Document 3eN1wa72az2LNN1dQkmpRnGvO

26) OECD 206-OPPTS 850.2300, Pilot chronic avian toxicity (Northern Bobwhite), ldlife International, Ltd. ECOTOXICOLOGY & ANALYTICALTESTING SERVICES Contains No CBI DEC 0 9 2003 July 1 1,2003 Dr. John Newsted Entrix East Lansing Office 4295 Okemos Road, Suite 101 Okcmos, MI 48864 Dear John: The following is a summary ofthe findings for the study: T-7485: A Pilot Reproduction Studywith the Northern Bobwhite (WLI ProjectNo.454-I15;3M EnvironmentalLab ProjectNo. E03-0214). Adult northern bobwhite were exposcd to T-7485 at nominal dietary concentrations of 75,200,550 and 1500ppm a.i. for a period o r 6 weeks. A control group, fed non-treated diet, was maintained concurrently with the treatment groups. Each treatment and the control group consisted of.five pairs of birds, housed with one male and one female per pen. At thc end of Week 6 , all surviving birds were euthanized and subjected to gross necropsy. Effects on adult health, body weight gain, feed consumption and egg production were evaluated. Egg, blood and liver samples were collccted for possible analyses. The adult birds werc observed daily for mortality, abnormal behavior and signs of toxicity. Body weights were measured at test initiation, on Weeks 2 and 4, and at adult termination. Feed consumption for cach pen was measured over a seven-day period each week throughout the test. Necropsies were performed on all birds. Liver weights were recorded for all surviving birds. Liver and blood samplcs (when available)were collccted at the time of necropsy for analysis. During the test, the number of eggs laid in each pcn was recorded to evaluate egg production. Following enumeration, eggs laid during the first five weeks of the tcst were disposed of by incineration. All eggs laid during the final week of exposure were stored refngerated. Two eggs (when available) were indiscriminately chosen from each pcn for possible analysis. All blood, liver and egg samples were shipped to 3M Corporation for possible analysis. Mortal i ties There were no treatment-related mortalities in any of the trcatment groups. While no mortalities occurred in the control group or in the 75,200,550 ppm a.i. treatment groups during the course ofthe test, one incidental mortality occurrcd in the 1500ppm ai. treatment group. The single mortality in the 1500 ppm a.i. treatment group was a hen (Pen 322) that was noted with an apparent neck injury on Day 2 of the test and was subsequently euthanized, three days following experimental start. Priorto euthanasia,the hen exhibited ventral head curl, wing droop, ataxia (loss of coordination),a ruffled appearance and lethargy. At necropsy the bird was thin, with a body weight of 162 grams. Extcrnally, feather loss was noted on the hcad and neck. Internally, there was a loss of muscle mass and a small bruise around the left ear. The liver contained scattered 8598 Commerce Drive . Easton, Maryland 21601 .Te1.410-822-8600. Fax 4 10-822-0632 . E-mail ecotox@wildlifeinternational.com ~ ~~ European Office: Bergkatnpweg I . 7231 CLWarnsveld .The Netherlands -7el.3 I(0) 575 - 573848 ' Fax. 3I (0)575 - 5748I3 ' E-mail:aleopold@worldonline.nl Dr. John Newsted July 11,2003 Page 2 white foci. The abdominal cavity showed evidence of egg yolk peritonitis and the ovary was regressing. Since the bird was euthanized within three days of experimental start and the mortality was clearly not treatmentrelated, the hen was replaced. The replacement bird was from the same lot that had acclimated with the test birds for use as a potential replacement. The data reported for Pen 322 are that of the replacement bird. Clinical Observations Scvcral birds wcrc noted with foot lesions and feather loss as a result of pen wear and/or pen mate aggression during the course of the test. An incidental clinical sign, lameness, was noted for one bird (Pcn 320) in the 550 ppm a.i. treatment group during Week 2 of the test. All other birds were normal in appearance and behavior for the duration of the test. Adult Body Weight and Feed Consumption When compared to the control group, there wcrc no apparcnt trcatmcnt-related effects on adult male or female body weight at the 75, 200 or 550 pprn a.i. test concentrations, or on female body weight at the 1500 ppm a.i. test concentration. There was a slight, hut consistent, reduction in adult male body weight at the 1500 pprn a.i. test concentration during each weight interval