and 

Aranyi, C. Effects of inhalation of 

ethylene dichloride on pulmonary 

defenses of mice and rats. Toxicology 

and Applied Pharmacology 91: 491-496. 

1987. 

15. U.S. EPA, OPPTS. "Burden 

Estimates for the Enforceable Consent 

Agreement for Ethylene Dichloride." 

(January 31, 2002). 

List of Subjects 

Environmental protection, Hazardous 

chemicals. 

Dated: May 13, 2003. 

Stephen Johnson, 

Assistant Administrator for Prevention, 

Pesticides and Toxic Substances. 

[FR Doc. 03-13721 Filed 6-2-03; 8:45am] 

BILLING CODE 6560-50--S 

ENVIRONMENTAL PROTECTION 

AGENCY 

[OPPT -2002-0067; FRL-7287-4] 

TSCA Section S(e); Notification of 

Substantial Risk; Policy Clarification 

and Reporting Guidance 

AGENCY: Environmental Protection 

Agency (EPA). 

ACTION: Notice. 

SUMMARY: EPA is hereby finalizing 

revisions to certain parts of EPA's 

"Statement of Interpretation and 

Enforcement Policy; Notification of 

Substantial Risk" (policy statement) 

issued March 16, 1978, concerning the 

reporting of "substantial risk" 

information pursuant to section 8(e) of 

the Toxic Substances Control Act 

(TSCA). EPA is making these revisions 

after having considered public 

comments that were solicited in 1993 

and 1995. Specifically, the revisions 

address the reporting of information on 

the release of chemical substances to, 

and the detection of chemical 

substances in, environmental media, the 

reporting deadline for written 

"substantial risk" information, and the 

circumstances under which certain 

information need not be reported to EPA 

undersection8(e) ofTSCA. EPA is 

republishing the policy statement in its 

entirety in this document, including 

both those portions of the policy 

statement that are revised and those 

portions that are not affected by any 

revisions. Since the policy statement 

was published in 1978, this 

republication is intended to ensure that 

a single reference source for the TSCA 

section 8(e) policy and guidance is 

easily available to the regulated 

community and other interested parties. 

FOR FURTHER INFORMATION CONTACT: For 

general information contact: Barbara 

Cunningham, Director, Environmental 

Assistance Division (7408M), Office of 

Pollution Prevention and Taxies, 

Environmental Protection Agency, 1200 

Pennsylvania Ave., NW., Washington, 

DC 20460-Q001; telephone number: 

(202) 554-1404; e-mail address: TSCAHotline@epa.gov. 

For technical information contact: 

Richard Hefter, Chief, High Production 

Volume Chemicals Branch, Risk 

Assessment Division, Office Pollution 

Prevention and Taxies, Environmental 

Protection Agency, 1200 Pennsylvania 

Ave., NW., Washington, DC 2046Q0001; telephone number: (202) 564-

7649; e-mail address: 

hefter .richard@epa.gov. 

SUPPLEMENTARY INFORMATION: 

I. General Information 

A. Does this Action Apply to Me? 

You may be potentially affected by 

this action if you manufacture, process, 

import, or distribute in commerce 

chemical substances and mixtures. 

Potentially affected entities may 

include, but are not limited to: • Chemical manufacturers, processors, 

and distributors (NAICS 325) • Petroleum refiners and distributors 

(NAICS 324) 

• Manufacturers of plastic parts and 

components (NAICS 325211) • Paints and coatings and adhesive 

manufacturing (NAICS 3255) • Cleaning compounds and similar 

products manufacturing (NAICS 3256) • Electronics manufacturing (NAICS 

334 and 335) • Automobiles manufacturing (NAICS 

3361) 
36196 Federal Register I Vol. 74, No. 139 I Wednesday, July 22, 2009 I Notices 

(Amendment) (Final Rule); in 40 CFR 

part 1042, subparts C, D, G and H; was 

approved 07/08/2009; OMB Number 

2060-0287; expires 07/31/2012. 

