Document 3N7VBnVkjM6XLxMvE35q2X6ba
23) OECD 205-OPPTS 850.2200, Acute avian toxicity (Northern Bobwhite), 454-112
T-74 85 : A DIETARY LC50 STUDY WITH THE NORTHERN BOBWHITE
SANITIZED
WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-1 12 3M LAB PROJECT NO.: E00-1429
DEC 0 9 2003
U.S.Environmental Protection Agency
Series 850 - Ecological Effects Test Guidelines (draft) OPPTS Number 850.2200
OECD Guideline 205
AUTHORS:
Sean P. Gallagher Jennie Grimes
Joann B. Beavers Raymond L. VanHoven
STUDY INITIATION: June 4,2002
STUDY COMPLETION: January 10,2003
1
SUBMITTED TO
3M Corporation Environmental Laboratory
Building 2-3E-09 935 Bush Avenue St. Paul, Minnesota 55 106
WildlifeInternational, Ltd.
8598 Commerce Drive Easton, Maryland 2 1601
(410) 822-8600
Page 1 of 55
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Project Niimbcr 454- I I2
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
SPONSOR: 3M Corporation
TITLE: T-7485: A Dietary LC50 Study with the Northern Bobwhite
WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454- 112
3M LAB PROJECT NO.: E00-1429
STUDY COMPLETION: January 10,2003
This study was conducted in compliance with Good Laboratory Practice Standards as published by the U S . Environmental Protection Agency, 40 CFR Part 160 and 792, 17 August 1989; which is basically in compliance with OECD Principles of Good Laboratory Practice, (ENV/MC/CHEM (98) 17), Paris 1998; and Japan MAFF, 11 NohSan Notification No. 6283, Agricultural Production Bureau, 1 October 1999.
STUDY DIRECTOR:
Sean P. Gallagher Senior Biologist, Avian Toxicology
DATE
SPONSOR APPROVAL:
//3&.B
DATE
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Projcct Number 454- I I2
QUALITY ASSURANCE STATEMENT
Thls study was examined for compliance with Good Laboratory Practice Standards as published
by the U.S.Environmental Protection Agency, 40 CFR Part 160 and 792, 17 August 1989; OECD
Principles of Good Laboratory Practice, (ENV/MC/CHEM (98) 17), Paris 1998; and Japan MAFF, 11 NohSan Notification No. 6283, Agricultural Production Bureau, 1 October 1999. The dates of all audits and inspections and the dates that any findings were reported to the Study Director and Laboratory Management were as follows:
ACTIVITY
DATE CONDUCTED
Test Substance
Preparation and Analytical
Sample Preparation
June 6,2002
Matrix Fortlfications
June 7,2002
Body Weight
Tissue Sampling
Biological Data & Draft Report
June 11,2002
June 28,2002
I
August 3 - August 8,2002
Analytical Data & Draft Report
Final Report
August 2,5 & 6,2002 January 9-10,2003
DATE REPORTED TO:
STUDY DIRECTOR MANAGEMENT
June 7,2002 June 7,2002 June 13,2002 June 28,2002
August 8,2002
August 6,2002 January 10,2003
June 18,2002 June 12,2002 June 18,2002 June 28,2002
August 16,2002
August 6,2002 January 10,2003
Quality AssurancgRepresentative
Wildlife International, Ltd.
-4REPORT APPROVAL
Project Numbcr 454- 1 I2
SPONSOR: 3M Corporation TITLE: T-7485: A Dietary LC50 Study with the Northern Bobwhite WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-1 12 3M LAB PROJECT NO.: E00-1429
STUDY DIRECTOR:
Sean P. Gallagher
I
Date
Senior Biologist, Avian Toxicology
CHEMISTRY PRINCPAL INVESTIGATOR: 1
RayhdndJ!. VanHoven, Ph.D. Scientist, Chemistry
REPORT APPROVED BY:
?? P
Joann B. Bhvers Director of Avian Toxicology
Date
I /I3163
Date
Willard B. Nixon, PkD. Director of Chemistry
WildlifeInternational. Ltd.
-5TABLE OF CONTENTS
Project Nutnbcr 454-1 12
TITLE PAGE ........................................................................................................................
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT...............................
QUALITY ASSURANCE STATEMENT ...........................................................................
REPORT APPROVAL .........................................................................................................
TABLE OF CONTENTS......................................................................................................
Tables and Appendices .......................................................................................................
SUMMARY ...........................................................................................................................
INTRODUCTION ................................................................................................................
OBJECTIVE .........................................................................................................................
MATERIALS AND METHODS.......................................................................................... Test Substance .................................................................................................................... Treatment Groups ............................................................................................................... Duration of the Test ............................................................................................................ Test Birds............................................................................................................................ Animal Diet ........................................................................................................................ Diet Preparation .................................................... f ............................................................ Diet Sampling ..................................................................................................................... Analytical Method .............................................................................................................. Housing and Environmental Conditions............................................................................. Observations ....................................................................................................................... Animal Body WeightsReed Consumption......................................................................... Gross Necropsy................................................................................................................... Statistical Analyses ........................................ :....................................................................
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Page 9 Page 9 Page 9 Page 10 Page 10 Page 10 Page 11 Page 11 Page 11 Page 13 Page 14 Page 14 Page 14
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RESULTS ............................................................................................................................. Page 15 Diet Analysis ...................................................................................................................... Page 15
Mortalities and Clinical Observations ................................................................................ Page 15 Body Weight and Feed Consumption ................................................................................. Page 16 Gross Necropsy ................................................................................................................... Page 16
Liver Weight ....................................................................................................................... Page 17
CONCLUSION..................................................................................................................... Page 17
REFERENCES ..................................................................................................................... Page 18
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TABLES AND APPENDICES
Project Number 454-1 12
TABLE 1: TABLE 2: TABLE 3: TABLE 4:
Cumulative Mortality from a Northern Bobwhite Dietary Toxicity
Study with T-7485........................................................................................
Page 19
Mean Body Weight (g) from a Northern Bobwhite Dietary Toxicity
Study with T-7485 ........................................................................................ Page 20
Mean Feed Consumption (ghirdday) from a Northern Bobwhite
Dietary Toxicity Study with T-7485 ............................................................ Page 21
Mean Liver Weight (8) and Mean Relative Liver Weight
from a Northern Bobwhite Dietary Toxicity
Study with T-7485........................................................................................
Page 22
APPENDIX I: APPENDIX 11:
Certificate of Analysis ..............1............................................................
Diet Formulation....................................................................................
Page 23 Page 27
APPENDIX TI1 Diet Preparation ...................._...:'............:...............................................Page 28
APPENDIX IV: Analytical Methods and Results ............................................................ Page 29
APPENDIX V: Cumulative Mortality by Pen from a Northern Bobwhite Dietary Toxicity Study with T-7485...................................................... Page 42
APPENDIX VI: Individual Body Weights (g) from a Northern Bobwhite Dietary Toxicity Study with T-7485 ...................................................... Page 43
APPENDIX VII: Feed Consumption (ghirdday) by Pen from a Northern Bobwhite Acute Dietary Toxicity Study with T-7485 .................................................................................
Page 50
APPENDIX VIII: Individual Liver Weights (8) from a Northern Bobwhite
Acute Dietary Toxicity Study with T-7485 ........................................... Page 52
APPENDIX IX: Changes to Protocol .............................................................................. Page 54
APPENDIX X: Personnel Involved in the Study ............................................................ Page 55
wildlife Intermtional, Ltd.
-7SUMMARY
Project Nunibcr 454-1 12
SPONSOR: 3M Corporation
TEST SUBSTANCE: T-7485
WILDLIFE INTERNATIONAL, LTD. PROJECT NO.: 454-1 12
STUDY: T-7485: A Dietary LC50 Study with the Northern Bobwhite
RESULTS:
The dietary LC50 value for northern bobwhite exposed to T-7485 was determined to be greater thar, 10,000 ppm a i , the highest concentration tested. With the exception of a single incidental mortality at the 1780 ppm a.i. concentration, no mortalities occured at any of the test concentrations tested. The no mortality concentration was 10,000 pprn a.i.
There were no overt sikns of toxicity or treatment-related effects on feed consumption or liver weights at any of the concentrations tested. Additionally, there were no apparent treatment-related effects on body weight among birds in 'the 1000, 1780 or 3160 ppm a.i. treatment group. However, there was a concentration responsive reduction in body weight gain among birds in the 5620 and 10,000 ppm a.i. treatment groups during
the exposure period. Based upon a reduction in body weight gain at the
5620 ppm a.i. test concentration, the no-observed-effect concentration was 3 160 pprn a.i.