that may have been treatment-related. Mean body weight measurements are presented in Table 1, and individual body weight measurements are presented in Appendix I. Due to excessive wastage by some birds, feed consumption was variable between pcns. However, when compared to the control group, there appeared to be no treatment-related effects on feed consumption at the 75, 200 or 550 pprn a.i. test conccntrations. There werc slight, but consistent, reductions in feed consumption at the 1500ppm a.i. treatment group during each feeding interval that may have been related to treatment. Mean feed consumption measurements are shown in Table 2, and feed consumption measurements by pen are presented in Appendix 11. & Production When compared thc control group, there were no apparent treatment-related effects upon egg production at the 75 and 200 ppm a.i. test concentrations. While there was a slight reduction in egg production at the 75 ppm a.i. conccntration, the reduction was not dose-responsive and was primarily due to two hens (Pens 309 and 3 10) which laid poorly during the final week of the test. Subsequent gross necropsy of these two hens revealed egg remnants in the abdominal cavity. However, there were reductions in mean egg production at the 550 and 1500 pprn a.i. test concentrations for which a treatment-related effect could not be precluded. Mean cgg production by concentration is presented in Table 3. Individual egg production data are presented in Appendix 111. Gross Necropsy At the end of Weelc 6 (Day 42), all adult birds were euthanized and subjected to gross necropsy. All findings observed were considered to be incidental to treatment. Necropsy findings are reported in Table 4 and Appendix IV. Liver WeiFht When compared to the control group, there were no apparent treatment-related effects on liver weight at the 75,200 or 550 ppm a i . test concentrations, or among males at the 1500 ppm a.i. test concentration. There was a slight reduction in mean female liver wcight at the 1500 ppm a.i. test concentration. Liver weights are presented in Table 5 and Appendix V. Dr. John Newsted July 1I , 2003 Page 3 Results of Diet Analvses The procedures followed to analyze the dietary samples are presented in Appendix VI. None of the control samples showed any indication of the presence of the test substance or of the presence of co-eluting substances at the characteristic retention time of the test substance. Diet samples were collected on Week 1, Day 0 of the test from the 75, 200,550, and 1500 ppm a.i. tcst lcvels to evaluate the homogeneity of the tesl * substance in the diet. The means and standard deviations for the 75,200, 550, and 1500 ppm a.i. test levels were 75.6 i 4.85 ppm ai., 218 8.87 ppm a i , 561 1. 17.2 ppm a.i., and 1530 41.2 ppm a i , respectively. The corresponding coefficients of variation were 1.32%, 4.06%, 3.06%, and 2.69% (Appendix VI, Table 4). The mean measured T-7485 concentrations from thc 75,200,550, and 1500 ppm a.i. test levels were 101%, 109%, 102%, and 102%, respectively, of the nominal concentrations. A typical ion chromatogram of a test sample is shown in Appendix VI, Figure 7. Diet samples collected on Week 6, Day 0 of the test from the 75,200,550, and 1500 ppm a.i. test levels * were analyzed to verify the concentration ofthe tcst substance in thc dict. Thc mean and standard deviation for the 75, 200, 550, and 1500 ppm a.i. test concentrations were 71.7 1.72 ppm a i , 192 f 3.21 ppm a i , 519 t 15.6ppma.i., and 1410 f 25.2 ppm a i , respectively. The corresponding coefficients ofvariation were2.40%, 1.68%, 3.01%, and 1.78% (Appendix VI, Table 5). The mean mcasurcd T-7485 concentrationsfrom the 75, 200, 550, and 1500 ppm a.i. test levels were 96%, 96%, 94%, and 94%, respectively, of the nominal concentrations. Analysis of diet samples collected from feeders after being held at ambient temperature for seven days averagcd 128%, 116%, 115%, and 109% ofthe Day 0 values for the 75,200,550, and 1500 ppm a.i. test concentrations, respectively (Appendix VI, Table 6). Results of Blood, Liver and Egg Analysis Blood, liver and egg samples were transferred to 3M Corporation for possible analysis. Conclusion Northern bobwhite were exposed to T-7485 at dietary concentrations of 0,75,200,550 and 1500 ppm a.i. for 6 weeks. No treatment-related mortalities or overt signs of toxicity were observed at any of the concentrations tested. Additionally, there were no