EPA ICR Number 1745.06; Criteria for 

Classification of Solid Waste Disposal 

facilities and Practices (Renewal); in 40 

CFR part 257, subpart B; was approved 

07/10/2009; OMB Number 2050-0154; 

expires 07/31/2012. 

EPA ICR Number 2027.04; NESHAP 

for Flexible Polyurethane Foam 

Fabrication (Renewal); in 40 CFR part 

63, subpart MMMMM; was approved 

07/10/2009; OMB Number 2060-0516; 

expires 07/31/2012. 

EPA ICR Number 2056.03; NESHAP 

for Miscellaneous Metal Parts and 

Products (Renewal); in 40 CFR part 63, 

subpart MMMM; was approved 07/10/ 

2009; OMB Number 2060-0486; expires 

07/31/2012. 

EPA ICR Number 1656.13; Risk 

Management Program Requirements and 

Petitions to Modify the List of Regulated 

Substances under Section 112(r) of the 

Clean Air Act (Renewal); in 40 CFR part 

68; was approved 07/10/2009; OMB 

Number 2050-0144; expires 07/31/2012. 

EPA ICR Number 2317.01; Generator 

Standards Applicable to Laboratories 

Owned by Eligible Academic Entities 

(Final Rule); in 40 CFR part 262, subpart 

K; was approved 07/10/2009; OMB 

Number 2050-0204; expires 07/31/2012. 

OMB Comments Filed 

EPA ICR Number 2355.01; 

Restructuring of Stationary Source 

Audit Program (Proposed Rule); OMB 

filed comment on 06/30/2009. 

EPA ICR Number 1975.06; NESHAP 

for Stationary Reciprocating Internal 

Combustion Engines (40 CFR part 63, 

subpart ZZZZ) (Proposed Rule); OMB 

filed comment on 07/04/2009. 

EPA ICR Number 2348.01; NESHAP 

for Paints and Allied Products 

Manufacturing (40 CFR part 63, subpart 

CCCCCCC) (Proposed Rule); OMB filed 

comment on 07107/2009. 

EPA ICR Number 2332.01; NESHAP 

for Aluminum, Copper, and Other 

Nonferrous Foundries (Proposed Rule); 

OMB filed comment on 07/10/2009. 

Dated: July 16, 2009. 

John Moses, 

Director, Collection Strategies Division. 

[FR Doc. £9-17397 Filed 7-21-09; 8:45am] 

BILLING CODE 6560-50-P 

ENVIRONMENTAL PROTECTION 

AGENCY 

[EPA-HO-OPPT -2003-001 0; FRL-8426-6] 

1 ,2-Ethylene Dichloride; Completion of 

EPA Program Review 

AGENCY: Environmental Protection 

Agency (EPA). 

ACTION: Notice. 

SUMMARY: EPA issued a testing consent 

order that incorporated an enforceable 

consent agreement (ECA) for 1,2-

Ethylene Dichloride (EDC) in June 2003, 

using authorities under section 4 of the 

Toxic Substances Control Act (TSCA). 

The companies subject to the ECA 

agreed to conduct toxicity testing in a 

tiered testing program that included 

development of pharmacokinetics and 

mechanistic data and a computational 

dosimetry model for route-to-route 

extrapolations. The testing program was 

designed to satisfy the toxicological data 

needs for EDC identified in a TSCA 

section 4 proposed test rule for a 

number of hazardous air pollutant 

chemicals. The modeling is intended to 

allow toxicological studies conducted 

using oral exposures to be interpreted so 

that they could also be used to predict 

the effects of inhalation exposures. This 

notice announces the completion of the 

program review component of the ECA 

for EDC. This notice also states EPA's 

findings and conclusion regarding the 

adequacy ofthe derived models to 

perfprm satisfactory route-to-route 

extrapolations, responds to comments 

on the Tier I Program Review Testing, 

and establishes revised deadlines for 

completion of Tier II testing and 

computational route-to-route dosimetry 

modeling for extrapolations listed under 

Tier II of the ECA for EDC. 