TEST DATES:
Hatch - May 27,2002 Acclimation - May 27,2002 to June 6 , 2 0 0 2 Experimental Start (EPA) -June 6, 2002 Experimental Start (OECD) - June 6,2002 Experimental Termination- July 19, 2002
NOMINAL TEST CONCENTRATIONS:
0, 1000, 1780, 3160, 5620 and 10,000 ppm a.i.
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SUMMARY
(contined)
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TEST ANIMALS:
Northern Bobwhite (Colinus virgznianus)
AGE TEST ANIMALS: 10 days of age at test initiation
SOURCE TEST A.NIMALS:
Wildlife International, Ltd. Production Flock 8598 Commerce Drive Easton, Maryland 2 1601
STUDY COMPLETION: January 10,2003
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INTRODUCTION This study was conducted by Wildlife International, Ltd. for 3M Corporation at the Wildlife International, Ltd. avian toxicology facility in Easton, Maryland. The in-life portion of the test was conducted from June 6, 2002 to June 28, 2002: Raw data generated at Wildlife International, Ltd. and a copy of the final report are filed under Project Number 454-1 12 in archives located on the Wildlife International, Ltd. site.
OBJECTIVE The objective of this study was to evaluate the toxicity of T-7485 to the Northern Bobwhite (Colinus virginianus) administered through the diet for five days. An LC50 value will be calculated, if possible.
MATERIALS AND METHODS The methods used in conducting this study are based upon procedures specified in the U.S. Environmental Protection Agency Series 850 - Ecological Effects Test Guidelines OPPTS Number 850.2200 (1); OECD Guideline 205, Guideline forFesting of Chemicals, Avian Dietary Toxicity Test (2); and upon ASTM Standard E857-87, "Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species" (3).
Test Substance
The test substance was received from 3M Corporation on March 27, 2000 and was assigned
Wildlife International, Ltd. Identification Number 52 16 upon receipt. The test substance was white
powder identified as: Potassium Perfluorobutane Sulfonate;
.,ot No.: 2; CAS No.
29420-49-3. The reported purity of the test substance was 97.3%, with an expiration date of January
17, 2007 (Appendix I). The test substance was stored under ambient conditions.
Treatment Groups The test consisted of a geometric series of five test concentrations and a control group. Thirty
northern bobwhite chicks were assigned to the control group and twelve northern bobwhite chicks were assigned to each of the treatment groups. The birds were sorted by weight, then chosen indiscriminately from within each represented weight class for placement into control and treatment groups. The birds were housed in brooding pens containing five or six chicks each. Nominal dietary
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concentrations used in this study were 0, 1000, 1780, 3160, 5620 and 10,000 parts per million active ingredient (ppm a i ) of T-7485. The dietary concentrations were established based upon known toxicity data and information supplied by the Sponsor.
Each group was fed the appropriate test or control diet for five days. During the exposure period the control group received untreated feed. Following the five-day exposure period all groups were given untreated basal diet for three days. On Day 8, half of the surviving treatment and control birds were euthanized and subjected to gross necropsy. Livers were weighed and liver and blood samples were collected for potential analysis. The remaining birds were maintained on basal ration until Day 22. On Day 22, these birds were euthanized, liver weights were recorded and samples of liver tissue and blood were collected.
Duration of the Test
The primary phases of this test and their approximate durations were:
I.
Acclimation - 10 days.
2. Exposure - 5 days.
/
3. Post-exposure observation - 3 or 17 days
Test Birds All northern bobwhite (Colinus virginianw) were 10 days of age and appeared to be in good
health at initiation of the test. The birds were obtained from Wildlife International, Ltd. Production
Flock, Easton, MD and were hatched on May 27, 2002. Birds ranged in weight from 17 to 24 grams at test initiation. The birds used in this study were immature and could not be differentiated by sex. All birds were from the same hatch, pen-reared and phenotypically indistinguishable from wild birds. All birds were acclimated to the caging and facilities from the day of hatch until initiation of the test.
Animal Diet Throughout acclimation and testing all test birds were fed a game bird ration formulated to
Wildlife International, Ltd.'s specifications (Appendix 11). The chicks were given a vitamin supplement (Durvet water soluble vitamin and electrolyte concentrate) in their water from the day they were hatched until the initiation of the test. Water from the town of Easton public water supply,
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Project Nuinbcr 454-1 12
and fecd were provided ad libitum during acclimation and testing. The birds receivcd no form of antibiotic medication during acclimation or testing.
Diet Preparation The test substance was mixed directly in basal ration into which corn oil had been added. The
concentration of corn oil in the control and treated diets was 2%. Mixing was done with a Hobart mixer (Model Number AS200T). All dietary test concentrations were adjusted to 100% active ingredient based upon the reported purity of the test substance. All dietary concentrations and the LC50 value are reported as ppm a i in the diet. Nominal dietary test concentrations used in this study were 1000, 1780, 3160, 5620 and 10,000ppm a.i. (Appendix 111).
Diet Sampling Samples of the test diets were collected to verify the test concentrations administered and to
confirm the stability and homogeneity of the test substance in the diets. Homogeneity of the test substance in the diet was evaluated by collecting six samples from the 1000 ppm a i a.i. test diet and six samples from the 10,000 ppm a.i. test diet at pfeparation on Day 0. Homogeneity samples were collected from the top, middle and bottom of the left and right sections of the mixing vessel. The homogeneity samples also served as verification samples. One verification sample was collected from the control diet and two verification samples were collected from each remaining treatment diet at preparation on Day 0. At the end of the exposure period (Day 5 ) , one sample was collected from the control diet and two samples were collected from each treatment diet to determine stability of the test substance in the diet under test conditions. The stability samples were collected from feed remaining in the feeders after being at ambient test pen conditions for five days. Samples were transferred immediately to Wildlife International, Ltd. chemistry laboratory.
Ana1yticaI Method A liquid chromatography mass spectrometry (HPLC/MS/MS) based method was developed at
Wildlife International, Ltd. for the analysis of the T-7485 in avian diet. The analytical method consisted of sample weighing, extraction with methanol, filtration, dilution with methanol and microfiltration. Dilutions into the calibration range of the HPLCiMSiMS methodology were performed with a solution of 50% methanol and 50% NANOpure@water. Samples were then analyzed by direct
injection. A method flowchart is provided in Appendix IV,Figure 1.
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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 API 3000LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Chromatographic separations were achieved using a Keystone PRISM RP column (50 mm x 2.0 mm, 3-pm particle size) fitted with a Keystone Javelin C I 8guard column (20 mm x 2.0 mm i.d.). The instrument parameters are summarized in Appendix
IV,Table 1.
All primary and secondary stock preparations were adjusted for the purity of the test substance (97.3%). A 1.00 mg a.i./mL primary stock solution of T-7485 in methanol was prepared by weighing 0.1027 g of the test substance and bringing to a final volume of 100 milliliters with methanol. Secondary stock solutions (100 and 10.0 mg a.i./L) of T-7485 in methanol were prepared by serial volumetric dilution of the primary stock.
Calibration standards were prepared in a soldtion of 50% methanol and 50% NANOpure@water by appropriate dilutions of the 100 mg a.i./L stock solution of T-7485 in methanol. Calibration standards of T-7485, ranging in concentration from 0.100 to 0.500 mg a.i./L, were analyzed with each sample set. The calibration standard series was injected at the beginning and end of the run, 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 W , 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 are presented in Appendix IV, Figures 3 and 4, respectively. Examples of equations used in calculations are presented in Appendix IV, Table 2.
The instrument limit of detection (LOD) was set based upon the injection volume (5.00 yL) and the lowest standard concentration 0.100 mg a.i./L. The LOD was set at 0.500 ng on-column. The method limit of quantitation (LOQ) for these analyses was set at 150 ppm a.i. based upon the lowest matrix fortification level analyzed concurrently with the samples. The 0.100 mg a.i./L T-7485
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standard was equivalent to a calculated value of 40 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, three 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 IV, Table 3). A representative ion chromatogram of a
matrix blank sample is presented in Appendix IV,Figure 5.
Avian diet samples were fortified at 150,3000, and 12000 ppm a.i. and analyzed concurrently with the samples to determine the mean procedural recovery. The method yielded mean procedural recoveries of 87.7%, 99.0% and 98.2% (Appendix IV, Table 3). Sample measured concentrations were not corrected for the mean procedural recoveries. A representative ion chromatogram of a
matrix fortification sample is presented in Appendix N ,Figure 6.
Housing and Environmental Conditions During acclimation and testing, all birds @re housed indoors in batteries of thermostatically
controlled brooding pens manufactured by Beacon Steel Products Co. (Model No. B735Q). Each pen had floor space that measured approximately 72 X-90 cm. Ceiling height was approximately 23 cm. External walls, ceilings and floors were constructed of galvanized steel wire and sheeting. Birds were sorted by weight, then chosen indiscrimina,tely from within each represented weight class for assignment to pens. Each group of birds was identified by pen number and test concentration. Individual birds were identified by colored leg bands.
