Document V3GQ7VJaM1RNX6veZb5wNv4wq

T-7485: A REPRODUCTION STUDY WITH THE NORTHERN BOBWHITE FINAL REPORT WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454-116 3M ENVIRONMENTAL LAB PROJECT NO. E03-0583 FIFRA Guideline 71-4 OECD Guideline 206 AUTHORS: Sean P. Gallagher Raymond L. Van Hoven Joann B. Beavers Mark Jaber STUDY INITIATION DATE: September 5, 2003 STUDY COMPLETION DATE: May 27, 2005 Submitted to 3M Corporation Environmental Laboratory 935 Bush Avenue St. Paul, Minnesota 55106 Wildlife International, Ltd. 8598 Commerce Drive Easton, Maryland 21601 (410) 822-8600 Page 1 of 439 Contains No CBI W i l dlife International, Ltd. Project Number 454-1 16 GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT SPONSOR: 3M Corporation TITLE: T-7485: A Reproduction Study with the Northern Bobwhite WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454-1 16 STUDY COMPLETION: May 27,2005 The study was conducted in compliance with Good Laboratory Practice Standards as published by the U.S. Environmental Protection Agency, 40 CFR Part 792, 17 August 1989; OECD Principles of Good Laboratory Practice (ENVIMCICHEM (98) 17); and Japan MAFF, 11 Nohljan Notification No. 6283, Agricultural Production Bureau, 1 October 1999, with the following exceptions: Periodic analysis of avian feed and water for potential contaminants wens performed using a certified laboratory and standard U.S. EPA analytical methods. STUDY DIRECTOR: Sean P. Gallagher I Senior Biologist, Avian Toxicology SPONSOR'S REPRESENTATIVE: DATE Contains No CBI Contains No CBI Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 5 - TABLE OF CONTENTS Title Page ......................................................................................................................................... 1 Good Laboratory Practice Compliance Statement........................................................................... 2 Quality Assurance Statement ........................................................................................................... 3 Report Approval............................................................................................................................... 4 Table of Contents............................................................................................................................. 5 Summary .......................................................................................................................................... 9 Introduction.................................................................................................................................... 10 Objectives ...................................................................................................................................... 10 Experimental Design...................................................................................................................... 10 Materials and Methods................................................................................................................... 11 Test Substance......................................................................................................................... 11 Test Organisms........................................................................................................................ 12 Identification ........................................................................................................................... 12 Avian Feed and Water ............................................................................................................. 12 Diet Preparation....................................................................................................................... 13 Diet Sampling.......................................................................................................................... 13 Analytical Method for Diets.................................................................................................... 14 Analytical Method for Serum, Liver and Eggs........................................................................ 16 Study Phases............................................................................................................................ 16 Housing and Environmental Conditions ................................................................................. 16 Observations............................................................................................................................ 17 Necropsy and Tissue Collection.............................................................................................. 17 Adult Body Weight and Feed Consumption ........................................................................... 18 Egg Collection and Storage ..................................................................................................... 18 Candling and Incubation.......................................................................................................... 18 Hatching and Brooding ........................................................................................................... 19 Egg Shell Thickness Measurements and Egg Content Collection .......................................... 20 Statistical Analyses.................................................................................................................. 20 Results and Discussion .................................................................................................................. 21 Analytical Results for Diets..................................................................................................... 21 Mortalities ............................................................................................................................... 22 Clinical Observations .............................................................................................................. 23 Gross Necropsy ...................................................................................................................... 24 Adult Body Weight ................................................................................................................. 24 Adult Feed Consumption......................................................................................................... 25 Reproductive Results............................................................................................................... 26 Egg Shell Thickness ................................................................................................................ 27 Offspring Body Weights.......................................................................................................... 27 Liver Weights .......................................................................................................................... 27 Serum, Liver and Egg Analytical Results ............................................................................... 27 Histopathology ........................................................................................................................ 28 Conclusion ..................................................................................................................................... 29 References...................................................................................................................................... 30 Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 6 - TABLE OF CONTENTS PAGE 2 TABLES AND FIGURES Table 1. Mean Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with T-7485................................................................................. 31 Figure 1. Mean Adult Male Body Weight (g) from a Northern Bobwhite Reproduction Study with T-7485 ................................................................ 