FOR FURTHER INFORMATION CONTACT: For 

general information contact: Colby 

Lintner, Regulatory Coordinator, 

Environmental Assistance Division 

(7408M), Office of Pollution Prevention 

and Taxies, Environmental Protection 

Agency, 1200 Pennsylvania Ave., NW., 

Washington, DC 20460-0001; telephone 

number: (202) 554-1404; e-mail address: 

TSCA-Hotline@epa.gov. 

For technical information contact: 

John Schaeffer, Chemical Control 

Division (7405M). Office Pollution 

Prevention and Taxies, Environmental 

Protection Agency, 1200 Pennsylvania 

Ave., NW., Washington, DC 20460-

0001; telephone number: (202) 564-

8173; e-mail address: 

schaeffer.john@epa .gov. 

SUPPLEMENTARY INFORMATION: 

I. General Information 

A. Does this Action Apply to Me? 

This action is directed to the public 

in general, and may be of particular 

interest to those persons who are or may 

be required to conduct testing of 

chemical substances under TSCA. Since 

other entities may also be interested, the 

Agency has not attempted to describe all 

the specific entities that may be affected 

by this action. If you have any questions 

regarding the applicability of this action 

to a particular entity, consult the 

technical person listed under FOR 

FURTHER INFORMATION CONTACT. 

B. How Can I Get Copies of this 

Document and Other Related 

Information? 

1. Docket. EPA has established a 

docket for this action under docket 

identification (ID) number EP A-HQOPPT-2003-0010. All documents in the 

docket are listed in the docket index 

available at http://www.regulations.gov. 

Although listed in the index, some 

information is not publicly available, 

e.g., Confidential Business Information 

(CBI) or other information whose 

disclosure is restricted by statute. 

Certain other material, such as 

copyrighted material, will be publicly 

available only in hard copy. Publicly 

available docket materials are available 

electronically at http:// 

www.regulations.gov, or, if only 

available in hard copy, at the OPPT 

Docket. The OPPT Docket is located in 

the EPA Docket Center (EPA/DC) at Rm. 

3334, EPA West Bldg., 1301 

Constitution Ave., NW., Washington, 

DC. The EPA/DC Public Reading Room 

hours of operation are 8:30 a.m. to 4:30 

p.m., Monday through Friday, excluding 

Federal holidays. The telephone number 

of the EPA/DC Public Reading Room is 

(202) 566-1744, and the telephone 

number for the OPPT Docket is (202) 

566-0280. Docket visitors are required 

to show photographic identification, 

pass through a metal detector, and sign 

the EPA visitor log. All visitor bags are 

processed through an X-ray machine 

and subject to search. Visitors will be 

provided an EPA/DC badge that must be 

visible at all times in the building and 

returned upon departure. 

2. EleCtronic access. You may access 

this Federal Register document 

electronically through the EPA Internet 

under the "Federal Register" listings at 

http://www.epa.gov/fedrgstr. 
Federal Register/Val. 74, No. 139/Wednesday, July 22, 2009/Notices 36197 

II. Background 

A. What is the EPA Program Review for 

EDC? 

In the Federal Register of September 

5, 2006 (71 FR 52329) (FRL-8088-3), 

EPA announced that it was conducting 

the program review component of the 

ECA for the EDC alternative testing 

program, and solicited public comment 

on data received under the Tier I 

Program Review testing segment of the 

ECA for EDC (CAS No. 107-06-2). 