apparent treatment-related effects upon body weight or upon fccd consumption at the 75,200 or 550 pprn a.i. test concentrations. There were no apparent treatment-related reductions in egg production at the 75 or 200 ppm a.i. test concentrations. However, there were slight, but consistent, reductions in male body weight and feed consumption in the 1500 ppm a.i. treatment group that may have been related to treatment. There were also reductions in mean egg production at the 550 and 1500 ppm a.i. tcst concentrations and in female liver weight at the 1500 ppm a.i. test concentration that may have been related to treatment. Based upon the results of this study, the conservative no-observed-effect concentration for northern bobwhite exposed to T-7485 in the diet was 200 pprn a.i. Sincerely, ' SPG/cmh/rptllS Y Sean P. Gallagher Senior Avian Biologist WildlifeInternational, Ltd. Table 1 Mean Adult Body Weight (g) from a Northern Bobwhlte Pilot Reproduction Study with T-7485 PROJECT NO.: 454-1 15 Expcrimental Group Sex Week 0 Change Week 0-2 Week 2 Change Week 2-4 Control Male 212 3 215 0 Female 229 6 236 3 75 Male 213 8 22 1 2 ppm a i . Female 234 6 240 1 200 Male 215 1 2 16 1 ppm a.i. Female 23 1 7 238 -1 550 Male 2 18 10 229 5 ppm a.i. Female 227 2 229 -8 1500 Male 215 1 217 -9 ppm a.i. Female 230 4 234 -5 The means for body weights and body weight changeswere calculatedand rounded separately. Week 4 214 239 223 24 1 217 236 233 22 1 208 229 Change Week 4-6 2 1 1 -25 2 -3 -8 1 0 3 Week 6 217 240 224 216 219 234 226 222 208 232 Total Change 4 11 11 -18 4 3 7 -5 -8 2 Wildlve International, ltd. Table 2 Mean Feed Consumption (ghirdday) from a Northern Bobwhte Pilot Reproduction Study with T-7485 PROJECT NO.: 454-115 Experimental Group Weeks ppm a.i. 1 2 3 4 5 6 Control 22 26 23 29 27 27 75 23 25 23 28 26 28 200 22 25 23 27 28 27 550 22 25 22 26 25 23 1500 19 22 20 22 23 24 Wild1$e InternationaI, Ltd. PROJECT NO.:454-1 15 Table 3 Egg Production Data by Week from a Northern Bobuhte Pilot Reproduction Study with T-7485 Experimental Group Weeks ppm a i 1 2 3 4 5 6 Totals EggdFemaleDay Control 25 24 33 34 32 34 182 0.87 75 25 21 31 33 30 20 166 0.79 200 29 31 31 33 32 33 189 0.90 550 20 23 24 24 26 21 138 0.66 1500 16 24 27 26 22 27 142 0.69 WildlifeInternational, Ltd. Table 4 Summary of Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with T-7485 Surviving Birds Euthanized at Test Termination PROJECT NO.: 454-1 15 Finding Males ppm a.i. Control 75 200 550 1500 Numbcr of birds 5 5 Feather loss 0 0 Spleen - slightly enlarged Liver - friable A b d o m i ~ cl avity - slight egg yolk peritonitis 0 0 0 0 Abdominal cavity - yolk remnants Ovary - regressing Right oviduct - partially developed Testes - small (one or both <1.25 cm) 0 0 1 2 0 Not rcrnarkable 4 3 3 3 5 Females ppm a.i. Control 75 200 550 1500 1 1 2 4 WildlifeInternational, Ltd. -8- PROJECT NO.:454-1 15 Table 5 Mean Liver Weights from a Northern Bobwhite Pilot Reproduction Study with T-7485 Experimental Group Control 75 ppm a.i. 200 ppm a.i. 550 ppm a.i. 1500 ppm a.i. Males Mean 3.624 * Liver Weights (g) Females SD Mean SD 0.756 6.514 f 1.406 3.757 rt 0.511 6.197 f 0.948 3.743 f 0.428 6.646 f 1.181 3.916 f 0.600 6.222 f 1.328 3.655 f 0.525 5.886 f 1.110 WildlifeInternational, Ltd. Appendix I - Table 1 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with T-7485 PROJECT NO.: 454-1 15 Control Males Change Change Change Total Pen Week 0 Week 0-2 Week2 Week24 Week4 Week46 Week 6 Change 301 194 -2 192 -1 191 2 193 -1 302 2 10 1 21 1 1 212 1 213 3 303 198 2 200 1 20 1 -3 198 0 I \o 304 227 3 230 -2 228 0 228 1 I 305 232 9 24 1 -1 240 11 25 1 19 Mean 212 3 215 0 214 2 217 4 SD 17 4 20 1 20 5 24 8 The means for body weights and body weight changeswere calculated and rounded separately. Wildlife International, Ltd. PROJECT NO.: 454-1 15 Appendix I - Table 2 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with T-7485 Control Females Change Change Pen Week 0 Week 0-2 Week 2 Week 2-4 Week 4 ~~ 30 1 236 302 206 303 242 304 219 305 243 5 241 10 216 10 252 1 220 6 249 3 244 10 226 6 258 -5 215 1 250 Mean 229 6 236 3 239 SD 16 4 17 6 18 The means for body weights and body weight changes were calculated and rounded separately. Change Week4-6 -2 9 -1 1 0 1 4 Week6 242 235 257 216 250 240 16 Total Change 6 29 15 -3 7 11 12 WildlifeInternational, Ltd. PROJECT NO.: 454-115 Appendix I - Table 3 Adult Body Weight (8) from a Northern Bobwhite