32 Figure 2. Mean Adult Female Body Weight (g) from a Northern Bobwhite Reproduction Study with T-7485 ................................................................ 33 Table 2. Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with T-7485 ................................................................ 34 Figure 3. Mean Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with T-7485 ................................................................ 35 Table 3. Summary of Reproductive Performance from a Northern Bobwhite Reproduction Study with T-7485 ................................................................ 36 Figure 4. Mean Reproductive Performance from a Northern Bobwhite Reproduction Study with T-7485 ................................................................ 37 Table 4. Summary of Reproductive Performance, Normalized as Percentages (%), from a Northern Bobwhite Reproduction Study with T-7485 ......... 38 Figure 5. Mean Reproductive Performance, Normalized as Percentages (%), from a Northern Bobwhite Reproduction Study with T-7485 ..................................... 39 Table 5. Mean Egg Shell Thickness Measurements (mm) from a Northern Bobwhite Reproduction Study with T-7485................................................................................. 40 Table 6. Mean Body Weight (g) of Hatchlings and 14-Day Old Survivors from a Northern Bobwhite Reproduction Study with T-7485 ..................................... 41 Table 7. Summary of Gross Pathological Observations from a Northern Bobwhite Reproduction Study with T-7485 ................................................................ 42 Table 8. Mean Liver Weights (g) from a Northern Bobwhite Reproduction Study with T-7485 ....................................................................................................... 43 Contains No CBI Wildlife International, Ltd. - 7 TABLE OF CONTENTS PAGE 3 APPENDICES Project Number 454-116 Appendix I. Appendix II. Appendix III. Appendix IV. Appendix V. Appendix VI. Appendix VII. Appendix VIII. Appendix IX. Appendix X. Appendix XI. Appendix XII. Appendix XIII. Appendix XIV. Appendix XV. Appendix XVI. Diet and Supplement Formulations................................................................... 44 Diet Preparation ................................................................................................ 46 Reproductive Parameters .................................................................................. 47 Individual Gross Pathological Observations from a Northern Bobwhite Reproduction Study with T-7485...................................... 50 Adult Body Weight (g) from a Northern Bobwhite Reproduction Study with T-7485...................................................................... 54 Feed Consumption (g/bird/day) from a Northern Bobwhite Reproduction Study with T-7485 ..................................................... 62 Reproductive Performance by Pen from a Northern Bobwhite Reproduction Study with T-7485 ..................................................... 66 Reproductive Performance by Week and Pen from a Northern Bobwhite Reproduction Study with T-7485.....................................100 Egg Shell Thickness Measurements (mm) per Pen by Week from a Northern Bobwhite Reproduction Study with T-7485 .........................131 Mean Hatchling Body Weight (g) per Pen by Week from a Northern Bobwhite Reproduction Study with T-7485 .........................135 Mean 14-Day Old Survivor Body Weight (g) per Pen by Week from a Northern Bobwhite Reproduction Study with T-7485 ...............139 Adult Liver Weights (g) from a Northern Bobwhite Reproduction Study with T-7485............................................................................................143 Offspring Liver Weights (g) from a Northern Bobwhite Reproduction Study with T-7485............................................................................................147 Histopathology Report .....................................................................................149 Certificate of Analysis .....................................................................................190 The Analysis of T-7485 in Avian Diet.............................................................193 Contains No CBI Wildlife International, Ltd. - 8 - Project Number 454-116 TABLE OF CONTENTS PAGE 3 APPENDICES (continued) Appendix XVII. Analytical Determination of T-7485 in Northern Bobwhite Tissues................ 209 Appendix XVIII. Diagram of Test Layout .................................................................................... 437 Appendix XIX. Changes to Study Protocol ................................................................................ 438 Appendix XX. Personnel Involved in Study ............................................................................. 439 Contains No CBI Wildlife International, Ltd. - 9 SUMMARY Project Number 454-116 STUDY: T-7485: A Reproduction Study with the Northern Bobwhite SPONSOR: 3M Corporation WILDLIFE INTERNATIONAL, LTD. PROJECT NUMBER 454-116 TEST DATES: Study Initiation - September 5, 2003 Experimental Start (OECD) - September 16, 2003 Experimental Start (EPA) - September 17, 2003 Photostimulation - November 5, 2003 First Eggs Set - December 11, 2003 Adult Termination - February 12, 2004 Biological Termination - April 6, 2004 Analytical Termination - December 15, 2004 Experimental Termination - December 15, 2004 TEST ANIMALS: Northern bobwhite (Colinus virginianus) AGE TEST ANIMALS: Approximately 18 weeks of age at the initiation of the test SOURCE TEST ANIMALS: K & L Quail 26 Thompson Flat Road Oroville, CA 95965 U.S.A. NOMINAL TEST CONCENTRATIONS: 0, 100, 300, and 900 ppm a.i. RESULTS: There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 100, 300 or 900 ppm a.i. test concentrations. The noobserved-effect concentration for northern bobwhite exposed to T-7485 in the diet during the study was 900 ppm a.i., the highest concentration tested. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 10 - INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. avian toxicology facility in Easton, Maryland 21601. The biological portion of the test was conducted from September 17, 2003 until April 6, 2004. Raw data generated at Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454-116 in archives located on the Wildlife International, Ltd. site. OBJECTIVES The objective of this study was to evaluate the effects upon the adult northern bobwhite (Colinus virginianus) of dietary exposure to T-7485 over a period of approximately 21 weeks. Effects on adult health, body weight, and feed consumption were evaluated. In addition, the effects of adult exposure to T-7485 on the number of eggs laid, fertility, embryo viability, hatchability, offspring survival, and egg shell thickness were evaluated. EXPERIMENTAL DESIGN Northern bobwhite (64 males and 64 females) were randomly distributed into one control group and three treatment groups. The test concentrations were selected in consultation with the Sponsor, based upon the results of a pilot reproduction study (Wildlife International, Ltd. Project Number 454-115. Group 1 2 3 4 T-7485 Treatment Groups Nominal Concentration (ppm a.i.) (Control) 0 100 300 900 Pens per Group 16 16 16 16 Birds per Pen Males Females 1 1 1 1 1 1 1 1 Each treatment and control group contained 16 pairs of birds with one male and one female per pen. Three treatment groups were fed diets containing either 100, 300 or 900 ppm a.i. of T-7485 for approximately 21 weeks. The control group was fed diet comparable to the treatment groups, but without the addition of the test substance. Contains No CBI Wildlife International, Ltd. - 11 - Project Number 454-116 All adult birds were observed daily throughout the test for signs of toxicity or abnormal behavior. Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8, and at adult termination and feed consumption was measured weekly throughout the test. At the beginning of Week 8, the photoperiod was increased to induce egg production. Following the start of egg production, eggs were set weekly for incubation. Weekly, eggs were selected by indiscriminate draw for egg shell thickness measurement and all remaining eggs were candled prior to incubation to detect egg shell cracks or abnormal eggs. Eggs were also candled twice during incubation to detect infertile eggs or embryo mortality. On Day 21 of incubation, the eggs were placed in a hatcher and allowed to hatch. Once hatching was completed, hatchlings were removed from the hatcher and the group body weight of the hatchlings by pen was determined. At 14 days of age, the average body weight by parental pen of all surviving offspring was determined. Upon completion of the test, statistical analyses were performed to determine statistically significant differences between groups. Egg contents from those eggs selected for eggshell thickness measurements were also collected for potential analysis. Samples of sera and liver were collected from all adults surviving until test termination and from selected offspring for potential analysis. Additionally, samples of liver, kidney and gonad were also collected from adults and selected offspring for histopathological examination. MATERIALS AND METHODS The study was conducted according to the procedures outlined in the protocol, "T-7485 A Reproduction Study with the Northern Bobwhite". The protocol was based on procedures outlined in the Environmental Protection Agency's Registration Guidelines Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, Subsection 71-4; OECD Guideline 206; and the ASTM "Standard Practice for Conducting Reproductive Studies with Avian Species" (1,2,3). Test Substance The test substance, T-7485, was received from 3M Corporation on July 18, 2003 and was assigned Wildlife International, Ltd. identification number 6407 upon receipt. The test substance was a white crystalline solid and was identified as: PFBS; T-7485; L-7038; Lot # 5; CAS # 29420-49-3. The test material had a reported purity of 98.2% and an expiration date of August 31, 2008 (Appendix XV). The test substance was held under frozen conditions in locked storage at the Wildlife International, Ltd. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 12 - facilities in Easton, Maryland. Concentrations of the test substance in the diet were adjusted to 100% active ingredient. Therefore, dietary concentrations are expressed as parts per million active ingredient (ppm a.i.) in the diet. Test Organisms Seventy-eight pairs of pen-reared northern bobwhite were purchased from K & L Quail, 26 Thompson Flat Road, Oroville, CA 95965, U.S.A. At the start of acclimation, the bobwhite were apparently healthy and phenotypically indistinguishable from wild type. The birds were from the same hatch, approaching their first breeding season and had not been used in any previous testing. At the start of acclimation, a random number generating function in a spreadsheet program was used to randomize pen assignment for each bird. Immediately prior to test initiation, all potential study birds were examined for physical injuries and general health. Birds that did not appear healthy, either due to injury or inability to acclimate to laboratory conditions, or were outside the weight range for the test, were excluded from the study. All birds were approximately 18 weeks of age at test initiation (first day of exposure to test diet) and ranged in weight from 171 to 220 grams at test initiation. Sex of the birds was determined by a visual examination of the plumage. Identification Adult birds were identified by individual leg bands, each pen was identified with a unique number, and groups of pens were identified by project number and concentration. All eggs laid during the study were marked with the pen number using a permanent ink marking pen for identification. Hatchlings were identified by leg bands so that they could be traced to their parental pen of origin. Avian Feed and Water All adult birds and their offspring were given feed and water ad libitum during acclimation and testing. The basal diet fed to both adults and offspring was formulated to Wildlife International, Ltd. specifications by Agway Inc. (Appendix I, Table 1). The basal ration contained at least 27% protein and 2.5% fat, and no more than 3.8% fiber. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 13 - The basal diet contained approximately 1.1% calcium, derived from feedstuffs and the 0.9% limestone used in the formulation of the basal diet by Agway. While this level of calcium is sufficient for growth and maintenance rations, additional calcium is required in the ration of breeding birds for egg shell formation. Therefore, an additional 5% (w/w) of limestone (approximately 38.5% Ca) was added to the basal diet for the adults. This raised the calcium level in the diet for the breeding birds to approximately 3%, slightly above the minimum recommended for quail (2.3%) (4). Offspring received basal diet without test substance and without the addition of 5% supplemental limestone. Water was supplied by the town of Easton public water supply. All offspring received a watersoluble vitamin and electrolyte mix in their water (Appendix I, Table 2). Neither the adults nor offspring received any form of medication in their feed during the test. Feed and water were analyzed periodically in accordance with Wildlife International, Ltd. Standard Operating Procedures. Diet Preparation Test diets were prepared by mixing T-7485 into a premix that was used for weekly preparation of the final diet. Control diet and each of the three treated diets were prepared weekly beginning on September 17, 2003 and presented to the birds on Wednesday of each week. Dietary concentrations were adjusted for purity of the test substance and are presented as parts per million active ingredient (ppm a.i.). Details of the weekly preparation of test and control diets are shown in Appendix II. Diet Sampling Homogeneity of the test substance in the diet was evaluated by collecting six samples from each of the treated diets and one sample from the control diet on Day 0 of Week 1. Samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. Control and treatment group diet samples were also collected from the feed troughs on Day 7 of Week 1 to assess stability of the test substance under actual test conditions. Additionally, a single sample was collected from the control and two samples were collected from each treatment group diet during Weeks 4, 8, 12, 16 and 20 of the test to measure/verify test concentrations. The diet samples were transferred immediately to the Wildlife International, Ltd. analytical chemistry facility, where they were stored frozen prior to analysis. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 14 - Analytical Method for Diets A liquid chromatography mass spectrometry (LC/MS/MS) based method was developed and verified at Wildlife International, Ltd. for the analysis of the T-7485 in avian diet (5). The verified analytical method consisted of sample weighing, extraction with methanol, vacuum filtration, micro filtration, and dilution into the calibration range of the LC/MS/MS methodology. The method was slightly modified in the present test to expedite sample processing and to reduce solvent volume. The change was centrifugation of an aliquot of each sample extract instead of vacuum filtration of the entire extract volume. Dilutions into the calibration range of the LC/MS/MS methodology were performed with methanol (intermediate dilutions, if necessary) and/or a solution of 50% methanol and 50% NANOpure or HPLC-grade reagent water (v/v). Samples were then analyzed by direct injection. A method flowchart is provided in Appendix XVI, Figure 1. Concentrations of T-7485 in extracts of the samples were determined by reverse-phase high performance liquid chromatography using a Hewlett-Packard Model 1100 High Performance Liquid Chromatograph (HPLC) with a Perkin-Elmer SCIEX API 3000LC Mass Spectrometer equipped with a Perkin-Elmer SCIEX TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone PRISM RP column (50 mm 2.0 mm, 3-m particle size) fitted with a Keystone Javelin C18 guard column (20 mm x 2.0 mm I.D.). The instrument parameters are summarized in Appendix XVI, Table 1. All primary and secondary stock preparations were adjusted for the purity of the test substance (98.2%). A 1.00-mg a.i./mL primary stock solution of T-7485 in methanol was prepared by weighing 0.1018 g of the test substance and bringing to a final volume of 100 milliliters with methanol. Secondary stock solutions of T-7485 in methanol (100- and 10.0-mg a.i./L) were prepared by serial volumetric dilution of the primary stock. Calibration standards were prepared in a solution of 50% methanol and 50% HPLC-grade reagent water (v/v) by appropriate dilutions of the 100- or 10.0-mg a.i./L stock solutions of T-7485 in methanol. Two sets of calibration standards of T-7485 were prepared. The first set, ranging in concentration from 0.0500 to 0.500 mg a.i./L, was analyzed with the Week 1, Days 0 and 7 sample sets. The second set, ranging in concentration from 0.00500 to 0.0250 mg a.i./L, was analyzed with all subsequent sample sets. The second set of lower-level calibration standards was selected for use in the remainder of the Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 15 - study to allow for greater sample dilution. The larger dilution minimized matrix loading into the LC/MS/MS system. A calibration standard series was injected at the beginning and at the end of each analytical sequence, and one standard was injected, at a minimum, after every five samples. Linear regression equations were generated using peak area responses versus the respective concentrations of the calibration standards. A representative calibration curve is presented in Appendix XVI, Figure 2. The concentration of T-7485 in the samples was determined by substituting the peak area responses of the samples into the applicable linear regression equation. Representative ion chromatograms of low and high-level calibration standards from the 0.00500 to 0.0250 mg a.i./L calibration standard set are presented in Appendix XVI, Figures 3 and 4, respectively. Examples of equations used in calculations are presented in Appendix XVI, Table 2. The instrument limit of detection (LOD) was set based upon the injection volume (5.00 or 10.0 L) and the lowest standard concentration (0.0500 or 0.00500 mg a.i./L) used in the study. The LOD was calculated as 0.250 ng on-column for Week 1, Days 0 and 7 sample analyses and was calculated as 0.0500 ng on-column for all subsequent sample sets. The method limit of quantitation (LOQ) for these analyses was set at 50 ppm a.i. based upon the lowest matrix fortification level analyzed concurrently with the samples. For Week 1, Days 0 and 7 analyses, the 0.0500 mg a.i./L low-level standard was equivalent to a calculated value of 20 ppm a.i. T-7485 in the matrix blank extract. For all subsequent sample intervals, the 0.00500 mg a.i./L low-level standard was equivalent to a calculated value of 25 ppm a.i. T-7485 in the matrix blank extract. Measured values greater than or equal to the ppm a.i. equivalent were reported. Along with the sample analyses, seven matrix blanks were analyzed to determine possible interferences. No interferences were observed at or above the ppm a.i. equivalent of the lowest standard during the sample analyses (Appendix XVI, Table 3). A representative ion chromatogram of a matrix blank sample is presented in Appendix XVI, Figure 5. Avian diet samples were fortified with T-7485 using the dry-mix technique at 50.0, 200, and 1000 ppm a.i. (seven fortifications at each level) and analyzed concurrently with the samples to determine mean procedural recoveries. The method yielded mean procedural recoveries of 95%, 91%, 112%, 109%, 105%, 106% and 103%. These values correspond to each sample set analyzed during the definitive study (Appendix XVI, Table 3). Sample measured concentrations were not corrected for the mean procedural Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 16 - recoveries. A representative ion chromatogram of a matrix fortification sample is presented in Appendix XVI, Figure 6. Analytical Method for Serum, Liver and Eggs Preparation and analysis of serum, liver and egg samples were conducted by the 3M Environmental Laboratory using methods validated by 3M Environmental Laboratory. In summary, the samples were weighed, homogenized in acetonitrile, extracted using a Zymark Rapid Trace Automated SPE System, and analyzed using liquid chromatography/mass spectrometry (LC/MS). The full description of the analytical method is presented in Appendix XVII. Study Phases The primary phases of the study and their approximate durations were: 1. Acclimation - Approximately 5 weeks. 2. Pre-photostimulation - 8 weeks. 3. Pre-egg laying (with photostimulation) - Approximately 3 weeks. 4. Egg laying - Approximately 10 weeks. 5. Post-adult termination (final incubation, hatching, and 14-day offspring rearing period) - 6 weeks. Housing and Environmental Conditions Housing and husbandry practices were conducted so as to adhere to the guidelines established by the National Research Council (6). The adult birds were housed indoors in batteries of pens manufactured by Georgia Quail Farm Manufacturing (GQFM Model No. 0330), measuring approximately 27 X 51 cm. The pens had sloping floors that resulted in ceiling height ranging from 20 to 25 cm. The pens were constructed of galvanized wire mesh and galvanized sheeting. A diagram of the test layout is presented in Appendix XVIII. Each pen was equipped with feed and water troughs. Weekly, sufficient feed for the feeding period was placed in the trough for each pen and presented to the birds. During the feeding period additional feed was weighed and added to the troughs as needed. Water troughs were changed and water added as necessary to provide potable water (generally every 2-3 days). Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 17 - Only birds associated with this study were maintained in the study room in order to avoid excessive disturbances. The average temperature in the adult northern bobwhite study room during the course of the test was 23.2 1.0C (SD) with an average relative humidity of 34 17% (SD). The air handling system in the study room was designed to vent up to 15 room air volumes every hour and replace them with fresh air. The photoperiod in the adult northern bobwhite room was maintained by a time clock. The photoperiod during acclimation and the first seven weeks of the test was eight hours of light per day. The photoperiod was increased to 17 hours of light per day at the beginning of Week 8 to induce egg laying and was maintained at that length until the adult birds were euthanized. Throughout the test, the birds received a mean of approximately 203 lux (~ 19 ft. candles) of illumination provided by fluorescent lights that closely approximated noon-day sunlight. Observations The test birds were acclimated to the facilities and study pens for approximately five weeks prior to initiation of the test. During acclimation, all birds were observed daily. Birds exhibiting abnormal behavior or debilitating physical injuries were not used for