Comments were to inform EPA's 

decision on whether or not additional 

data and/or model development are 

needed before Tier II testing and 

computational route-to-route dosimetry 

modeling extrapolations could proceed 

for the Tier II endpoints listed in the 

ECA for EDC. Details of the testing 

program for EDC are available in the 

ECA and in the Federal Register of June 

3, 2003 (68 FR 33125) (FRL-7300-6), in 

which EPA announced it had entered 

into an ECA and issued a testing 

consent order for EDC. The ECA for EDC 

was developed in response to EPA's 

request for ECA proposal for health 

effects testing of a number of hazardous 

air pollutants (HAPs or HAP chemicals), 

including EDC (see the proposed test 

rule in the Federal Register of June 26, 

1996 (61 FR 33177) (FRL-4869-1), and 

the proposed test rule, as amended, in 

the Federal Register of December 24, 

1997 (62 FR 67466) (FRL-5742-2); 

February 5, 1998 (63 FR 5915) (FRL5769-3); and April21, 1998 (63 FR 

19694) (FRL-5780-6)). The HAPs 

rulemaking proposed testing for health 

effects by the inhalation route of 

exposure. In the proposed rule, EPA 

also invited the submission of proposals 

that included pharmacokinetics studies 

and model development that would 

permit the extrapolation of testing 

administered by other exposure routes, 

such as the oral route, to predict for 

inhalation exposures. On November 22, 

1996, Dow Chemical Company, Vulcan 

Materials Company (no longer in 

existence), Occidental Chemical 

Corporation, Oxy Vinyls, LP, Georgia 

Gulf Corporation, Westlake Chemical 

Corporation, PPG Industries, Inc., and 

Formosa Plastics Corporation, U.S.A. 

(the "Companies"), under the auspices 

of the HAP Task Force, submitted a 

proposal for alternative testing of EDC 

that included physiologically based 

pharmacokinetics (PBPK) and model 

development to support route-to-route 

extrapolation of testing to be conducted 

under the ECA by the oral route. EPA 

considered this proposal sufficient to 

enter into ECA negotiations with the 

Companies and other interested parties 

(62 FR 66626; December 19, 1997) 

(FRL-5763-1). The ECA for EDC that 

resulted was announced in the Federal 

Register of June 3, 2003 (68 FR 33125). 

Since the route-to-route extrapolation of 

test results was a new approach, EPA 

and the Companies included a program 

review step within the testing program. 

The testing program consists of Tier I 

HAPs Testing; Tier I Program Review 

Testing; EPA Program Review; and Tier 

II Testing. 

Tier I HAPs Testing consisted of 

endpoint testing conducted by 

inhalation exposure for acute toxicity, 

with bronchoalveolar lavage (BALl and 

histopathology, and acute neurotoxicity. 

The Tier I Program Review Testing 

consisted of studies to develop PK/ 

MECH data, analyze glutathione 

metabolism, and perform model 

simulation. These studies were 

conducted to extend the computational 

dosimetry model of D'Souza eta!. (1987, 

1988; Refs. 1 and 2) to improve the 

model's application to the specific 

health effects endpoints for EDC listed 

in the ECA. The studies also enabled 

EPA to validate the model and verify the 

model's ability to adequately perform 

quantitative route-to-route 

extrapolations of dose response. Further 

description of the Tier I HAPs Testing 

and the Tier I Program Review Testing 

is provided in the Federal Register of 

September 5, 2006 (71 FR 52329). That 

notice, as well as the final study reports, 

can be accessed in the docket (EPAHQ-OPPT-2003-0010) as explained in 

Unit LB. 

As specified in the ECA, the EPA 

program review is required before the 

Tier II Testing segment is undertaken. In 

the Federal Register of September 5, 

2006 (71 FR 52329), EPA announced 

that it was conducting the program 

review component of the ECA for EDC, 

and solicited public comment on data 

received under the Tier I Program 

Review Testing segment of the ECA. 

Comments were to inform EPA's 

decision on whether or not additional 

data and/or model development were 

needed before Tier II Testing and 

computational route-to-route dosimetry 

modeling extrapolations can proceed for 

the Tier II endpoints listed in the ECA 

for EDC. 

B. What were the Public Comments on 

the Tier I Program Review Testing for 

EDC? 

EPA received two public comments in 

response to its solicitation for comments 

on the Tier I Program Review Testing. 