Pilot Reproduction Study with T-7485 75 ppm a.i. Males Change Change Change Total Pen Week 0 Week 0-2 Week 2 Week 2-4 Week4 Week44 Week6 Change 306 205 7 212 0 212 -4 208 3 307 23 1 15 246 9 255 2 257 26 308 21 1 5 216 0 2 16 2 218 7 I c3 c 309 183 7 190 -7 183 0 183 0 I 3 10 235 5 240 8 248 4 252 17 Mean 213 8 22 1 2 223 1 224 11 SD 21 4 23 7 29 3 31 11 The means for body weights and body weight changes were calculated and rounded separately. Wildlife International, Ltd. PROJECT NO.: 454-1 15 Appendix I - Table 4 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with T-7485 75 ppm a.i. Females ~ ~ ~~~ Change Change Change Total Pen Week 0 Week 0-2 Week 2 Week 2-4 Week 4 Week 4-6 Week 6 Change ~ 306 236 -6 230 3 233 -4 229 -7 307 208 17 225 -1 224 -1 223 15 308 277 0 277 4 281 -68 213 -64 cI. 309 2 17 11 228 2 230 -28 202 -15 h, I 3 10 232 7 239 -3 236 -24 212 -20 Mean 234 SD 27 6 240 9 21 1 24 1 3 23 ~~ The means for body weights and body weight changes were calculated and rounded separately. -25 27 ~~ ~ 2 16 -18 10 29 - WildlifeInternationa1, Ltd. PROJECT NO.: 454-1 15 Appendix I - Table 5 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with T-7485 200 ppm a i Males Change Change Pen Week 0 Week 0-2 Week 2 Week 2-4 Week 4 311 224 2 226 0 226 3 12 229 3 232 5 237 3 13 222 1 223 -4 219 3 14 189 -5 184 4 188 3 15 21 1 5 216 -2 2 14 Mean 215 SD 16 1 216 4 19 1 2 17 4 18 The means for body weights and body weight changes were calculated and rounded separately. Change Week 4-6 1 2 4 1 1 2 1 Week 6 227 239 223 189 215 219 19 Total Change 3 10 1 0 4 4 4 WiId1ife InternationaI, Ltd. PROJECT NO.: 454-1 15 Appendix I - Table 6 Adult Body Wcight (g) from a Northern Bobwhite Pilot Reproduction Study with T-7485 200 ppm a.i. Females Pen 311 3 12 313 3 14 315 Mean SD Week 0 229 234 209 237 244 23 1 13 Change Week 0-2 3 14 7 0 11 7 6 Week 2 232 248 216 237 255 238 15 Change Week24 4 -2 -7 -7 6 -1 6 Week4 236 246 209 230 26 1 236 19 The means for body weights and body weight changes were calculated and rounded separately. Change Week4-6 -29 5 5 9 -3 -3 15 Week6 207 25 1 214 239 258 234 22 Total Change -2 2 17 5 2 14 3 15 Wildlve International, Ltd. PROJECT NO.:454-115 Appendix I - Table 7 Adult Body Weight (g) from a Northern Bobwhte Pilot Reproduction Study with T-7485 550 ppm a i Males Change Change Pen Week0 Week0-2 Week 2 Week 2-4 Week 4 3 16 216 16 232 8 240 3 17 2 16 8 224 4 218 3 18 220 8 228 14 242 3 19 208 13 221 1 222 320 232 6 238 7 245 Mean 218 10 229 5 233 SD 9 4 7 8 12 The means for body weights and body weight changes were calculated and rounded separately. Change Week 4-6 10 -3 -40 -8 2 -8 19 Week 6 250 215 202 2 14 247 226 22 Total Change 34 -1 -18 6 15 7 19 Wildlife International, Ltd. PROJECT NO.: 454-1 15 Appendix I - Table 8 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with T-7485 550 ppm a.i. Females Change Change Pen Week 0 Week0-2 Week2 Week 2-4 Week 4 3 16 248 -5 243 -4 239 317 214 3 217 -8 209 318 232 20 252 -50 202 319 213 -8 205 2 207 320 227 -1 226 20 246 Mean 227 2 229 -8 22 1 SD 14 11 19 26 20 The means for body weights and body weight changes were calculated and rounded separately. Change Week 4-6 -3 1 10 36 -3 -7 ~~ ~~ Week 6 ~~ ~ 208 219 238 204 239 1 222 25 16 Total Change -40 5 6 -9 12 -5 21 Wild1ife Intemational, Ltd. PROJECT NO.: 454-1 15 Appendix I - Table 9 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with 1'-7485 1500 ppm a i Males Change Change Pen Week0 WekO-2 Week2 Week2-4 Week4 321 23 1 3 234 -3 23 1 322 193 5 198 2 200 323 207 4 211 0 21 1 3 24 23 1 -1 224 -42 182 325 215 1 2 16 -1 215 Mean 215 SD 16 1 217 -9 208 5 14 19 18 The means for body weights and body weight changes were calculated and rounded separately. Change Week4-6 0 0 -4 4 -1 0 3 Week6 23 1 200 207 186 214 208 17 Total Change 0 7 0 -45 -1 -8 21 Wildrife International, Ltd. PROJECT NO.:454-115 Appendix I - Table 10 Adult Body Weight (g) from a Northern Bobwhite Pilot Reproduction Study with T-7485 1500 ppm a.1. Females Change Change Pen Week 0 WakO-2 Week2 Week 2-4 Week 4 321 230 4 234 -5 229 322 204 -6 198 10 208 323 230 14 244 -32 212 324 221 11 232 -1 23 1 325 265 -2 263 1 264 Mean 230 4 234 -5 229 SD 22 8 24 16 22 The means for body weights and body weight changes were calculated and rounded separately. Change Week46 5 -2 18 -1 1 7 3 11 Week6 234 206 230 220 27 1 232 24 Total Change 4 2 0 -1 6 2 3 WildlifeInternational, ltd. Appenlx I1 - Table 1 Feed