Through Day 12 of the test, the average temperature in the brooding compartment of the pens was 39C k1"C (SD). Brooding was discontinued on Day 12, when birds were of sufficient size to
thermoregulate. Average ambient room temperature for this study was 27.1"C * 1.7"C (SD) with an average relative humidity of 54% * 11% (SD). The photoperiod (maintained by a time clock) \vas
sixteen hours of light per day during acclimation and throughout the test. The light source was fluorescent lights which closely approximate noon-day sunlight. The birds were exposed to an average of approximately 180 lux of illumination. Housing and husbandry practices were based on guidelines established by the National Research Council (4).
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Observations During acclimation -all birds were observed daily. Birds exhibiting abnormal behavior or
physical injury were not used for the study. Following test initiation and continuing until termination, all birds were typically observed at least twice daily. A record was maintained of all mortality, signs of toxicity, and abnormal behavior.
Animal Body Weightsmeed Consumption Individual body weights were measured at the initiation of the test, and on Days 5, 8, 15 and
22 for all remaining birds. Average feed consumption values during the exposure period (Days 0-5) and the post-exposure observation period (Days 6-8) were determined by pen for each treatment group and the control group. Additionally, feed consumption was determined for Days 8-15 and 1622 for the remaining treatment and control birds. Feed consumption was determined by measuring the change in the weight of the feed presented to the birds over a given period of time. The accuracy of feed consumption values may have been affected by the unavoidable wastage of feed by the birds.
Gross Necropsy
1
All test birds that died during the course of the test were subjected to a gross necropsy. On
Day 8 of the test, half of the birds in each treatment group and half of the control birds were
euthanized and subjected to gross necropsy. On Day 22 of the test, all remaining birds were
euthanized and subjected to gross necropsy. Prior to euthanasia, blood was collected from each bird
when possible. During necropsy, the liver was removed and weighed. Blood and liver samples were
transferred to the sponsor for analysis if deemed appropriate. .
Statistical Analyses There were no treatment related mortalities observed in this study, therefore, it was not
possible to perform the calculation (5,6,7) of an LC50 value using the computer program of Stephan (8). The LC50 value is estimated to be greater than 10,000 ppm a.i., the highest concentration tested. Differences in the mean body weights, body weight change, absolute liver weights and relative liver weights between the control group and the treatment groups were determined by an Analysis of Variance (ANOVA). Bonferoni's t-Test, as run with Toxstat Software (9,lO) was used to compare the five treatment group means with the control group mean and assessed the statistical significance of the differences observed.
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RESULTS Diet Analysis
Diet samples collected from the 1000 and 10000 ppm a.i. test concentrations were analyzed to evaluate the homogeneity of the test substance in the diet. The mean and standard deviation for the
1000 and 10000 ppm a.i. test concentrations were 938 f 60.6 pprn a i and 9440 * 627 ppm a.i.,
respectively. The corresponding coefficients of variation were 6.46% and 6.65%, respectively
(Appendix IV,Table 4). A typical ion chromatogram of a test sample is shown in Appendix IV,
Figure 7.
Samples collected during Day 0 of the test to verify test substance concentrations for the 1000,
1780, 3160, 5620, and 10000 pprn a.i. diets had mean measured concentrations of 938, 1700, 2940,
5420, and 9440 pprn a i , respectively. These values represented 93.8, 95.5, 93.0, 96.4, and 94.4% of
nominal concentrations, respectively (Appendix IV,Table 5). Analysis of diet samples collected
from feeders after being held at ambient temperature for 5 days averaged 116%, 108%, 98.0%, 112%,
and 111% of the Day 0 values for the 1000, 1780, 3160,5620 and 10000 pprn a.i. test concentrations,
respectively (Appendix IV, Table 5).
t
Mortalities and Clinical Observations There were no mortalities in the control group (Table 1 and Appendix V). One control bird
was noted limping on Days 5 and 6 of the test, apparently due to an injury. Otherwise, all control
birds were normal in appearance and behavior throughout the test. In addition, there were no
treatment related mortalities at the 1000, 1780, 3160, 5620 and 10,000 ppm a i test concentrations.
There were no overt signs of toxicity noted in any of the treatment groups. Toe piclung, a cannibalistic form of aggression, was observed in all treatment groups during the course of the test. Two to five birds in each treatment group were noted with toe, foot or ankle lesions as a result of the
t
pen-matc aggression. Lameness associated with foot lesions was noted for several birds. In some instances, lethargy was noted in birds suffering extensive lesions. At the 1780 ppm a.i. test concentration, one bird was found dead on the morning of Day 3. Upon examination, the bird was noted with lesions on both ankles and dried blood on the breast feathers. Necropsy revealed a pale spleen, liver and kidneys. Due to the time of death and the presence of autolysis, liver and blood samples were not collected from this bird. This mortality was not considered treatment-related.
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One bird in the 1780 ppm a.i. treatment group and one bird in the 5620 ppm a i . treatment group was noted with slight lateral head curl during the last week of the test. This condition is
usually associated with head or neck injury and was not considered treatment related. With the exception of incidental observations associated with physical injury, all birds in all treatment groups were normal in appearance and behavior throughout the test.
Body Weight and Feed Consumption When compared to the control group, there were no apparent treatment-related effects on
body weight among birds in the 1000, 1780 or 3160 ppm a.i. treatment groups throughout the test
(Table 2 and Appendix VI). Any differences observed between the control group and the 1000 and 3 160 ppm a.i. treatment groups were not statistically significant. There was a statistically significant
(p<O.OI) reduction in body weight gain among birds in the 1780 ppm a.i. treatment group during the exposure period (Days 0-5). However, the reduction was not concentration responsive and therefore not considered treatment-related. There was a statistically significant (p<O.O1) concentration
responsive reduction in body weight gain amongbirds in the 5620 and 10,000 ppm a i treatment
groups during the exposure period that was considered treatment-related. The reduction was also reflected in significantly (p<O.OI) lower mean body weights in the 5620 and 10,000 ppm a i .
treatment groups at the Day 5 and Day 8 body weight intervals.
There were no apparent treatment related effects on feed consumption at the 1000, 1780, 3 160, 5620 and 10,000 ppm a.i. test concentrations (Table 3 and Appenhx VII). When compared to the control group, a slight reduction in feed consumption was noted for all test concentrations during the exposure period. In addition, these reductions were not concentration responsive, and in most cases were not associated with a corresponding reduction in body weight gain. Therefore, the slight differences observed were not considered to be treatment-related.
Gross Necropsy Gross necropsy revealed two birds each in the 1780 and 3 160 ppm a.i. treatment groups and
three birds each in the 1000, 5620 and 10,000 ppm a.i. treatment groups with toe, foot or ankle lesions. In addition, one bird at the 1000 ppm a.i. test concentration was noted with an encapsulated
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feather mass in the abdominal cavity and a small spleen. All findings were considered incidental to treatment. The necropsy results for all other birds were not remarkable.
Liver Weight When compared to the control group, there were no apparent treatment-related effects on
mean absolute liver weight at the 1780 or 3160 ppm a.i. test concentrations. There was a slight reduction in Day 8 mean absolute liver weight for the 5620 ppm a.i. concentration that was not statistically significant ( p 0 . 0 5 ) . However, there were statistically significant (p<O.O1) reductions in the mean absolute liver weights at the 1000 and 10,000 ppm a i . test concentrations for Day 8 liver weights. While there were reductions in mean absolute liver weight at the 1000, 5620 and 10,000 ppm a.i. test concentrations at Day 8, when liver weight was examined as a percentage of body weight (relative liver weight) these differences were no longer apparent or statistically significant. For Day 22 absolute and relative liver weights, all test concentrations were comparable to the control group and not statistically different. Mean absolute liver weights and mean relative liver weights are presented in Table 4. Individual liver weights are presented in Appendix VIII.
t
CONCLUSION talue for northern bobwhite exposed to T-7485 was determined to be a i , the highest concentration tested. With the exception of a single
incidental mortality at the 1780 pprn a.i. concentration, no mortalities occured at any of the test
concentrations tested. The no mortality concentration was 10,000ppm a.i.
There were no overt signs of toxicity or treatment-related effects on feed consumption or liver weights at any of the concentrations tested. Additionally, there were no apparent treatment-related effects on body weight among birds in the 1000, 1780 or 3160 ppm a.i. trreatment group. However, there was a concentration responsive reduction in body weight gain among birds in the 5620 and 10,000 ppm a.i. treatment groups during the exposure period. Based upon a reduction in body weight gain at the 5620 pprn a.i. test concentration, the no-observed-effect concentration was 3 160 ppm a.i.