Comments from People for the Ethical 

Treatment of Animals (PET A) were on 

behalf of themselves and the following 

organizations: The Physicians' 

Committee for Responsible Medicine, 

the Humane Society of the United 

States, the Doris Day Animal League, 

and Earth Island Institute. PET A 

expressed support for the use of PBPK 

modeling to limit additional animal 

testing through the use of route-to-route 

extrapolation to existing studies. 

However, PETA also stated that they 

disagreed that additional Tier II tests 

(i.e., for reproductive effect or 

subchronic neurotoxicity) are needed; 

contending that existing studies for 

these effects are adequate. EPA 

disagrees, and its basis for requiring this 

additional testing is discussed in 

previous Federal Register documents, 

cited in Unit II.A. A second comment, 

from a private citizen, was focused on 

opposition to the manufacture of 

chlorine compounds in general, 

including EDC, and not the testing 

program. 

C. What are the Conclusions of the EPA 

Tier I Program Review Testing for EDC? 

The companies have completed the 

Tier I and Tier I Program Review testing 

segments of the ECA for EDC. The 

companies have also examined 

additional PBPK models and other 

available information in order to more 

fully update the model developed by 

D'Souza eta!., 1987, 1988; as specified 

in the ECA agreement. The results of 

this work, the updated model, and 

model simulations have been discussed 

with EPA (Refs. 3 through 6) and have 

also been recently published as a peerreviewed article in the scientific 

literature (Ref. 7). It is EPA's conclusion 

that the PBPK model developed by the 

test sponsors under the EDC ECA is 

acceptable for route-to-route 

extrapolations and that Tier II testing 

and extrapolation reporting can proceed 

as per the schedule set forth below (Ref. 

8). Specifically, EPA concludes that: 

1. The PK/MECH data report and Tier 

I toxicity studies have been conducted 

in accordance with the protocols and 

specifications as described in Appendix 

C of the ECA. 

2. The available study records are 

sufficient to allow an evaluation of the 

quality of the studies performed. 

3. The EDC PBPK model is 

appropriately chemical-specific, and 

suitably based on the current 

understanding of the kinetics of EDC. 

4. The species, dose level, exposure 

regimens, and vehicles used are relevant 

for the toxicity data that are the object 

of the Tier II extrapolations. 

5. The Tier I Program Review PK/ 

MECH data, along with additional data, 

show that periodicity was demonstrated 

and that the various data sets bearing on 

the issue of periodicity can be properly 
36198 Federal Register/Val. 74, No. 139/Wednesday, July 22, 2009/Notices 

interpreted and managed in the studies 

that support the model. 

6. Refinements of the model related to 

absorption, tissue distribution, and 

metabolism were accomplished, or 

suitably explained, including the role of 

extrahepatic metabolism as it impacts 

the model dose metrics and route-toroute extrapolation; appreciably 

improving prior PBPK models of ED C. 

It is EPA's decision that the HAP Task 

Force can proceed with the Tier II 

Testing under the schedule set forth in 

Table 1. of this Federal Register 

document. 

TABLE 1.-REOUIRED TESTING, TEST STANDARDS, AND REPORTING REQUIREMENTS FOR EDC 

Testing segment Required testing Test standard Deadline for final report' (months) 

Tier II testing andfor extrapolation reporting 

Subchronic toxicity route-to-route 

extrapolation of dose-response 

(oral Tier II testing to inhalation) 

of a study reported by Daniel, et 

al., (1994) 

EGA appendix C.2 and C.6 12 

Subchronic neurotoxicity (oral) 

1

40 CFR 799.9620 (as annotated in 18 

EGA appendix D.2) 

Subchronic neurotoxicity route-to- EGA appendix C.3 and C.6 21 

route extrapolation of dose-response (oral Tier II testing to inhalation) 

Reproductive toxicity (oral) 40 CFR 799.9380 (as annotated in 25 

EGA appendix D.3) 

Reproductive toxicity route-to-route EGA appendix C.4 and C.6 28 

extrapolation of dose-response 

(oral data to inhalation, including 

Tier II testing and extant studies 

reported by Alumot, et al.. 