Consumption (ghirdday) from a Northern Bobwhite Pilot Reproduction Study with T-7485 PROJECT NO.: 454-1 15 Control Pen 1 2 3 4 5 6 301 24 26 25 30 30 28 302 17 20 21 24 22 23 303 21 24 24 27 25 26 304 21 23 22 27 26 27 305 26 36 25 39 33 33 MtXl 22 26 23 29 27 27 SD 3 6 2 6 4 3 WiZdlijie InternationaI, Itd. Appendix I1 - Table 2 Feed Consumption (ghirdday) from a Northern Bobwhte Pilot Reproduction Study with T-7485 PROJECT NO.: 454-1 15 75 ppm a.i. Pen 1 2 Weeks 3 4 5 6 I 306 20 21 21 28 27 29 N 0 307 24 27 27 34 29 31 I 308 22 26 22 26 26 33 309 25 27 22 26 20 25 310 20 24 22 25 26 22 Mean 23 25 SD 2 3 23 28 2 4 26 28 3 5 WildlifeInternational, ltd. Appendix I1 - Table 3 Feed Consumption (ghirdday) from a Northern Bobwhte Pilot Reproduction Study with T-7485 PROJECT NO.: 454-1 15 200 ppm a.i. Weeks Pen 1 2 3 4 5 6 I 311 20 25 24 24 27 22 N c 312 24 27 23 30 27 31 I 313 21 24 23 26 29 27 3 14 18 23 22 26 25 24 3 15 25 25 24 29 29 30 Mean 22 25 23 27 28 27 SD 3 1 1 2 2 4 WiZdZijieInternational, ltd. Appendix I1 - Table 4 Feed Consumption ( @ i d d a y ) from a Northern Bobwhite Pilot Reproduction Study with T-7485 PROJECT NO.: 454-115 550 ppm a.i. Weeks Pen 1 2 3 4 5 6 I 3 16 21 23 24 23 18 18 N N 3 17 21 24 14 27 27 25 I 318 28 28 27 25 26 25 3 19 17 22 22 27 29 23 320 22 25 23 27 26 26 Mean 22 25 22 26 25 23 SD 4 2 5 2 4 3 Wildlife International, ltd. Appendix I1 - Table 5 Feed Consumption (ghirdday) from a Northern Bobwhte Pilot Reproduction Study with T-7485 PROJECT NO.: 454-115 1500 ppm a.i. Weeks Pen 1 2 3 4 5 6 I 32 1 22 23 24 27 28 30 h, W 322 20 20 21 23 23 I 323 18 23 19 19 22 23 324 18 20 15 18 19 22 325 17 23 22 24 22 23 MGUl 19 22 20 22 23 24 SD 2 2 4 4 3 3 ~ ~ ~~ - Data not reported. On day 3 of the test, the female in Pen 322 was replaceddue to possible neck injury. Wildzife International, Ltd. PROJECT NO.: 454-1 15 Appendix 111- Table 1 Eggs Laid per Pen per Week from a Northern Bobwhltc Pilot Reproduction Study with T-7485 Control ~ Pen 1 2 3 4 5 6 Totals Eggs/Female/Day 301 6 5 7 7 7 7 39 0.93 302 4 4 7 6 6 7 34 0.81 303 5 4 7 7 6 6 35 0.83 304 5 5 6 7 6 7 36 0.86 305 5 6 6 7 7 7 38 0.90 I N P Totals 25 24 33 34 32 34 182 0.87 I WildlifeIntemationai, Ltd. Appendix I11 - Table 2 Esgs Laid per Pen per Week from a Northern Bobwhite Pilot Reproduction Study with T-7485 75 ppm a.i PROJECT NO.:454-1 15 Pen 1 2 3 4 5 6 Totals EeeslFemaleDav 306 6 5 6 7 7 6 37 0.88 307 3 3 5 5 5 6 27 0.64 308 5 5 6 7 6 7 36 0.86 309 6 7 7 7 4 0 31 0.74 3 10 5 7 7 7 8 1 35 0.83 Totals 25 27 31 33 30 20 166 0.79 Wildlije International, Ltd. Appendix 111- Table 3 Eggs Laid per Pen per Week from a Northern Bobwhite Pilot Reproduction Study with T-7485 200 ppm a.i. PROJECT NO.: 454-1 15 Pen 1 2 3 4 5 6 Totals EeedFemale/Dav 311 6 7 6 6 7 7 39 0.93 312 5 6 6 6 7 7 37 0.88 313 6 6 7 7 6 6 38 0.90 314 5 5 5 7 5 6 33 0.79 3 15 7 7 7 7 7 7 42 1 .oo I N OI Totals 29 31 31 33 32 33 189 0.90 I WildlqeInternational, Ltd. PROJECT NO.:454-115 Appendx 111- Table 4 Eggs Laid pcr Pen per Week from a Northern Bobwhite Pilot Reproduction Study with T-7485 550 ppm a.i. Weeks Pen 1 2 3 4 5 6 Totals Egg s/Female/Day 3 16 5 5 6 7 5 1 29 0.69 3 17 5 5 3 2 6 6 27 0.64 318 5 7 7 6 3 3 31 0.74 319 3 3 5 5 6 5 27 0.64 320 2 3 3 4 6 6 24 0.57 I N 4 Totals 20 23 24 24 26 21 138 0.66 I Wildlife International, Ltd. PROJECTNO.: 454-115 Pen 1 321 5 322 323 4 324 3 325 4 Totals 16 Appendix I11 - Table 5 Eggs Laid per Pen per Week from a Northern Bobwhite Pilot Reproduction Study with T-7485 1500 ppm a.i. Weeks 2 3 4 5 6 Totals 5 7 6 7 7 37 2 3 3 4 4 16 G 6 6 0 6 28 5 5 4 5 5 21 6 6 7 6 5 34 24 27 26 22 27 142 EggsiFemalelDay 0.88 0.42 0.67 0.64 0.81 0.69 - Data not reported. On day 3 of the test, the female in Pen 322 was replaceddue to possible neck injury. WildlifeInternational, Ltd. - 29 - PROJECTNO.: 454-1 15 Appendix IV - Table 1 Individual Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with T-7485 Birds Euthanized at Test Termination Control Finding Spleen - slightly enlarged Not remarkable Males Pen Number 301 302 303 304 305 X x X X X Feather loss Not remarkable Finding Females Pen Number ~ 301 302 303 304 305 X X X X X WildlifeInternational, Ltd. - 30 - PROJECT NO.: 454-1 15 Appendix IV - Table 2 Individual Gross Pathological Observations from a Northern Bobwhte Pilot Reproduction Study with T-7485 Birds Euthanized at Test Termination 75 ppm a.i. Feathcr loss Not remarkable Finding Males Pen Number 306 307 