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REFERENCES
Projcct Numbcr 454-1 12
1 U.S. Environmental Protection Agency, 1996. Series 850 - Ecological Effects Test Guidelines (draft), OPPTS Number 850.2200: Avian Dietary Toxicity Test.
2. Organization for Economic Cooperation and Development. 1984. Avian Dietary Toxicity Test. OECD Guideline for Testing of Chemicals. Guideline 205. Paris.
3. American Society for Testing and Materials. 1987. Standard Practice for Conducting Subacute Dietary Toxicity Tests with Avian Species. ASTM Standard E857-87. Annual Book of ASTM Standards,'Vol. 11.04. Philadelphia, PA.
4. National Research Council. 1996. Guide for the Care and Use of Laboratory Animals. Washington, D.C. National Academy Press. 125 pp.
5. Stephan, C.E. 1977. Methods for Calculating an LC50. Pages 65-84 InAquatic Toxicology and
Hazard Evaluations, American Society for Testing and Materials. Pub. No. STP 634. Philadelphia, PA.
6. Finney, D.J. 1971. Statistical Methods in Biological Assay, 2nd ed., Griffin Press, London.
f
7. Thompson, W.R. 1947. Bacteriological Reviews, Vol2, No.2: 115-145.
8. Stephan, C.E. 1978. U.S. EPA, Environmental Research Laboratory, Duluth, MN. Personal Communication
9. Dunnett, C.W. 1955. A Multiple Comparison's Procedure for Comparing Several Treatments
with a Control. Jour. Amer. Sfafis.Assoc. 50:1096:1121
10. Gulley, D.D. 1990. TOXSTAT Release 3.2. The University of Wyoming.
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TABLE 1
Cumulative Mortality from a Northern Bobwhite Dietary Toxicity Study with T-7485
Eq~erimentnl Group
(pp1na.i.) Day0
No. Dead Per NO. Exposed Exposure Period
Day 1 Day2 Day3 Day4
Day5
No. Dead Per No. Exposed Post-Exposure Period
Day6 Day7 Day8 I Day22'
Control 0
0130 0130 0130 . 0130 0130 0130 *
0130 0130
0130
011.5
Treatment 1000 I780
3160
5620 10,000
0112 0112 0112 0112 0112 0112 0112 0112 0112 1/12 1/12 1/12 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112 0112
0112 0112
0112
016
1/12
1/12
1/12
016
0112 Of12
0112
016
0112
0112
011 2
016
0112
0112
0112
016
The LC50 value was estiniaied to be greater than 10,000ppm ai.,the highest concentration tested.
' - I-lalf of the birds were euthanized on Day 8 for necropsy and tissue collection. '- N o morialities occui-red i n any of the control or treatment groups from Day 8 to Day 22.
wiId1ife IntermtionuI, Ltd.
- 20 -
Project Number 453-I I2
TABLE 2
Mean Body Weight (8) from a Northern Bobwhite Dietary Toxicity Study with T-7485
Experimental Grou~
(ppni a i . )
Exposure Period Day0 Day5 Change'
Day 8
Post-Exposure Period Change' Day 15 Change' Day 22
Change'
T- n t a I -I-Change
Control
0
Mean 20
31
11
SD
2
4
3
41
9
67 ' 27
95
28
75
6
3
10
4
13
5
12
Treatment
I000
Mcan 30
29
9-
36
7
64
23
88
24
68
SD
2
4
3
7
4
9
4
9
2
8
\
1780
Mean 20
28
7**
SD
2
4
3
36
8
65
29
92
27
72
5
3
8
2
9
2
9
3160
Mean 20
30
9
SD
2
4
3
37
8
68
28
95
26
75
7
3
8
3
8
2
7
5620
Mean 20 25** 5**
33**
8
63
28
91
28
71
SD
2
4
3
6
2
5
4
7
3
6
10,000
Mean 20 23** 3**
SD
2
6
4
30**
7
8
3
64
29
88
25
68
9
5
11
4
10
'Mean change is calculated separately from the mean body weights using individual body weights (see Appendix VI).
** - Significantly different from the control at p<O.01 (Bonferroni's t-Test).
wiId1if e Internationa1, Ltd.
-21 TABLE 3
Mean Feed Consumption (ghirdday) from a Northern Bobwhite Dietary Toxicity Study with T-7485
Project Number 451-1 12
Experimental Group (ppm a.i)
Exposure Period Days 0-5
Control Mean
9
SD
1
Treatment
1000
Mean
7
1780
Mean
6
3160
Mean
6
5620
Mean
6
10000
Mcnn
5
Days 6-8 13 3
`r,
9 8 10 12 8
Post-Exposure Period Days 9-15
18
5
Days 16-22
22 1
16
23
11
19
14
19
11
17
11
19
wildlife International, Ltd.
Project Number 454-1 12
- 22 -
TABLE 4
Mean Liver Weight ( 6 )and Mean Relative Liver Weight' from a Northern Bobwhite Dietary Toxicity Study with T-7485
Experi iiieiita1
Day 8
Day 22
Group
Liver
Relative
Liver
Relative
(ppm n.i.)
Weight (g)
Liver Weight
Weight (g)
Liver Weight
Control 0
Mean SD
1.017 0.127
2.43 9 0.253
Treatment
1000
Mean
0.723**
2.326
SD
0.153
' 0.%4
1780
Mean
1.014
SD
0.188
2.855 0.469
3160
Mean
0.930
SD
0.160
2.793 0.540
5620
Mean
0.866
SD
0.256
2.845 0.544
1 0000
Mcan SD
0.705** 0.214
2.764 0.271
'Relative Liver Weight = Liver Weight (g)/Body Weight (g) x 100
** - Significantly different from the control at p<O.O1 (Bonferroni's t-Test)
2.540 0.507
2.320 0.224
2.385 0.316
2.53 1 0.386
2.423 0.337
2.289 0.360
2.68 1 0.337
2.694 0.538
2.588 0.187
2.667 0.261
2.670 0.40 1
2.598 0.239
wild1f e Internntional, Ltd.
- 23 -
APPENDIX I Certificate of Analysis
Projcct Numbcr 454- I 12
Wildlife InternationnI , Ltd. __ ~ -24-
Projcct Numbcr 454- 1 12
Purity' T a t Name
Amearance Identification '
NMR.
hdetals (ICP/MS) I . Calcium
2. Magnesium
3. Sodium' 4. Potassium' 5. Nickel
6. Iron 7. Manganese
Total % Impurity (NMR)
Total % Impurity (LCMS)
Related Compounds (POAA)
Total % Imuuritv (GUMS)
r
I.
Residual Solvents (TGA)
Punty by DSC
Inorganic Anions (IC) 1. , Chloride 2. Fluoride 3. Bromide 4. Nitrate
5 . Nitrite
6. Phosphate 7 . Sulfate
Organic Acids' (IC)
i
i
Elemental Analysis': I . Carbon 2. Hydrogen
1 3 Nitrogen
' 4. Sulfur 5 Fluorine
Speclflcatfons White. crvstalline solid
1
1
I
Theoret~caVl alue = 14.2% Theoretical Value = 0.0% Thcoretical Value = 0.0% Theoretical Value = 9.5% Theoretical Value = 50.6%
Result 97.3 Y.
Conforms
Positive
1. 0.003 wt/wt.% 2. 0.001 wt/wt.% 3. 0.768 wt./wt.% 4. 10.718 wt./wt.% 5. <0.001 wtlwt.% 6. 0.002 wt/wt.% 7. <0.001 wtlwt.%
2.07wt/wt.% 5.56 wt./wt.% None Quantified None Ouanbfied None Detected Not Applicable
I . 0.016 wt./wt.% 2. <0.005wt./wt.% 3. 40.040 wtJwt.%
4. co.009 wt./wt.% 5 . 4.006 wt/wt.% 6 . c0.030 w-t./wt.% 7 . C0.040wt./wt.%
I . 40.1 wt./wt.% 2. <0.1 wtfwt.% 3 . co.1 wt./wt.%o 4. <0.25 wt./wt.%
I . 1 1.o wtJwt.%
2. 0.282 wt./wt.% 3. 0.465 wt./wt.% 4. 1I .2 wr./wt.% 5 . 48.2 wt./M.Yo
COA023032-71 Revision 2.doc
Page 1 o f 3
WildlifeInternational, Ltd.
Project Nimber 454-1 12
CEntrtz Analqtical Laboratories, Inc.