(1976), Rao, et al., (1980), and 

Lane, et al., (1982)) 

'Number of months after the date of publication of this Federal Register document, which announces that EPA has concluded the EPA Program Review, when the final report is due. In addition, every 6 months from the effective date of the Order until the end of the EGA testing program, interim reports describing the status of all testing to be performed under this EGA must be submitted by the Companies to EPA. 

III. References 

1. D'Souza, R.W., Francis, W.R., Bruce 

R.D., and Andersen, M.E. 

Physiologically based pharmacokinetic 

model for ethylene dichloride and its 

application in risk assessment. P. 286-

301, In: Pharmacokinetics in Risk 

Assessment. National Academy Press. 

Washington, D.C. (1987). 

2. D'Souza, R.W., Francis, W.R., and 

Andersen, M.E. Physiological model for 

tissue glutathione depletion and 

increased resynthesis after ethylene 

dichloride exposure. Journal of 

Pharmacology and Experimental 

Therapeutics. 245(2):563-568. (1988). 

3. EPA, Office of Prevention, 

Pesticides and Toxic Substances, 

Chemical Control Division. Letter from 

Jim Willis, Director, CCD to Dr. Peter 

Voytek, HAP Task Force. RE: EPA Tier 

I Program Review for EDC. January 10, 

2007. 

4. EPA and HAP Task Force. 

Technical Consultation Meeting on 1, 2-

Ethylene Dichloride Program Review. 

February 12, 2007. 

5. HAP Task Force. Letter from Peter 

E. Voytek, Manager, HAP Task Force to 

Jim Willis, Director, Chemical Control 

Division, Office of Pollution Prevention 

and Taxies, with Enclosure: Response to 

Issues Raised in EPA's Tier 1 Data 

Evaluation Meeting. May 23, 2007. 

6. Sweeny, L. M. and Gargas, M.I. 

Physiologically based pharmacokinetic 

model development and simulations for 

ethylene dichloride (1,2-dichlorethane) 

in rats. Prepared by the Sapphire Group, 

Dayton Ohio for the HAP Task Force, 

Millwood, Virginia. Revised Draft 

Report. March 11,2009. 

7. Sweeny, L. M., Saghir, S. A., and 

Gargas, M.I. Physiologically based 

pharmacokinetic model development 

and simulations for ethylene dichloride 

(1 ,2-dichlorethane) in rats. Regulatory 

Toxicology and Pharmacology. 51:311-

323. (2008). 

8. EPA. Email from Rob Dewoskin, 

PhD, DABT, US EPA/NCEA (National 

Center for Environmental Assessment) 

to John Schaeffer. Review of Final report 

- EDC ECA Program Review Completion. 

April 22, 2009. 

List of Subjects 

Environmental protection, 1,2-

Ethylene Dichloride, EDC, Hazardous 

chemicals. 

Dated: July 10, 2009. 

Jim Willis, 

Director, Chemical Control Division, Office 

of Pollution Prevention and Taxies. 

[FR Doc. E9-17170 Filed 7-21-09; 8:45am] 

BILLING CODE 656()..5()-S 

ENVIRONMENTAL PROTECTION 

AGENCY 

[EPA-HQ-ORD; FRL-8930-3] 

Flexible Approaches to Environmental 

Measurement-The Evolution of the 

Performance Approach 

AGENCY: Environmental Protection 

Agency (EPA). 

ACTION; Notice. 