308 309 3 10 X X X X X Females Finding Pen Number 306 307 3 08 309 3 10 Fcather loss Abdominal cavity - slight egg yolk peritonitis Abdominal cavity - yolk remnants Ovary - regressing Not remarkable X X X X X X X Wildlijie International, Ltd. -31 - PROJECT NO.: 454-1 15 Appendix IV - Table 3 Individual Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with T-7485 Birds Euthanized at Test Termination 200 ppm a i . Finding Feather loss Testes - small (-1.25 cm) Not remarkable Males 311 X Pen Number 3 12 3 13 3 14 ~ 3 15 X X X X Finding Feather loss Right oviduct - partially developed Not remarkable Females 311 3 12 313 3 14 315 X X X X X X Wildlife International, Ltd. - 32 - PROJECT NO.:454-1 15 Appendix IV - Table 4 Individual Gross Pathological Observations fiom a Northern Bobwhite Pilot Reproduction Study with T-7485 Birds Euthanized at Test Termination 550 ppm a.i. Finding Testes - small (-1.0 cm) Right testis - small (-1.25 cm) Not remarkable Males Pen Number 3 16 317 3 18 3 19 320 X X X X X Feather loss Liver - friablc Not remarkable Finding Females Pen Number 316 3 17 318 319 320 X X X X X WildlifeInternational, Ltd. -33 - PROJECT NO.: 454-1 15 Appendix IV - Table 5 Individual Gross Pathological Observations from a Northern Bobwhite Pilot Reproduction Study with T-7485 Birds Euthanized at Test Termination 1500 ppm a.i. Not remarkable Finding Males Pen Number 32 1 322 323 324 325 X X X X X Feather loss Not remarkablc Finding Females 321 322 323 324 325 X X X X X WildlveInternutional, Ltd. - 34 - PROJECT NO.: 454-1 15 Appendix V - Table 1 Liver Weights by Pen from a Northern Bobwhite Pilot Reproduction Study with T-7485 Experimental Group Control 75 ppm a.i. 200 ppm a.i. 550 ppm a.i. 1500 ppm a.i. Male Liver Pen Weights (g) 301 3.832 302 2.995 303 3.110 304 3.336 305 4.848 ' 306 3.706 307 12.315 308 4.099 309 3.053 3 10 4.168 311 4.426 3 12 3.239 313 3.647 3 14 3.719 315 3.685 3 16 4.612 3 17 4.474 318 3.387 3 19 3.326 320 3.781 32 1 4.036 322 3.427 323 3.272 324 4.375 325 3.164 I- Abnormallylarge liver. Data was not included in mean calculation. Group MGUl SD 3.624 f 0.756 3.757 f 0.511 3.743 k 0.428 3.916 f 0.600 3.655 f 0.525 WildlifeInternational, Ltd. -35 - PROJECT NO.: 454-1 15 Appendix V - Table 2 Liver Weights by Pen from a Northern Bobwhite Pilot Reproduction Study with T-7485 Experimental Group Control 75 ppm a.i. 200 ppm a.i. 550 ppm a i . 1500 ppm a.i. Female Liver Pen Weights (6) 301 7.880 302 4.994 303 6.461 3 04 5.255 305 7.978 306 6.786 307 6.370 308 4.707 309 7.166 3 10 5.957 311 5.875 3 12 5.994 313 6.236 314 6.399 3 15 8.726 3 16 5.301 3 17 7.044 3 18 8.175 319 5.253 320 5.338 321 5.825 322 4.324 323 6.269 3 24 5.618 325 7.392 Group Mean SD 6.514 f 1.406 6.197 f 0.948 6.646 1.181 6.222 2 1.328 5.886 f 1.110 Wildlije International, Ltd. - 36 - Project Number 454-115 Appendix VI Table 1 Typical HPLCRMSIMS Opcrational Parameters INSTRUMENT: ANALYTICAL COLUMN: Hewlett-Packard Model 1100 High Performance Liquid Chromatograph with a Perkin-Elme; API 3000 LC Mass Spectronieter 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-pn) GUARD COLUMN: Kcystone Javelin CIScolumn (20 nun x 2 nun I.D.) OVEN TEMPERATURE: 4OoC STOP TIME: 3.OO minutes FLOW RATE: 200 pL/niinute MOBILE PHASE: 10%NANOpure Water@with 0.1% Ammonium Formate: 90% Methanol INJECTION VOLUME: 5.0 pL T-7485 RETENTION TIME: Approximately 1.7 minutes T-7485 MONITORED MASS: 299.0 + 93.9 Wildlife International, Ltd. - 37 - Project Number 454-1 15 Appendix VI Table 2 Example Calculations Sample number 454-1 15-9, nominal concentrationof 200 ppm a i in avian feed Peak Area = 58141 1 Y-intercept = 2962 1.3262 Slope = 2682496.7500 Primary Dilution: Initial Weight (Wi) = 10.0 g Original Final Volume (VI)= 100 mL Secondary Dilution: Initial Volume (V,) = 0.100 mL Final Volume (Vf) = 10.0 mL Overall Dilution Factor (Vl/W, x Vf/v;) = 1000 mL/g = 1000 L/Kg eak area - y-intercept) T-7485 at instrument (nig a.i./L) = (' slope - (581411 - 29621.3262) - 2682496.7500 = 0.2057 mg a.i./L T-7485 in sample (mg a.i./Kg) = T-7485 at instrument (nig a.i./L) x overall dilution factor (WKg) 0.2057 nig a.i. 1000 L T-7485 in sample (mg a.i./Kg) = X- Kg = 205.7 mg a.i./Kg rpm = 205.7 ppm a.i. T-7485 Percent of Nominal Concentration= T-7485 ma.1. insam le nomin:l x 100 ---220050.7 x 100 = 103% Instrument Software: MacQuan, version 1.6 WiId1ife Internationa1, Ltd. Project Number 454-1 15 Appendix VI Table 3 Matrix Blank and