the test. During the study, all adult birds were observed daily for signs of toxicity or abnormal behavior. Additionally, all offspring were observed daily from hatching until euthanasia. A record was maintained of all mortalities and clinical observations. Necropsy and Tissue Collection Adult birds that died or were euthanized during the course of the study were subjected to a gross necropsy. At the conclusion of the exposure period, all surviving adult birds were euthanized with carbon dioxide gas. Prior to euthanasia, blood samples were drawn from birds. The blood was separated and serum was collected for potential analysis. Livers were also weighed and collected. A portion of liver was collected for histopathological examination and the remaining liver was collected for potential analysis. Samples of kidney and gonad were also collected for histopathological examination. All histopathological samples were fixed in 10% buffered formalin and shipped to EPL in Sterling, Virginia for histopathology. Additionally, similar samples were collected from a single indiscriminately selected offspring from each pen (if available) at 16 days of age. Offspring were sampled from the last lot of offspring produced. Following sample collection, all carcasses were disposed of by incineration. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 18 - Adult Body Weight and Feed Consumption Adult body weights were measured at test initiation, on Weeks 2, 4, 6, 8, and at adult termination. Body weights were not measured during egg laying because of the possible adverse effects handling may have on egg production. Feed consumption for each pen was measured weekly throughout the test. Feed consumption was determined by weighing the freshly filled feeder on Day 0, recording the amount of any additional diet added during the week, and weighing the feeder and remaining feed at the end of the feeding period (Day 7). An attempt was made to minimize feed wastage by the birds by using externally mounted feeders designed with a "feed-saver" lip. The amount of feed wasted by the birds was not quantified, since the wasted feed was normally scattered and mixed with water and excreta. Therefore, feed consumption is presented as an estimate of total feed consumption. Egg Collection and Storage Eggs were collected daily from all pens, when available. The eggs were stored in a cold room until incubation. The cold room was maintained at a mean temperature of 13 0C (SD) with a mean relative humidity of approximately 84 6% (SD). Groups of eggs were identified by an alphabetic lot code. All eggs laid in a weekly interval were considered as one lot. Candling and Incubation At the end of the weekly interval, all eggs were removed from the cold room, counted and eggs selected by indiscriminate draw for egg shell thickness measurement. The remaining eggs were candled with a Speed King (Model No. 32) egg-candling lamp to detect egg shell cracks or abnormal eggs. Cracked or abnormal eggs were recorded and discarded. All eggs to be incubated were fumigated with formaldehyde gas in an airtight cabinet with a circulating fan for approximately two hours, to reduce the possibility of pathogen contamination prior to incubation. Formaldehyde gas was generated by combining 20 g of potassium permanganate and 19 ml of 37% commercial grade formalin in a porcelain bowl at the base of the airtight cabinet. All eggs not discarded or used for egg shell thickness measurements were placed in a NatureForm Incubator (Model No. NMC 4000). In the incubator the temperature was maintained at an average 37.4 0.0C (SD) with an average relative humidity of 54 0%. The incubator was equipped with a pulsator Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 19 - fan and blades that produced a mild breathing air movement designed to eliminate intracabinet temperature and humidity variation during incubation. In order to prevent adhesion of the embryo to the shell membrane, the incubator was also equipped with an automatic egg rotation device, designed to rotate the eggs from 50 off of vertical in one direction to 50 off of vertical in the opposite direction (total arc of rotation was 100) every two hours through Day 21 of incubation. Eggs were candled on Day 11 or 12 of incubation to determine embryo viability and on Day 21 to determine embryo survival. Hatching and Brooding On Day 21 of incubation, the eggs were placed in a Petersime Hatcher (Model No. S6H) and allowed to hatch. Pedigree baskets constructed of galvanized steel wire mesh were used to keep hatchlings separated by parental pen of origin. Eggs were not rotated in the hatcher. The average temperature in the hatching compartment was 37.2 0.0 C (SD), and the average wet bulb temperature was raised to 33.3 0.0 C (SD) (relative humidity of approximately 77%). All hatchlings, unhatched eggs, and egg shells were removed from the hatcher on Day 25 or 26 of incubation. The group body weight of the surviving hatchlings by pen was determined. Hatchlings were leg banded for identification by pen of origin and then routinely housed according to the appropriate parental concentration grouping in brooding pens until 14 days of age. The hatchlings were fed untreated diet without the addition of 5% supplemental limestone. At 14 days of age, the average body weight by parental pen of all surviving chicks was determined. The chicks were euthanized with carbon dioxide and disposed of by incineration. While the final lot of offspring (Lot J) was weighed at 14 days of age, chicks from this lot were maintained alive until 16 days of age for tissue sampling. Lot J offspring were disposed of by incineration following sampling. Hatchlings were housed in batteries of brooding pens manufactured by Beacon Steel Company (Model B735Q). Each pen measured approximately 72 X 90 X 23 cm high. The external walls and ceilings of each pen were constructed of galvanized wire mesh and galvanized sheeting. Floors were of galvanized wire mesh. Thermostats in the brooding compartment of each pen were set to maintain a temperature of approximately 38 C from the time of hatching until the birds were 14 days of age. The average ambient room temperature was 25.5 1.1C (SD) with an average relative humidity of 15 6% (SD). The photoperiod for the hatchlings was maintained by a time clock at 16 hours of light per day. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 20 - Egg Shell Thickness Measurements and Egg Content Collection Weekly throughout the egg laying period, one egg was collected, when available, from each of the odd numbered pens during odd numbered weeks (1,3,5, etc.) and from each of the even numbered pens during the even numbered weeks (2,4,6, etc.). Prior to being opened, eggs were rinsed with water to remove any excreta or feed adhering to the shell. The eggs were opened at the waist, the contents were removed and placed in a Whirl Pak and stored frozen for potential analysis. The empty shells were thoroughly rinsed with water, and allowed to air dry for at least one week at room temperature. The average thickness of the dried shell plus the membrane was determined by measuring five points around the waist of the egg using a micrometer. Measurements were made to the nearest 0.002 mm. Statistical Analyses Upon completion of the test, an analysis of variance (ANOVA) was performed to determine statistically significant differences between groups. Dunnett's multiple comparison procedure (7,8) was used to compare the three treatment means with the control group mean and assess the statistical significance of the observed differences. Sample units were the individual pens within each experimental group, except adult body weights where the sample unit was the individual bird. Percentage data were examined using Dunnett's method following arcsine square root transformation (see Appendix III for reproductive parameters). Each of the following parameters was analyzed statistically: 1. Adult Body Weight - Individual body weight was measured at test initiation, Weeks 2, 4, 6, 8 and at termination. Statistical comparisons were made between the control group and each treatment group at each weighing interval by sex. 2. Adult Feed Consumption - Feed consumption expressed as grams of feed per bird per day was examined by pen weekly during the test. Statistical comparisons were made between the control and each treatment group. 