3048 Research Drive Slate College, PA 16801
www.cenlrelab.com
Phone:(814)231-8032
Fax:(814)231-1253or (814)231-1580
INTERIM CERTIFICATE OF ANAL YSIS
Revision 2 Centre Analytical Laboratories COA Reference #: 023-032
3M Product: PFBS, Lot 2 Test Control Reference #: TCR-00017-71
Date ofLast Analysis: 01/17/01
Expiration Date: 01/17/07
Storage Cdnditions: < -10 "C
Re-assessment Date: 01117/07
- - 'Purity = 100% (Total NMR impurities, 2.07% +Total LCMS impurities, (5.56%
4.99%*) + Total Metal impurities, 0.006% + Total inorganic anions, 0.016%)
- Total impurity from all tests Purity = 100% 2.662%
= 2.662% 97.3%
L * 1.93% of the N M R total impurity value is due to the presence of the . This impurity is Is0 observed in the LCMS analysis at
3.99%. The impurity value is taken from the R analysis as it is deemed the more quantitative technique in the absence of standards (See impurity profile on page 3).
2Potassiurn and sodium are present as countenbns and thus are excluded from the punty calculation.
3.
'Theoretical value calculations based on the empirical formula, C4F9SO3Xi (MW=338.2)
COAO23032-7 I Revision 2 . d N
Page 2 of 3
Wildlife InternntionaI,Ltd.
- 26 -
Projcct Nurnbcr 454-1 12
CentrE Analytical laboratories. Inc.
3048 Research Drive Slate College, PA 16801
www.centrelab.com
(814) 231-8032
Fax: (814) 231-1253or (814) 231-1580
INTERIM CERTIFICATE OF ANALYSIS
Revision 2 Centre Analytical Laboratories COA Refereuce #: 023-032
3M Product: PFBS, Lot 2 Test Control Reference #: TCR-00017-71
LCh1.S Purity Profile:
Peak #:
1
2 3
Total
Retention Time (min) 1
Mas@)
I Identity I Area
1 186918
1 G29660
I PFBS 30835100
3265 1678
% Impurity 0.57 4.99
5.56
hMR Purity Profile:
I '---_ Component* CF,-CF~-CF~-CF~-SOJ', Total Impurity
iMolecular Weight 298.9
Relative Weight Percent '
97.9
0.05
1.93
0.13 0.01 2.07
*A11 components are assumed to be associated with K' cation.
Note: NMR detected impurities in boldface type.
This work was conducted under EPA Good Laboratory Practice Standards (40 CFR 160).
Prepared By:
L I
Date
2 . d&f, Scientist, Centre Analytical Laboratories
Reviewed By:
/;1
n Flaheny
PA4
Date
'Laboratory Manager, Centre Analytical Laboratories
COA023032-7 1 Revision 2.doc
Page 3 of 3
wild1$e International, Ltd.
- 27 -
Projcct Numbcr 454-1 12
APPENDIX I1
Diet Formulation Wildlife International, Ltd. Game Bird Ration'
INGREDIENTS
Fine Corn Meal
Soy Bean Meal, 48% Protein
Wheat Midds Protein Base Agway Special, 60% Protein Alfalfa Meal, 20% Protein Dried Whey Ground Limestone Eastman CalPhos
Methionine Premix + Liquid
Vitamin and Mineral Premix (see below) GL Ferm (Fermatco)' Salt Iodized Total
PERCENT (%)
44.83 30.65 6.50 6.00 4.00
3.00 2.50 0.90 0.60 0.35 0.32 0.25 0.10 100.00
VITAMIN AND MINERAL PREMIX
AMOUNT ADDED PER TON
Vitamin D3 Vitamin A kboflavin Niacin
Pantothenic Acid
Vitamin Biz
Folic Acid Biotin Pyridoxine Thiamine Vitamin E Vitamin K (Menadionc Mangancse Zinc Coppcr Iodine Iron Selenium
Dimethylpyrimidinol
Bisulfite)
2,000,000 I.C.U. 7,000,000 I.U.
6 grams 40 grams
10 grams
8 mgs 600 mgs
64 mgs 1.2 grams 1.2 grams
20,000 I.U. 5.8 grams
102 g a m s
47 g a m s 6.5 g a m s 1.5 grams 51 grams
182 mgs
'The guaranteed analysis is a minimum of 27% protein, a minimum of 2.5% crude fat and a maximum of 5% cnide fiber. 'Fcrmrntation By-Products (Source of Unidentified Growth Factors).
wildlife Intemntional, Ltd.
- 28 -
Project Niinibcr 454- I 12
APPENDIX I11 Diet Preparation
Weight and volume of constituents used to prepare test diets:
Nominal Concentrations
(ppm a.i.)
Test Substance
(9)
Basal Ration
(a)
0 1000 .I 7 8 0 3160 5620 10000
9.2496 16.4645 29.2291 5 1.9837 92.497 1
8820.0 8810.8 8803.5 8790.8 8768.0 8727.5
Diets were prepared as follows:
Corn Oil (mL)
195 195 195 195 195 195
5000.0 g of basal ration was weighed into a tared Hobart mixing bowl.
195 mL of corn oil was added to the ration and the bowl contents were mixed for approximately 5
minutes on a Hobart mixer.
1
The test substance was weighed in a tared weigh boat.
Approximately 100 g of basal ration was removed from the mixing bowl and placed in a Warin@ blender.
The test substance was added to the blender, the weigh boat was rinsed with additional ration
from the mixing bowl, with the rinse also being placed in the blender.
The blender contents were blended for approximately 1 minute and transferred to the mixing bowl. The blender was rinsed with uncontaminated ration from the mixing bowl, with the rinse also being returned to the mixing bowl.
The bowl was placed on a Hobart mixer and the contents were mixed for approximately five minutes. The remaining ration was weighed and added to tlic bowl and the contents were mixed for approximately tcn morc minutes.
The diet was transferred to a labeled paper feed bag.
The control diet was prepared by weighing 5000.0 g of basal ration into a tared Hobart mixing bowl. 195 mL of corn oil was added and the bowl was placed on the Hobart mixer and the
contcnts were niixcd for approximately five minutes. The remaining ration was weighed and addcd to the bowl. The contents of the bowl were mixed for approximately ten minutes. Diet was transferred to a labeled paper feed bag.
wiIdlife InternationaI, Ltd.
- 29 -
APPENDIX IV Analytical Methods and Results
Project Nuinbcr 454-1 12
wild1ife InternntionnI, L td.
- 30 -
Projcct Numbcr 454- I I2
Appendix IV
Table 1 Typical HPLC/MS/MS Operational Parameters
INSTRUMENT: ANALYTICAL COLUMN:
Hewlett-Packard Model 1 100 High Performance Liquid Chromatograph with a Perkin-Elmer API 3000 LC Mass Spectrometer equipped with a Perkin-Elmer TurboIonSpray ion source. Operated in multiple ion reaction monitoring (MRM) mode.
Keystone Prism RP column (50 mm x 2 mm I.D., 3-pm)
GUARD COLUMN:
Keystone Javelin CT8column (20 mm x 2 mm ID.)
OVEN TEMPERATURE:
40C
STOP TIME:
5.00 minutes
FLOW RATE: MOBILE PHASE:
200 pL/minute
10% NANOpur$Water@ with 0.1% Ammonium Formate: 90% Methanol
INJECTION VOLUME: T-7485 RETENTION TIME:
5.00 pL Approximately 2.1 minutes
T-7485 MONITORED MASS:
299.0 amu
wiIdlife InternntionaI, Ltd
-31 -
Project Number 454-1 12
Example Calculations
Appendix IV Table 2
Sample number 454- 112,113-7, nominal concentration of 1000ppm a.i. in avian feed Peak Area = 1157259 Y-intercept = 287382.5938 Slope = 3571005 Primary Dilution:
Initial Weight (Wi) = 10.0 g Original Final Volume (VI)=200 mL
Secondary Dilution: Initial Volume (Vi) = 0.0500 mL Final Volume (V;) = 10.0 mL
Overall Dilution Factor (V,/Wi x VdVJ = 4000 mL/g = 4000 L/Kg
eak area - y-intercept)
T-7485 at instrument (mg a.i./L) = ('
slope
- (1157259 - 287382.5938)
-
'357 1005
= 0.24359 mg a.i./L
T-7485 in sample (mg a.i./Kg) = T-7485 at instrument (mg a.i./L) x overall dilution factor (L/Kg)
0.24359 mg a.i. 4000 L
T-7485 in sample (mg a.i./Kg) = L
X-
Kg
= 974 mg a.i./Kg = 974 ppm a.i.
T-7485 ( m a.i.) in sam le Percent'of Nominal Concentration = T-7485 (ELm a.i.)non,in:l x 100
_-- 974 x 100 = 97.4%
1000
Instrument Software: MacQuan, version 1.6.
W i l d l f e International, Ltd.
- 32 -
Appendix IV
Table 3 Matrix Blank and Fortifications Analyzed Concurrently with the Samples
Project Number 454-1 12
Number
(454-112,113)
Sample Type
Interval
Concentration of T-7485 (ppm a.i.)