Fortifications Analyzed Concurrently with the Samples ale - Number 3 ' ' (454-115-) 5 e Concentration of Fortifed Measured'. Percent Recove Mean Percent of Nominal MAB-1 Matrix Blank Week 1, Day 0 0 < 25 _- MAS-I Matrix Fortification Week I, Day 0 50.0 47.0 94.0 MAS-2 Matrix Fortification Week 1, Day 0 500 496 99.1 MAS-3 Matrix Fortification Week 1, Day 0 2000 1920 95.9 __ I MAB-2 Matrix Blank Week 1, Day 7 0 < 25 w 00 MAS4 Matrix Fortification Week 1, Day 7 50.0 51.9 104 I MAS-5 Matrix Fortification Week 1, Day 7 500 522 104 MAS-6 Matrix Fortification Week 1, Day 7 2000 1900 94.9 MAE3 -3 Matrix Blank Week 6, Day 0 0 < 25 __ MAS-7 Matrix Fortification Week 6, Day 0 50.0 47.2 94.4 MAS-8 Matrix Fortification Week 6, Day 0 500 499 99.8 MAS-9 Matrix Fortification Week 6, Day 0 2000 1960 97.9 I 'The lowest level analyticalstandardwas equivalent to a calculated value of 25 ppm a i . in the matrix blank extract. Measured values greaterthan or equal to the ppm a.i.equivalent are reported. 'Measured and percent recovery values calculated in MacQuan ver. 1.6 and rounded to three significant figures. Manual calculations may vary slightly. WiId1ife XntemationaI, Ltd. Project Number 454-1 15 Appendix VI Table 4 Homogeneity of T-7485 in Avian Diet Nominal Concentration 75 Sample I D. Number 3 T-7185Concentration P P Location Sampled In Mixing Vess- el Measured' (ppm a.i.) Top Left 71.O Top Right 72.3 Middle Lett 73.9 Middle Right 72.8 Bottom Left 82.1 Bottom Right 81.3 200 9 Top Left 206 10 'Top Right 214 11 Middle Lett 223 12 Middle Right 215 13 Bottom Left 220 14 Bottom Right 232 550 15 Top Lett 542 16 Top Right 547 17 Middle Left 572 18 Middle Right 572 19 Bottom Lett 584 20 Bottom Right 549 1500 21 Top Left 1500 22 Top rzlght 1500 23 Middle Left 1500 24 Middle Right 1560 25 Bottom Left 1600 26 Bmht 1530 'Measured values were not corrected for a procedural recoveries based on samplesets (Table 3). Mean (R) Standard Deviation (SD) Coefficient of Variation (CV) z = 75.6 SD = 4.85 C V = 1.32 2 = 218 SD = 8.87 CV = 4.06 3 = 561 SD = 17.2 CV = 3.06 R = 1530 SD = 41.2 CV = 2.69 Mean Percent of Nominal 101 109 102 102 Wild1v e International, XItd. Project Number 454-1 15 Appendix VI Table 5 Verification of T-7485 Concentrations in Avian Diet Nominal - Concentration (ppm a.i.) Sample I.D. Number (454-1 1 5 ) Interval (Day 0 ofweek-) ' Measured T-7485 Concentrations (ppm ai.) Mean (X) Standard Deviation (SD) Coefficient of Variation (CV) Percent of Nominal Mean 0 41 42 < 25 < 25 < 25 75 43 70.5 x = 71.7 94 96 44 71 .O SD = 1.72 95 45 73.7 CV = 2.40% 98 , 200 46 6 193 x = 192 P 0 97 96 I 47 6 188 SD = 3.21 94 48 6 194 CV = 1.68% 97 550 49 6 50 6 51 6 52 1 x = 519 95 94 533 SD= 15.6 97 502 CV = 3.01% 91 1500 52 6 53 6 54 6 1390 x = 1410 93 94 1440 SD = 25.2 96 1410 CV = 1.78% 94 'The lowest level anal@cal standard was equivalent to a calculated valuc of 25 ppm a i . in the matrix blank extract. Measured values greater thanor equal to the ppm a.i. equivalent are reported. W i l d l i f eInternational, Ltd. Project Number 454- 1 15 Appendix VI Table 6 Ambient Stability of T-7485 in Avian Diet During the Northern Bobwhite Dietary LCSO Study Nominal Concentration (ppm a i ) 0 75 200 550 1500 Sample Number (454-1 15-) 1 2 3-8' 9-14' 15-20' 21-26' T-7485 Concentration Week 1, Day 0 Week 1, Day 7 Mean Meall Sample Mean 1 Measured' Measured Percent of Nominal Number 454-1 15-) Measured2 @pm a i . ) Measured < 25' < 25 - < 25 27 < 25 < 25 28 < 25 -1 75.6' 101 29 93.3 96.4 30 97.4 31 98.6 -I 218' 109 32 249 253 33 255 34 255 -- 1 561' 102 36 630 645 37 667 38 638 -I 1530' 102 39 1600 1670 40 1720 41 1690 Mean Percent of __ 128 116 115 109 'Day 0 values are from homogeneity samples presented in Table 4. 2Measuredvalues were not correctedfor mean procedural recoveriesbased on sample sets (Table 3). ? h e lowest level a n a l y t d standardwas equivalentto a calculated value of 25 ppm a i . in the matrix blank extract. Measured values greater than or equal to the ppm a i . equivalentare reported. WiZdlife InternationaI, Ltd. - 42 - Project Number 454-115 Appendix VI 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. Weigh 10 g saniples of the matrix blank, matrix fortification, and test samples into weigh boats and transfer to 8-02. French square glass bottles. Record weights. For each sample, measure 100m L s of methanol with a graduated cylinder and transfcr into the French square bottle. J Cap bottles and