3. Eggs Laid of Maximum Laid - The number of eggs laid per female divided by the largest number of eggs laid by any one female. This transformation was used to convert the number of eggs laid to a percentile value less than or equal to 100. 4. Eggs Cracked of Eggs Laid - The number of eggs determined by candling to be cracked divided by the number of eggs laid, per pen. 5. Viable Embryos of Eggs Set - The number of viable embryos at the Day 10-12 candling was divided by the number of eggs set, per pen. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 21 - 6. Live 3-Week Embryos of Viable Embryos - The number of live embryos at the Day 21 candling was divided by the number of viable embryos, per pen. 7. Hatchlings of 3-Week Embryos - The number of hatchlings removed from the hatcher was divided by the number of live 3-week embryos, per pen. 8. 14-Day Old Survivors of Hatchlings - The number of 14-day old survivors was divided by the number of hatchlings, per pen. 9. Hatchlings of Eggs Set - The number of hatchlings was divided by the number of eggs set, per pen. 10. 14-Day Old Survivors of Eggs Set - The number of 14-day old survivors was divided by the number of eggs set, per pen. 11. Hatchlings of Maximum Set - The number of hatchlings per female divided by the largest number of eggs set from any one female. This transformation was used to convert the number of hatchlings to a percentile value equal to or less than 100. 12. 14-Day Old Survivors of Maximum Set - The number of 14-day old survivors per pen divided by the largest number of eggs set. 13. Egg Shell Thickness - The average egg shell thickness of indiscriminately selected eggs per pen was measured. 14. Offspring's Body Weight - The group body weights of surviving hatchlings and 14-day old survivors were measured by parental pen group. 15. Liver Weight - Liver weights were measured for adults and selected offspring. Statistical comparisons were made between the control group and each treatment group by age and sex. RESULTS AND DISCUSSION Mature northern bobwhite received T-7485 at nominal dietary concentrations of 100, 300 or 900 ppm a.i. for 21 weeks. A control group was maintained concurrently with the treatment groups. Analytical Results for Diets Diet samples were collected on Week 1, Day 0 of the test from the 100, 300 and 900 ppm a.i. test levels to evaluate the homogeneity of the test substance in the diet. The mean and standard deviation for the 100, 300 and 900 ppm a.i. test levels were 105 5.13 ppm a.i., 299 12.0 ppm a.i. and 880 16.0 ppm a.i., respectively. The corresponding coefficients of variation were 4.91%, 4.00% and 1.82% (Appendix XVI, Table 4). The mean measured T-7485 concentrations from the 100, 300 and 900 ppm a.i. test levels were 105%, 100%, and 98%, respectively, of the nominal concentrations. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 22 - Diet samples collected on Weeks 4, 8, 12, 16 and 20, Day 0 of the test from the 100, 300 and 900 ppm a.i. test levels were analyzed to verify the concentration of the test substance in the diet. The mean and standard deviation for the 100, 300 and 900 ppm a.i. test concentrations were 107 7.74 ppm a.i., 314 5.97 ppm a.i. and 961 36.8 ppm a.i., respectively. The corresponding coefficients of variation were 7.21%, 1.90% and 3.83% (Appendix XVI, Table 5). The mean measured T-7485 concentrations from the 100, 300 and 900 ppm a.i. test levels were 107%, 105% and 107%, respectively, of the nominal concentrations. A typical ion chromatogram of a test sample is shown in Appendix XVI, Figure 7. Analysis of diet samples collected from feeders after being held at ambient temperature for 7 days averaged 96%, 105%, and 112% of the Day 0 values for the 100, 300 and 900 ppm a.i. test concentrations, respectively (Appendix XVI, Table 6). Mortalities There were no treatment-related mortalities in the 100, 300 or 900 ppm a.i. treatment groups during the course of the test. There were three adult mortalities that were considered to be incidental to treatment that occurred during the test, one in the control group and one each in the 100 and 300 ppm a.i. treatment groups. The female in Pen 1608 of the control group was found dead during Week 12 of the test. Gross necropsy revealed feather loss and an open lesion on the head and neck. The breast muscle, spleen, liver and kidneys were noted to be pale and the ovary was developing. The male in Pen 1617 of the 100 ppm a.i. treatment group was found dead at the beginning of Week 6 of the test, having exhibited no prior signs of toxicity. Gross necropsy revealed bruising on the cranium and intracranial bleeding between the orbits. The final mortality was the female in Pen 1639 of the 300 ppm a.i. treatment group that was euthanized at the end of Week 14 due to its moribund condition. Four days prior to being euthanized, this female had been noted with a neck lesion. Gross necropsy revealed a large head and neck lesion, exposing the cranium and neck muscle, with necrotic margins. The bird was emaciated, with a loss of muscle mass and prominent keel. The spleen was noted to be large and pale, the kidneys were pale, the cecal contents were firm and the ovary was regressing. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 23 - No other mortalities occurred during the course of the study. Due to the nature of the lesions observed at necropsy, none of the mortalities that occurred were considered to be related to treatment. Clinical Observations No overt signs of toxicity were observed at any of the concentrations tested. Incidental clinical observations noted during the test included those that normally are associated with injuries and penwear, and were noted for the control group and all treatment groups. Such observations included foot, leg, and head lesions, feather loss and ventral head curl. In addition, lameness, loss of coordination, ruffled appearance, wing droop and lethargy also were observed and were generally associated with incidental injuries. While some incidental observations were persistent once noted (e.g. feather loss), most were transient in nature and subsided once an injury had healed. In the control group, incidental clinical observations were noted for eight individuals during the course of the test. Three birds were noted with feather loss, four birds were noted with foot lesions and associated lameness, two birds were noted with either bruising or a lesion on the head and one bird was noted with a lesion and bruising on the upper back. Additionally, one bird was also noted with right wing droop, one bird was noted with a growth above the left eye, and one bird was noted with a transient loss of coordination. Except for incidental findings, all birds in the control group were normal in appearance and behavior throughout the study. In the 100 ppm a.i. treatment group, incidental clinical observations were noted for five individuals during the course of the test. Two birds were noted with feather loss, one bird was noted with foot lesions and associated lameness and ruffled appearance, and two birds were noted with either bruising and/or a lesion on the head. Additionally, one bird was also noted with twisted primary feathers