Fortified
Measured'.'
Percent Recovery'
Mean Percent of Nominal
MAB- 1
Matrix Blank
Day 0
0
<40
MAS-1
Matrix Fortification
Day 0
150
137
91.1
87.7
MAS-2
Matrix Fortification
Day 0
3000
2600
86.7
MAS-3
Matrix Fortification
Day 0
12000
10200
85.2
\
MAB-2
Matrix Blank
Day 5
0
<40
MAS-4
Matrix Fortification
Day 5
150
157
105
99.0
MAS-5
Matrix Fortification
Day 5
3000
2810
93.5
MAS-6
Matrix Fortification
Day 5
12000
11800
98.6
MAB-3' MAS-73
Matrix Blank Matrix Fortification
Day 0 Day 0
0
<40
1so
143
95.6
98.2
MAS-g3
Matrix Fortification
Day 0
3000
2990
99.6
MAS-9'
Matrix Fortification
Day 0
12000
11900
99.4
I The lowest level analytical standard was equivalent to a calculated value of 40 ppm a i in the matrix blank extract. Measured values greater than or equal to the ppm a i . equivalent are reported. 'Measured and percent recovery values calculated using MacQuan ver. 1.6 and rounded to three significant figures. Manual calculations may vary slightly. 'Quality control samples analyzed concurrent with re-extracted sample 454- 112,113-8 (Table 5 ) .
wildlijie International, Ltd.
- 33 -
Appendix IV
Table 4 Homogeneity of T-7485in Avian Diet
Nominal Concentration
(ppm a.i.) 1000
Sample I.D. Number
(454-1 12,113) 2 3 4 5 6 7
Location Sampled
In Mixing Vessel Top Left Top Right
Middle Left ~ Middle Right
Bottom Left Bottom Right --+
T-7485 Concentration Measured'
(ppm a.i.) 829 908 954 995 966 974
10000
14
Top Left
15
Top Right
16
Middle Left
17
Middle Right
18
Bottom Left
19
Bottom Right
10000 9230
10300
8720 8850
9510
'Measured values were not corrected for proceduralrecoveries based on sample sets (Table 3)
Project Number 454-1 12
Mean (X) Standard Deviation (SD) Coefficient of Variation (CV)
X= 938 SD =60.6 CV = 6.46
Mean Percent of Nominal
93.8
X= 9440
94.4
SD = 627
CV = 6.65
wildlife International, Ltd.
Project Number 454-1 12
- 34 -
Appendix IV
Table 5
Ambient Stability of T-7485 in Avian Diet During the Northern Bobwhite Dietary LC50 Study
~
T-7485 Concentration
Nominal Concentration
(pprn a i . )
Sample Number (454-1 12,113)
Week I , Day 0
Measured' (ppm a.i.)
Mean Measured (ppm a.i.)
Mean Percent of Nominal
Sample Number (454-1 12-)
Week I , Day 5
Measured' (pprn ai.)
Mean Measured (pprn a.i.)
Mean Percent of
Day 0
0
1
< 40?
20
< 403
1000
2-7'
938'
L 93.8
21
22
1100 1080
1090
116
1780
8
1 7704
1700
95.5
23
9
1630
24
1880
1830
108
1770
3160
IO
3000
2940
93 .O
25
3170
2880
98.0
11
2880
26
2590
5620
12
5420
5420
96.4
27
13
5410
28
6150
6060
112
5970
10000
14-19'
9440'
94.4
29
10300
10500
111
30
10700
~~
'Day 0 values are from homogeneity samples presented in Table 4. 'Measured values were not corrected for mean procedural recoveries based on sample sets (Table 3). 3The lowest level analytical standard was equivalent to a calculated value of 40 ppm a.i. in the matrix blank extract. Measured values greater than
or equal to the ppni a.i. equivalent are reported. 4Result is the mean of duplicate re-extraction of original sample. Original recovery was 78.2% of nominal concentration due to procedural error.
Re-extraction duplicate measured values were 1760 and 1780 ppm a i , representing 99.0% and 99.9% of the nominal concentration, respectively.
W i l d i f e International, Ltd.
- 35 -
Projcct Numbcr 454- 1 I2
Appendix IV Analytical Method Flowchart for the Analysis of T-7485 in Avian Diet
Prepare matrix fortification samples in the desired avian feed stock using the dry mix technique.
1
Weigh 10 g samples of the matrix blank, matrix fortification, and test samples into weigh boats and transfer to 8-oz. French square glass bottles. Record weights.
1
For each sample, measure 100 m L s of methanol with a graduated cylinder and transfer into the French square bottle.
1
Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm.
2 Filter into a 200-mL volumetric flask using a powder innel and glass wool. Rinse retained feed 3 times with methanol into filtrate. Bring to volume with methanol. i
Transfer an aliquot of each sample into a IO-mL B-D syringe fitted with a 0.45 pm Acrodisc" and filter into a
scintillation vial.
1
Prepare appropriate dilution(s) to bring final concentration into the calibration range of the LC/MS/MS methodology. Use 50% methanol:50% NANOpure" water dilution solvent.
1
Ampidate and submit samples for HPLC/MS/MS analysis
Figure 1. Analytical method flowchart for the analysis of T-7485 in avian diet.
wildlife International, Ltd.
- 36 -
Appendix IV
Area
2 1 00000180000015000001200000-
900000~ 600000
Projcct Number 454- 1 12
o i 3 0.0
I
~
1
1
~
1
1
,
,
1
1
1
,
,
,
,
,
,
/
,
,
,
,
,
1
0.06 0.12
0.18
0.29
0.30
0.36
0.42
0.48
Conc. mg a.i./L
Figure 2. Representative calibration curve for T-7485. Slope = 3571005, y-Intercept = 287382.5938, r = 0.99518.
WiId life InternationaI, Ltd.
- 37 -
Projcct Numbcr 454- I 12
Appendix IV
~~~
PFBS-1
STD 0.100 rng a.i./L
5292-OOl A-1
4.98 in 1 period
Fws
1
No lnternai Standard
Use Area
1: 4.97 MRM. 298 scans
299.0. Noise Thres.
100.0
Quant Thres.
50.0
Min. Width
12
Mult. Width
10
Base. Width
35
RT Win. (sea) 2 0
Smooth
1
Expected FIT
2.28
Area
614522
Height
89890
Start Time
1.98
End Time
2.46
Integration Width Retention Tme Integration Type
0.49 2.1 1
A - BE!
Tue, Jun 11, 2002 13:43
1Ool
intensity: 400000 cps
126
10
08 5 1 8 3
180 207
253
I
I
I
v
I
I
I
1
I
I
a
.
I
s
41
81
121 161 201 2 4 1 281 Scan
0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time
Figure 3 . Representative ion chromatogram of a I6w-level T-7485 standard, 0.100 mg a.i./L.
Wildlife Internntional, Ltd.
-38 -
Appendix IV
Project Nitmbcr 454- 1 I2
PFBS-5
STD 0.500 mg a.l./L
5292-00lA-5
4.98 in 1 period FfE No lnlernal Slandard Use Area
1: 4.97 MRM. 298 scans
299.0.
Noise Thres.
100.0
Quanl Thres. Min. Width Mult. Widlh
50.0 12 10
Base. Width RT Win. (secs) Smooth Expected RT
35 * 20 1
2.28
Area
1918 26
Height
247205
Start Time
1.98
End Time
2.56
Integralion Widiir
0.59
Retention Time Integralion Type
2.1 1
A - BB
Tue, Jun 1 1 , 2002 14:07
1ool
intensity: 400000 cps
126
7i 6
i
514
3
2
1
41
8 1 121 161 201 241 281 Scan
0.69 1.36 2.03 2.70 3.37 4.04 4.71 Time
I
Figure 4. Representative ion chromatogram of a hi1gh-level T-7485 standard, 0.500 mg a.i./L.
wildlife Internntiona1, L td.
- 39 -
Appendix IV
Project Niiinbcr 454-1 12
PFES-7
MAB-1
454-112.1 13-
Tue. Jun 11. 2002 1 4 1 9
4.98 in 1 period
Fms
No Internal Standard
Use Area
1: 4.97 MRM. 298 scans
299.0-
Noise Thres. Quanl Thres.
100.0 50.0
Mln. Width
12
Mull. Widlh
10
Base. Width
AT Win. (secs)
Smooth Expected AT
3 5 .' 20 1 2.28
Area
0
Height 0
Start Time End Time
0.00
0.00
lntegration Width
0.00
Retention Time Integration Type
0.00
ld
o
d
iniensily: 400000 cps
1
51
8
r
41
0.69
77 107126
192 233254 285
a
r
,
*
,
,
,
b
,
,
,
,
8 1 121 161 201 2 4 1 2 8 1 Scan
1.36 2.03 2.70 3.37 4.04 4.71 Time
Figure 5. Representative ion chromatogram of a rTfdtrix blank sample, (454-1 12,113-MAB-1). The arrow indicates the approximate retention time of T-7485. 10-g sample, Overall dilution = 400X.