place on shaker table. Allow the samples to shake for a minimum of 30 minutes at approximately 250 rpm. J Transfer an aliquot of each sample into a 20-mL scintillation vial and centrifuge at a setting of 1500 rpni for approximately 10 minutes. Transfer an aliquot of each sample into a 3-mL B-D syringe fitted with a 0.45pm Acrodisc@and filter into a new scintillation vial. Prepare appropriate dilution(s) to bring final concentrationinto the calibration range of the LC/MS/MS methodology. Use 50% methanol:50% NANOpure@water (v/v) dilution solvent. Ampulate and submit samples for HPLC/MS/MS analysis. Figure 1 . Analytical method flow-chart for the analysis of T-7485 in avian diet. wildlijie International, Ltd. -43 - Project Number 454-1 15 Appendix VI T-7485 2 9 9 . 0 - S 6 . 9 No Internal Standard Weighted (l/x) Intercept = 29621.3262 Slope = 2662496.7500 Correlation Coalt. = 0.99760 Area 12oooooj / 4 O~,,,,,,,,,,',,,,,,,,,,,,,,. 0.0 0.06 0.12 0.18 0.24 0.30 0.36 0.42 0.48 Conc. rng a.i./L Figure 2 . Representative calibration curve for T-7485. wildlife International, Ltd. - 44 - Project Number 454-1 15 Appendix VI T7485-1 STD 0.0500 rng 8.I.L 5292-002A-6 2.98 in 1 period T-7405 No lntemal Standard Use Area 1: 2.98 MRM. 179 scans 299.0.>98.9 Noise Thres. 2.0 Quant mres. 1 .o Min. Width 12 Mull. Width 10 Base. Width 100 RT Win. (sect) 20 Smooth 1 Expected AT 1 All Area 150065 Hoight 26829 Start T h e 1.56 End Tim 2.70 Integration Widlh 1.14 Retention Time Integration Type A - f .69 BE Tue, Apr 1. 2003 03:32 1 OD9 080 706050- 403c- 2D 10 o- 9 33 47 101 67 85 intensity: 280000 cps 155167 Figurc 3. Representative ion chromatogram of a low-level T-7485 standard, 0.0500 mg a.i./L. Wildlije International, Ltd. -45 - Project Number 454-1 15 Appendix VI T7485-6 5292-002A-5 STD 0.500 mg a.IJL 2.98 in 1 period T-7485 No Internal Standard Use Area 1: 2.98 MRM, 179 scans 299.0->96.9 Noise Thres. 2.0 Quant Thres. 1 .O Mln. Width 12 Mull. Width 10 Base. Width H T Win. (sacs) Smooth Expecled RT 100 20 1 1.69 Area 1299883 Height 249455 Start Time 1.61 End Time 2.91 Integration Wldth 1.31 Retention Time Integration Type -1.74 A BB Tue, Apr 1, 2003 0351 1 intensity: 280000 cps 1104 I:\ Figurc 4. Representative ion chromatogram of a high-level T-7485 standard, 0.500 mg a.i./L. wildlife International, Ltd. - 46 - Project Number 454-1 15 Appendix VI T7485-8 454-1 15- MAE-1 Tue, Apr 1, 2003 03:59 2.98 in 1 period T-7485 No Internal Slandard Use Area I : 2.98 MRM, 179 S C ~ S 299.0->96.9 Noise Thres. 2.0 Quam Thres. 1. O Min. Width 12 Mult. Width 10 Base. Width 100 RT Win. (secs) 20 Smooth 1 ExpCCtOd FIT 1.69 Area 1064 Iieighl 130 Start Time 1.66 End Time Integralion Width Retenlion Time Integralion Type -2.16 0.52 1.73 A BV intensily: 280000 cps lO0l 90 ac 70 60 5c 40 3a 20 10 6 1927 31 0.52 51 61 1.02 82 103115 134 91 1.52 121 2.03 153 151 2.53 171 Scan Time Figure 5 . Representative ion chromatogram of a matrix blank sample, (454-1 15-MAB-I). Thc arrow indicates the approximate retention time of T-7485. 10-g sample, Overall dilution = 500X. W i l d 1ife International, Ltd. - 47 - Project Number 454-1 15 Appendix VI ~74as-10 454-115- MAS-2 Tue, Apr I , 2003 04:07 2.98 in 1 period T-7485 No Internal Standard Use Area 100- 1: 2.98 MRM, 178S C ~ 90- 299.0-298.9 Noise Thres. 2.0 80 Quanl Thres. 1 .O 70 Min. Widlh 12 Mull. Width 10 60 Base. Width 100 RT Win. (secs) 2 0 50- 104 ESxmpoeoctthedRT 11.69 40- Area 694486 30. Heighl 132848 Start Time 159 20 End Time 2.91 10. 7 lnlegrallon Width 1.32 Retention Time lnlegralion Type 1.74 A BB Figure 6. Representative ion chromatogram of a matrix fortification (454-1 15-MAS-2). Nominal concentration 500 ppni a i . , 10-g sample, Overall dilution = 2000X. WildlveInternational, Ltd. -48 - Project Number 454-1 15 Appendix VI T7485-22 454-1 15- 9 Tue, Apr 1, 2003 Od55 2.98 in 1 period 7-7485 No lntmai Standard Use Area 1: 2.98 MRM, 179 scans 299.0->98.9 Noi9e Thns. 2.0 Quant Thres. 1 .O Min. Width 12 Mull. Width 10 Ease. Width 100 RT Win. (secs) 2 0 Smooth ExpectedRT 1 1.69 Area 581411 Height 1 1 2323 Start Time 1.59 End Time 2.83 Integration Width 1.24 Retention T i m Integration Type A -1.73 BB 1001 20. 10. o, 12 2 7 4 0 72 31 61 0 52 102 intensity: 280000 cps 103 I\ 91 1.52 121 203 151 2.53 scan TI me Figure 7 . Representative ion chromatogram of an avian diet sample on Day 0, 454-1 15-9 Nominal concentration 200 ppm a i . , 10-g sample, Overall dilution = IOOOX.