on the left wing, presumably due to a wing injury. Except for incidental findings, all birds in the 100 ppm a.i. treatment group were normal in appearance and behavior throughout the study. In the 300 ppm a.i. treatment group, incidental clinical observations were noted for seven individuals during the course of the test. One bird was noted with feather loss and four birds were noted with foot lesions, one of which was also noted with associated lameness. One bird was noted with swelling on the head, as well as a head lesion. Additionally, one bird was noted with a ventral head curl, a condition that is thought to be caused by a neck injury. One female in the 300 ppm a.i. treatment group Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 24 - was noted with head and neck lesions that resulted in her being euthanized. Prior to her being euthanized in a moribund condition, this bird exhibited depression, a reduced reaction to external stimuli (sound and movement), a ruffled appearance and lethargy. Except for incidental findings, all birds in the 300 ppm a.i. treatment group were normal in appearance and behavior throughout the study. In the 900 ppm a.i. treatment group, incidental clinical observations were noted for 11 individuals during the course of the test. Five birds were noted with feather loss and six birds were noted with foot lesions, three of which were also noted with associated lameness. One hen with foot lesions was also noted with a ruffled appearance and lethargy that were attributed to the injury. One bird was noted with bruising on the back and another bird was noted with bruising on the head and neck. Lesions were noted on the back of one bird and on the head of another bird. Two birds were noted with lesions around one eye, one of these birds was also noted with a ruffled appearance that was attributed to the eye lesion. Except for incidental findings, all birds in the 900 ppm a.i. treatment group were normal in appearance and behavior throughout the study. Gross Necropsy All surviving adults were subjected to gross necropsy following adult termination. All findings observed were considered unrelated to treatment. Necropsy findings are reported in Table 7 and Appendix IV. Adult Body Weight There were no apparent treatment-related effects upon adult body weight at any of the concentrations tested. No statistically significant differences between the control group and the 100, 300 or 900 ppm a.i. treatment groups were observed at any of the body weight intervals. Mean body weight measurements are presented in Table 1, and Figures 1 and 2. Individual body weight measurements are presented in Appendix V. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 25 - Adult Feed Consumption There were no apparent treatment-related effects upon feed consumption at the 100, 300, and 900 ppm a.i. test concentrations. No statistically significant differences between the control group and the 100 and 900 ppm a.i. treatment groups were observed at any of the feed consumption intervals. When compared to the control group, there was a slight, but statistically significant (p < 0.01), increase in feed consumption at the 300 ppm a.i. test concentration during Week 16 of the test. However, this difference was slight and was neither consistent nor concentration responsive. Since the difference represented an increase in the consumption of the treated diet, it was not considered to be treatment related. Mean feed consumption measurements are shown in Table 2 and Figure 3. Feed consumption measurements by pen are presented in Appendix VI. Estimated test substance intakes, daily dietary dose, for northern bobwhite were calculated by treatment group for the pre-egg production period, the egg production period and the overall adult period using the following formula: Daily Dietary Dose = Test Concentration (mg a.i./kg) x Daily Feed Consumption (g/bird/day) (mg a.i./kg body weight/day) Body Weight (g/bird) The mean body weight value is the mean of both male and female body weights. For the pre-egg production interval the body weights were averaged over Weeks 0, 2, 4, 6 and 8. For the egg-production interval body weights were averaged over Weeks 8 and 20 (adult termination). The accuracy of the estimated mean daily dietary dose may be impacted by differences in individual feed consumption, both within and between pens, and feed wastage. The estimated daily dietary doses are presented in the table below. Contains No CBI Wildlife International, Ltd. - 26 - Project Number 454-116 Estimated Maximum Mean Daily Dietary Dose of T-7485 (mg a.i./kg body weight/day) Test Interval (test weeks) Test Concentration (ppm a.i.) Mean Body Weight (g) Mean Feed Consumption (g/bird/day) Estimated Daily Dietary Dose (mg a.i./kg/day) Pre-Egg Production 0 196 16 0.0 (Weeks 1 - 10) 100 196 17 8.5 300 196 17 25.2 900 197 17 76.4 Egg Production 0 212 22 0.0 (Weeks 11 - 21) 100 214 22 10.3 300 216 23 32.0 900 214 22 93.9 Over-all 0 200 19 0.0 (Weeks 1 - 21) 100 201 20 9.7 300 201 20 29.7 900 201 20 87.8 Reproductive Results There were no treatment-related effects upon reproductive performance at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in any of the reproductive parameters measured in the 100, 300, or 900 ppm a.i. treatment groups. While not statistically significant, there was a slight reduction in the total number of eggs laid at the 900 ppm a.i. test concentration. However, reduction was primarily attributable to a single hen (Pen 1662), that exhibited foot lesions and did not lay any eggs during the last five weeks of the study. If data from that pen is excluded, the number of eggs laid per hen at the 900 ppm a.i. test concentration is comparable to the control value. Summaries of the reproductive data are presented in Tables 3 and 4, and in Figures 4 and 5. Reproductive parameters by pen are presented in Appendix VII and VIII. Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 27 - Egg Shell Thickness There were no apparent treatment related effects upon egg shell thickness at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in egg shell thickness in the 100, 300 or 900 ppm a.i. treatment groups. Egg shell thickness data are presented in Table 5 and Appendix IX. Offspring Body Weights There were no apparent treatment related effects upon offspring body weight at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in the body weight of hatchlings or 14-day old survivors from the 100, 300 or 900 ppm a.i. treatment groups. Offspring body weight data are presented in Table 6, and Appendices X and XI. Liver Weights There were no apparent treatment related effects upon adult or offspring liver weights at any of the concentrations tested. When compared to the control group, there were no statistically significant differences in liver weights of adults or 14-day old offspring from the 100, 300 or 900 ppm a.i. treatment groups. Liver weight data are presented in Table 8 and Appendices XII and XIII. Serum, Liver and Egg Analytical Results At the conclusion of the exposure period, serum and liver samples were collected from all surviving adult birds. Serum and liver samples were also collected from indiscriminately selected offspring from each test pen from the last lot of offspring produced from the study (Lot J). Egg contents from those eggs selected for eggshell thickness measurements were collected for potential analysis. Following are summary tables of the results of serum, liver and egg content analyses for each test concentration. The full report for the analysis of serum, liver and egg contents is provided in Appendix XVII. Contains No CBI Wildlife International, Ltd. - 28 - Project Number 454-116 Summary of Mean Concentrations of T-7485 in Serum and Liver Test Concentration 100 ppm a.i. 100 ppm a.i. 100 ppm