Wildlife International, Ltd.
- 40 -
Appendix IV
Projcct Numbcr 454- I 12
pms-a
MAS-1
454-1 12,113-
Tue, Jun 11, 2002 14 2 5
4.98 in 1 period Fws
No Inlemal Standard U s e Area
1: 4.97 MRM. 298 scans
299.0
Noise Thres. Quanl Thres.
Min. Widlh Mull. Width Base. Width AT Win. (sea) Smooth Expected AT
100.0
50.0. 12 10
35 20
1 2.28
Area
1507578
Helohl
20301 7
Slai Time ~
1.98
End Time Integration Width Relention Time Integration Type
2.56
0.59
A
-2.1 1 88
intensily: 400000 cps 10
5 4 3 2 1
0.69
121 161 201 241 281 &an 1.36 2.03 2.70 3.37 4.04 4.71 Time
1
Figure 6. Representative ion chromatogram of a matrix fortification (454-1 12,113-MAS-I). Nominal concentration 150 ppm a i , 10-g sample, Overall dilution = 400X.
W i l d l f e International, Ltd.
-41 -
Appendix IV
Project Numbcr 454-1 12
PFBS-20
27
454-1 12-
Wed, Jon 12, 2002 16:21
m4.98 in 1 period
No Internal Standard
Use Area
1: 4.97 MRM. 298 scans
90
299.0.
Noise Thres. Quanl Tiyes.
100.0 50.0
Min. Width
12
Mult. Width
10 *
Base. Width
35
RT Win. (secs) 2 0
Smooth
1
ExpectedRT
2.28
Area
1399373
Height
156107
Start Time
2.01
2
End Time
2.60
1
Integration Width
0.59
Retention Time
Integration Type
2.16
A - BB
I
intensity: 400000 cps
I
1? 9
I
41 0.69
81 1.36
121 2.03
161 2.70
201 3.37
241 4.04
2 8 1 Scan 4.71 Time
Figure 7. Representative ion chromatogram of an avian diet sample on Day 5 , 543-1 12-27 Nominal concentration 5620 ppm a i , 10-g sample, Overall dilution = 20000X.
wildlife Internntional, Ltd.
-42 -
Project Number 453-1 12
Experiment a1 Group
APPENDIX V Cumulative Mortality by Pen from a Northern Bobwhite Dietary Toxicity Study with T-7485
No. Dead Per No. Exposed
Pen
Exposure Period
No. Dead Per No. Exposed Post-Exposure Period
Control 0 0 0 0 0 0
Treatment
1000 1000
1780 1780
3160 3 160
5620 5620
10000 10000
1
015
015
015
015
015
015
2
015
015
015
015
015
015
3
015
015
015
015
015
015
4
015
015
015
015
015
015
5
015
015 . 015
015
015
015
6
015
015
015
015
0/5
015
\
1
016
016
016
016
016
016
2
016
016
016
016
016
016
1
016
016
016
116
116
1I6
2
016
016
016
016
016
016
1
016
016
016
016
016
016
2
016
016
016
016
016
016
1
016
016
016
016
016
016
2
016
016
016
016
016
016
I
016
016
016
016
016
016
2
016
016
016
016
016
016
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
015
016
016
016
016
016
016
016
116
116
116
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
016
The LC50 value was estimated to be greater than 10,000 ppm a.i., the highest concentration tested. I - Halfofthe birds were euthanized on Day 8 for necropsy and tissue collection. - No mortalities occrirred in any of the control or treatment groups after Day 8.
wildlife International, Ltd.
-43 -
Project Number 454-1 12
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Dietary Toxicity Study with T-7485 Page 1
Experimental
Group (ppm a.i.) Bird
Total Day 0 Day 5 Change Day 8 Change Day 15 Change Day22 Change Change
0
1
17 30 13
2
20 30 10
3
19 31 12
4
21 34 13
5
23 36 13
Mean 20 32 12
SD
2
3
1
39 9
38
8
41 10
50 16
47 11
43 11
5
3
! 0
1
18 25
7
2
2
20 30 10
4270 10
3
21 34 13
43
9
4
19 36 17
48 .* 12 . -
5
22 37 15
49 12
Mean 20 32 12
41
9
SD
2
5
4
.9
4
0
1
18 29 1 1
42 13
2
19 32 13
43 1 1
3
20 32 12
45 13
4
22 30
8
33 3
_i
23 35 12
46 1 1
Mean 20 32 1 1
42 10
SD
2
7
2
5
4
-Indicates bird was euthanized on Day 8 for sampling
wildlife International, Ltd.
- 44 -
Projcct Nunibcr 454- 1 12
APPENDIX VI
Individual Body Weights (8)from a Northern Bobwhite Dietary Toxicity Study with T-7485 Page 2
Experimental
Group
Total
0
1
17 26
9
34
8
54
20
79
25
62
2
19 33 14
44 11
76
32
105
29
86
3
20 33 13
46 13
80
34
116
36
96
4
21 33 12
40
7
67
27
93
26
72
5
23 37 14
48 11
78
30
109
31
86
Mean 20 32 12
42 10
71
29
100
29
80
SD
2
4
2
6
2
11
5
15
4
13
0
1
18 25
7
32 1 7
57
25
78
21
60
2
19 26
7
33
7
59
26
83
24
64
3
20 33 13
42
9
69
27 103
34 . 83
4
20 29
9
38 ' 9
60
22
95
35
75
5
23 37 14
48 1 1
84
36
113
29
90
Mean 20 30 10
39
9
66
27
94
29
74
SD
2
5
3
.7
2
11
5
14
6
13
0
1
19 26
7
34
8
61
27
90
29
71
2
17 24
7
30
6
56
26
83
27
66
3
20 34 14
44 10
73
29
96
23
76
4
21 35 14
48 13
73
25
100
27
79
5
22 29
I
33 4
56
23
76
20
54
Mean 20 30 10
38
8
64
26
89
25
69
SD
2
5
4
8
3
9
2
10
4
IO
Group
Mean 20 31 1 1
41
9
67
27
95
28
75
Total
SD
2
4
3
6
3
10
4
13
5
12
wildlife International, Ltd.
- 45 -
Project Number 454- 1 12
APPENDIX VI
Individual Body Weights (8) from a Northern Bobwhite Dietary Toxicity Study with T-7485 Page 3
Experimental
Group
Total
1
17 26
9
1000
2
18 20
2
3
19 26
7
4
20 29
9
5
20 28
8
6
23 29
6
Mean
20 26
7
SD
2
3
3
35
9
24 4
33
7
33
4
29
1
32
3
31
5
4
3
!
1
17 28 11
39 11
63
24
87
24
70
1000
2
17 28 1 1
35 - 7
62
27
84
22
67
3
20 27
7
31
4
48
17
74
26
54
4
20 30 10
40 10
64
24
85
21
65
5
22
34
12
45
11
67
22
94
27
72
6
23 38 15
51 13
77
26
101
24
78
Mean 20 31 1 1
40
9
64
23
88
24
68
SD
2
4
3
7 3
9
4
9
2
8
Group
Mean
20
29
9
36
7
64
23
88
24
68
Total
SD
2
4
3
7 4
9
4
9
7
8
-Indicates bird was euthanized on Day 8 for sampling.
wildlife International, Ltd.
- 46 -
Project Nuiiibcr 454-1 12
APENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Dietary Toxicity Study with T-7485 Page 4
\
Experimental
Group (ppma.i.) Bird
Total Day0 Day5 Change Day8 Change Day 15 Change Day22 Change Change
1
17
a
a
a
a
a
a
a
B
a
1780
2
20 29
9
39 10
3
20 22
2
34 12
4
21 31 10
40
9
5
22 28
6
35
7
6
21 23
2
30
7
Mean 20 27 6
36
9
SD
2
4
4
4
2
f
1
17 24
7
34 10
62
28
88
26
71
1780
2
20 32 12
41
9
73
32 104
31
84
3
19 26
7
26
0
54
28
79
25
60
4
23 34 11
45 11
73
28 100
27
77
5
20 30 10
37
7
67
30
95
28
75
6
22
27
5
34
7
60
26
85
25
63
Mean 20 29
9
36
7
65
29
92
27
72
SD
2
4
3
7 4
8
2
9
2
9
Group
Mean 20 28
7
36
8
65
29
92
27
72
Total
SD
2
4
3
5
3
8
2
9
2
9
- Indicates bird was euthanized on Day 8 for sampling.