SUMMARY: Assuring the quality of 

environmental measurements is 

essential to the implementation of the 

Environmental Protection Agency's 

(EPA's or the Agency's) environmental 

programs, both regulatory and 

voluntary. In an October 6, 1997, Notice 

of Intent (FRL-5903-2), the Agency 

outlined a "Performance Based 

Measurement System (PBMS)" concept 
12!06/2011 12.08 FAX 202 457 8315 PATTOH BOGGS 

FROM :RS! FAX NO. :5408371602 

Novemberl7. 2011 

Addendum Modification 

To 

.Enfor~eable Consent Agreement 

For 

1,2-Ethylene Dichloride 

(CAS No. 107-06-2) 

Tier/ HAPs Tutittg 

. Docket No. OPPT-2003-0010 

141002/003 

Dec. e6 2811 01;32PM P2 

12/0S/2011 TIJE 12:03 (TX/RX HO 8030] lfl]002 
12/08/2011 12:08 FAX 202 457 8315 PATTON BOGGS f4l 003/0o::: 

FR0f'1 : F:S I FAX NO. :5408371602 D~c. 06 2011 01:34PM Pl 

We the undersigned agree to the change to the Enforceable .Consent Agreement for 1 ,2~Bthylene. 

bichloride (CAS No. 107-06-2) as descri.bed in the table below: 

Original Language (\n Table 1 ofthe ECA as amended ·OD July 22, 1.009 (74 FR 36196) nod 

by appro-val letter of May 12, 2010) 

Tier IJ Testing and/or Program Review 

ExtnJpolation Reporting 

Suhchronic neurotoldcity 

(o~al) 

0 ..... ~-:!--.,. .... ;. ......... ~~ ... ~-v-: .. J • .._ • .,....,_ ...u JO. .. n .. H.\.o 

extrapolation of dose-response 

(oral Tier IT testitig to inhalation) 

Reproductive> rvxicity 

(om) 

Reproductive toxicity rome-to route 

<-xtrapolation of doso-r~ponse 

(oral data to inhalation, including 

Tier 11 testing ~d e:ll:ttlrtt swdics 

reported by Alumot, c;l a!., 

( 1976}, lU!o, '!~ !''·· ( 1980), and 

Lane. ct at,, (1~~2)) 

Test Standard 

40 CFR 799.9620 

(as annowed In 

ECA appendix 0.2) 

40 CF'R 799.9380 

( ns m!llotatect in 

ECA at~pendlx 0.3) 

f.:cA npl'OIIdix C.4 and C.6 

Deadlldt for Fmal Rl!pori 

To be detennincd follmving. EPA 

review and IIPProval of COmbined 

.R~productlve Effocts/N~rotoxlcity 

protocol 

1 Jlft:Y montrtS IOllOWtng nnar .f6j;Orl .. - .. ···-"" " ........ . 

for Combined Reproductive Effilct$1 

Neurotoxic:ity SIUdy 

To be determined followiog EPA 

review and approval of Combined 

Reproductive Effeet!i/Neurototiciry 

proto eo! 

Th~ee months followini fino.! ~ort 

for Combined Reproductive Effc~ 

Neurotoxi.,ity sruciy 

Amended, !..anguj;\ge (clumge in Table 1· ofthe ECA ·as amended ou No-vember 17, 2011) 

Tiet< II Testing and/or Program Review 

Extrap<~latlon· ReportiDI! 

Reprooucti vc tox:lclcy/Neurotoxicity 

(oral) 

Rcpn)du.ctive tox:icity/Neuro>oxicity 

raute·to-rout.: extrapolation of dosc-rosponse 

(oral <1ata to inhalation, inoluding 

Ti"r U tesling and extant studies 

~pon:ed by Alumot, ct aL 

(1976), Rao, et<).l., (1980). lllld 

LMe, 1:t a!., (1 <;)~2)) 

'' 

Director 

Chemicai Control Division 

Test Standard Deadline for Final RJ!port 

Comblaed Reproductive: Effects 

/Neurotoxicity Protoool 

ECA appendix C.4. an(! C.6 

June: 28, 2013 

(New study plill'l due 

February 2~, 2012) 

Septernbvr 21,2013 

PeterE:YOYtek~.D: ~ ;yW 

bate 

Manager 

HAP Task Force 

12/08/2011 TUE 12:05 [D(!RX NO 8031] @001