a.i. 300 ppm a.i. 300 ppm a.i. 300 ppm a.i. 900 ppm a.i. 900 ppm a.i. 900 ppm a.i. Group Adult Male Adult Female Offspring Adult Male Adult Female Offspring Adult Male Adult Female Offspring Mean (SD) Concentration of T-7485 (ppm a.i.) in Serum Mean (SD) Concentration of T-7485 (ppm a.i.) in Liver 16.5 (10.3) 14.6 (8.4) 0.0369 (0.0148) 3.25 (2.26) 3.52 (2.31) 0.0211 (0.0103) 27.9 (9.5) 37.8 (23.5) 0.0567 (0.0324) 7.78 (3.32) 11.1 (6.03) 0.0515 (0.0620) 68.2 (21.3) 104 (84.6) 0.133 (0.0743) 15.7 (4.78) 29.6 (19.7) 0.111(0.0792) Summary of Mean Concentrations of T-7485 in Egg Contents Treatment Group 100 ppm a.i., Lot B Eggs 100 ppm a.i., Lot G Eggs 300 ppm a.i., Lot B Eggs 300 ppm a.i., Lot G Eggs 900 ppm a.i., Lot B Eggs 900 ppm a.i., Lot G Eggs Lot B = 2nd week of eggs set Lot G = 7th week of eggs set Mean (SD) Concentration of T-7485 (ppm a.i.) in Egg Contents 7.65 (2.05) 14.0 (5.50) 23.6 (8.58) 31.4 (19.1) 50.5 (27.9) 92.6 (31.8) Histopathology Samples of liver, kidney and gonad were collected from all adult birds for histopathological examination. Similar samples were also collected from indiscriminately selected offspring from each test pen, from the last lot of offspring produced from the study (Lot J). The spleen from one adult female in the 900 ppm a.i. test concentration was noted as slightly enlarged during gross necropsy, and was also Contains No CBI Wildlife International, Ltd. Project Number 454-116 - 29 - collected for histopathological examination. All histopathological samples were fixed in 10% buffered formalin and shipped to EPL in Herndon, Virginia for histopathology. No lesions considered related to exposure to T-7485 were noted in the liver, kidney, ovary and testes of the adults or their offspring. The few findings in various tissues of the adults and offspring from the control and treatment groups were considered to be incidental and unrelated to treatment. Testes of adult males for all treatment groups exhibited spermatogenesis that was characterized by the presence of numerous mature spermatozoa in the seminiferous tubules. While a few males in the treatment groups exhibited decreased testicular diameter and/or seminiferous tubule degeneration, this result was not doseresponsive and was most consistent with physicological regression. Additionally, similar findings were noted for several males in the control group. The pathology report provided by EPL is presented in Appendix XIV. CONCLUSION There were no treatment-related mortalities, overt signs of toxicity or treatment-related effects upon body weight or feed consumption at any of the concentrations tested. Additionally, there were no treatment-related effects upon any of the reproductive parameters measured at the 100, 300 or 900 ppm a.i. test concentrations. The no-observed-effect concentration for northern bobwhite exposed to T-7485 in the diet during the study was 900 ppm a.i., the highest concentration tested. Contains No CBI Wildlife International, Ltd. - 30 REFERENCES Project Number 454-116 1 U.S. Environmental Protection Agency. 1982. Pesticide Assessment Guidelines, FIFRA Subdivision E, Hazard Evaluation: Wildlife and Aquatic Organisms, subsection 71-4, Environmental Protection Agency, Office of Pesticide Programs. Washington, D.C. 2 Organization for Economic Cooperation and Development. 1984. Avian Reproduction Test. OECD Guideline for Testing of Chemicals. Guideline 206. Paris. 3 American Society for Testing and Materials. 1986. Standard Practice for Conducting Reproductive Studies with Avian Species. ASTM Standard E1062-86. Annual Book of ASTM Standards. Vol. 11.04. Philadelphia, PA. 15 pp. 4 Merck & Co., Inc. 1991. The Merck Veterinary Manual. Merck & Co. Rahway, NJ. 1832 pp. 5 Wildlife International, Ltd. 2003. Analytical Method Verification for the Determination of T-7485 in Avian Diet. Wildlife International, Ltd. Project No. 454C-129. 6 National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, DC. National Academy Press. 125 pp. 7 Dunnett, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments with a Control. Jour. Amer. Statis. Assoc. 50: 1096-1121. 8 Dunnett, C.W. 1964. New Tables for Multiple Comparisons with a Control. Biometrics 20: 482491. Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Wildlife International, Ltd. Project Number 454-116 INGREDIENTS Appendix I DIET AND SUPPLEMENT FORMULATIONS WILDLIFE INTERNATIONAL, LTD. GAME BIRD RATION1 PERCENT (%) Fine Corn Meal Soy Bean Meal, 47.5% Protein Wheat Midds Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos GL Ferm (Fermatco)2 Salt Iodized CM T-Premix 561 CM TM Premix 434 CHO CHL 60% 55# Liquimeth 40% Lysine 75% DL Methionine 055 Vit K 16 g/lb 50# Selenium 42.86 33.39 6.48 9.96 3.00 2.50 0.62 0.11 0.25 0.10 0.05 0.08 0.09 0.25 0.06 0.10 0.05 0.05 Total 100.00 VITAMIN AND MINERAL CONTENT AMOUNT ADDED PER POUND Vitamin D3 Vitamin A 0.600 kiu/lb 2.000 kiu/lb Riboflavin 2.1992 mg/lb Niacin 21.6932 mg/lb Pantothenic Acid 5.2241 mg/lb Vitamin B12 Folic Acid 6.0000 ug/lb 0.5169 mg/lb Biotin 65.9360 ug/lb Pyridoxine 1.4639 mg/lb Thiamine 0.9200 mg/lb Vitamin E 5.0000 iu/lb Vitamin K (Menadione Dimethylpyrimidinol Bisulfite) 3.2400 mg/lb Manganese 115.0203 mg/kg Zinc 194.6601 mg/kg Copper 24.3007 mg/kg Iodine 2.4410 mg/kg Iron 150.4117 mg/kg Selenium 0.4749 mg/kg Beta-Carotene 1.1413 mg/lb Calcium 0.9750 % Chloride 0.2164 % Choline 1882.4116 mg/kg Cobalt 0.1139 mg/kg Magnesium 0.2503 % Phos 30 0.3908 % Phos 50 0.4719 % Potassium 1.1148 % Sodium 0.1335 % Sulfur 0.3478 % Phosphorus 0.6800 % Ca:PHOS 1.4338 ratio 1 The guaranteed analysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of 3.8% crude fiber. 2 Fermentation By-Products (Source of Unidentified Growth Factors) Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI -216- Project Number 454-1 16 Rep01tEO3-0583 T-7465:A RepodudionStudy with the Northern B o M i Phase:Analytical Determinationd PFBS in Northem BotwhiteTissues Table 35. C4F&03' Data Summary.900 ppm Dose Group. Adult Male Quail Liver ....................... 28 Table 36. C4f:&Oi Data Summary,900 ppm Dose Group, offspring Quail Liver.......................... 29 Table 37. C4f:&e; Data Summary, Quail Egg Lot B ....................................................................... 29 Table 38. C4F&0J Data Summary, Quail Egg Lot G....................................................................... 30 Table 39.Summaryof Resub: C4F&OJ- Concentration in Serum................................................... 31 Table 40.Summary of Results: C, F&Oi Concentration in Liver ..................................................... 31 Table 41.Summary Table, C4F&Oi Concentration in Egg.............................................................. 32 3M EnvironmentalLaboratory ?age 706227 Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Report E03-0583 -240- Project Number 454-1 16 T-7485: A Reproduction Study with the Northern Bobwhite Phase:Analytical Determinationof PFBS in Northern Bobwhite Tkues Table 39.SUmMn of Results.CAS% ConcentrationIn Serun' I~ppmOfmm 0.0- 16 27.9 300ppm,Female I 15 I 37.8 900ppm,Femak I 16 I I04 0.0334' 26.3 I 27.8 I 0.0433 I 82.2 I 0.0278 - 0.0562 14.2- 42.9. 12.2- 102 - 0.0245 0.119 - _.__ 34.3 98.8 30.5- 370 I 100ppm,Male I 17 I 100 ppm, Female 100PpmoRspring 3.25 3.52 0.021 1 I 2.71 3.91 0.- I - 0.808 8.89 0.396- 8.19 < 0.0137-0.0301 300pp~F~em,ale 900 m.Male 11.1 0.051!Y 15.7 9.08 0.- 15.1 - 3.57-23.5 - <0.0138 0.200 - 6.73 23.7 900 ppm, Female 29.6 0.111 23.5 0.0835 -- 9.73-77.4 - 0.0340 0.313 The uncertainty for the liwr samples is 46% as calculatedin the validetion. "Mean and Medianreportedfor samples inwhich analytewas detected. For m e specificinformationsea tables in Attachment A 3M EnvironmentalLaboratory Page 31 of 227 Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI Contains No CBI