'' N o data available due to mortality
Wildlife International, Ltd.
-47 -
Project Nuinbcr 454-1 12
1
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Dietary Toxicity Study with T-7485 Page 5
Experimental
Group
Total
(ppm a.i.) Bird Day 0 Day 5 Change Day 8 Change Day 15 Change Day 22 Change Change'
1
18 26
8
29
3
3160
2
20 28
8
34
6
3
19 29 10
36
7
4
21 24
3
30
6
5
20 28
8
31
3
6
23 33 10
42
9
Mean 20 28
8
34
6
SD
2
3
3
5
2
1
1
17 25
8
32
7
59
27
88
29
71
3160
2
19 31 12
39
8
65
26
89
24
70
3
20 27
7
35 - 8
59
24
85
26
65
4
21 33 12
47 14
76
29
103
27
82
5
21 34 13
43
9
75
32 102
27
81
6
23
36
13
48
12
76
28
IO1
25
78
Mean 20 31 1 1
41 10
68
28
95
26
75
SD
2
4
3
6
3
8
3
8
2
7
Group Total
Mean 20 30
9
SD
2
4
3
~
~~
37
8
68
28
95
26
75
7
3
8
3
8
2
7
-Indicates bird was euthanized on Day 8 for sampling.
wildlife Internntiona1, Ltd.
- 48 -
Project Number 454-1 12
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Dietary Toxicity Study with T-7485 Page 6
Experimental
Group (ppma.i.) Bird
~~
Total Day0 Day 5 Change Day 8 Change Day 15 Change Day22 Change Change
1
18 23
5
33 10
57
24
84
27
66
5620
2
19 25
6
36 11
68
32
100
32
81
3
20 29
9
39 10
69
30
95
26
75
4
20 24
4
31
7
58
27
83
25
63
5
21 29
8
36
7
59
23
92
33
71
6
22 25
3
36 11
65
29
92
27
70
Mean 20 26
6
35
9
63
28
91
28
71
SD
1 3 2
3, 2
5
4
7
3
6
i
1
18 17 -1
24
7
5620
2
18 21
3
26
5
3
19 22
3
26 4
4
20 22
2
29
7
5
22 27
5
35
8
6
23 33 10
42
9
Mean 20 24
4
30
7
SD
2
6
4
7
2
Group
Mean 20 25
5
33
8
63
28
91
28
71
Total
SD
2
4
3
6
2
5
4
7
3
6
-Indicates bird was euthanized on Day 8 for sampling.
Wildlife International, Ltd.
-49 -
Projcct Numbcr 454- 1 12
APPENDIX VI
Individual Body Weights (g) from a Northern Bobwhite Dietary Toxicity Study with T-7485 Page 7
Experimental Group
(ppma.i.) Bird
Total Day0 Day5 Change Day8 Change Day 15 Change Day22 Change Change
1
18 25
7
34
9
66
32
95
29
77
10,000
2
17 18
1
27
9
47
20
68
21
51
3
22 22
0
33 11
62
29
90
28
68
4
21 28
7
36
8
63
27
82
19
61
5
22 31
9
41 10
73
32
96
23
74
6
23 25
2
37 12
71
34
98
27
75
Mean 21 25
4
35 10
64
29
88
25
68
SD
2
5
4
5
1
9
5
11
4
10
/
1
18 19
1
20
1
10,000
2
24 33
9
42
9
3
17 16 -1
21
5'
4
20 18 -2
21
3
5
21 21
0
25
4
6
21
19
-2
* 25
6
~~
Mean 20 21
I
26
5
SD
2
6
4
8
3
Group
Mean 20 23
3
30
7
68
Total
SD
2
6
4
8
3
5
9
4
11
IO
-Indicates bird \vas euthanized on Day 8 for sampling.
w i l d1fe Intemational, I ,td.
- 50 -
Projcct Numbcr 454-1 12
APPENDIX VI1
Feed Consumption (g/bird/day) by Pen from a Northern Bobwhite Dietary Toxicity Study with T-7485 Page 1
Experimental Group
(ppma.i) Pen
Exposure Period Post-Exposure Period Post-Exposure Period Post-Exposure Period
Days 0-5
Days 6-8
Days 9-15
Days 16-22
Control
1
9
13
0
2
9
9
3
7
10
4
8
13
12
22
5
9
17
22
23
6
11
14
18
20
I
Mean'
9
13
18
22
SD
1
3-
5
1
' Mean values calculatedusing Excel in full-precisionmode. Manual calculation may vary.
-No data available, birds euthanized on Day 8 for sampling.
wildlife International, Ltd.
-51 -
Projcct Number 454-1 12
APPENDIX VI1
Feed Consumption (ghirdday) by Pen from a Northern Bobwhite Dietary Toxicity Study with T-7485 Page 2
Experimental Group
(ppm a.i.)
1000
~
~
~~~
Exposure Period Post-Exposure Period Post-Exposure Period Post-Exposure Period
Pen
Days 0-5
Days 6-8
Days 9-15
Days 16-22
1
7
9
2
7
9
16
23
Mean'
7
9
16
23
1780
1
6
8
2
7
8
11
19
Mean'
6-
8
11
19
3160
1
6
11 1
2
6
9 ..
14
19
Mean'
6
10
14
19
5620
1
5
9
11
17
2
7
15
Mean'
6
12
11
17
10000
6
9
11
19
4
7
Mean'
5
8
I1
19
' Mean valuc.s calculated using Excel in full-precision mode. Manual calculation may vary.
-No data available. birds euthanized on Day 8 for sampling.
WildlifeInternational, Ltd.
-52-
Project Number 454-1 12
APPENDIX VI11 Individual Liver Weights (g) from a Northern Bobwhite Dietary Toxicity Study with T-7485
Page 1
Day 8
Replicate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
0 0.905 0.941 0.934 1.239 1.004 0.728 1.011 1.118 0.993 1.157 1.098 0.904 1.091 1.007 1.127
1000 0.725 0.506 0.707 0.802 0.639 0.960
Test Concentration (ppm a i . )
1780
2
1.251 0.760 0.987 1.134
3160 0.922 0.824 0.820 0.766 1.147
5620 0.755 0.478 0.743 0.957 1.130
0.937
1.103
1.134
1-
Mean'
1.017
0.723
1.014
0.930
0.866
SD
0.127
0.153
0.188
0.160
0.256
- Indicates bird was euthanized on Day 22 for sampling. 'Mean values calculated using Excel in full-precision mode. Manual calculation may vary. *No liver weight due to mortality.
10,000 0.545 1.100 0.513 0.653 0.652 0.769
0,705 0.214
Wildlife International, Ltd.
- 53 -
Project Number 454- 1 12
APPENDIX VI11 Individual Liver Weights (g) from a Northern Bobwhite Dietary Toxicity Study with T-7485
Page 2
Test Concentration (ppin ai.)
3 0
1000
1780
3160
5620
.I
2.225 2.290 2.072 2.514 2.81 3
2
3.575 2.357 2.562 1.995 2.490
3
3.444 2.741 1.973 2.,208 2.745
4
2.779 2.096 2.682 2.764 2.030
5
2.600 2.208 2.710 3.066 2.033
6
I .905 2.227 2.309 2.638 2.426
7
2.033
8
2.927
9
2.747
10
2.716
11
2.48 1
12
2.268
13
I .948
14
2.389
15
2.068
Mean'
2.540 2.320 2.385 2.531 2.423
10,000 2.402 1.851 2.075 2.196 2.298 2.913
2.289
- Indicates bird was euthanized on Day 8 for sampling.
/
wildlife International, Ltd.
--_ - ..
- 54 -
Appendix IX
Changes to Protocol
Project Nurnbcr 454- I 12
The study was conducted in accordance with the approved Protocol with the following change.
1. The protocol was amended to indicate tissue samples will be analyzed at the Sponsor's discretion and results of tissue analysis will not be reported. Addtionally, change the Sponsor's Representative to Sue Beach.
2. The protocol was amended to indicate statistical comparisons of body weight and liver weight data will be made.
3. The protocol was amended to change the sponsor's representativeto John Newsted.
' wildlife International, Ltd.
- 55 -
APPENDIX X
Personnel Involved in the Study
Project Numbcr 454-1 12
The following key Wildlife International, Ltd. personnel were involved in the conduct or management of this study:
(1) Mark Jaber, Wildlife Toxicologist (2) Joann B. Beavers; Director of Avian Toxicology (3) Linda R. Mitchell, Director of Ecotox Operations
(4) Sean P. Gallagher, Senior Biologist, Avian Toxicology ( 5 ) Willard B. Nixon, Ph.D., Director of Chemistry (6) Timothy Z. Kendall, Supervisor of Chemistry ( 7 ) Raymond L. Van Hoven, Ph.D., Scientist, Chemistry