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_ o 6rg N-Et FOSE alcohol Teratology - / &- 0^0324 ( 0 H A Z L E T O N LABORATORIES AMERICA, INC. v t < k s N < * a a 4s o . u f t . A PILOT RAT TERATOLOGY STUDY T-3352 FINAL REPORT Submittedto 3MCompany St. Paul, Minnesota May 11, 1983 OOQ32S HAZLETON LABORATORIES AMERICA. INC 9200 LEESBURG TURNPIKE VIENNA VIRGINIA S3 180 USA SUBJECT: Pilot Rat Teratology Study Project No. 154-159 We, the undersigned, hereby declare that the work was performed under our supervision, according to the procedures herein described. Study Director: Ph.D. Staff Scientist Department of Toxicology Laboratory Supervision: Teratology: GEORGEA. BURDOCK, Ph.D. Rodent Toxicology I Department of Toxicology RUTH S. DURLOO, B.S. Research Associate Department of Toxicology Report Preparation: Technical Writer Scientific Resources Department blm 000326 (jjjj)H A Z L E T O N LABORATORIES AMERICA. INC. O O UCCSU*>0 TU *M *IK l. V IIN N A . U .S .A TABLE OF CONTENTS Page SUMMARY 1 INTRODUCTION 3 CONTROL ANO TEST MATERIALS 3 TEST ANIMALS 4 METHODS Mating Period Groups and Dosage Levels Compound Preparation and Administration Maternal Observations and Records Cesarean Sacrifice and Tissue Preservation Statistical Analyses Specimen, Raw Data, and Final Report Storage 4 4 5 _6 '6 7 8 9 RESULTS - MATERNAL DATA Clinical Signs Mortality Body Weights Food Consumption Gross Pathology Uterine Weights 10 10 10 10 11 11 11 RESULTS - CESAREAN DATA Pregnancy Rates, Corpora Lutea, Uterine Implantations, and Implantation Efficiency Fetal Viability, Weight, and Sex Fetal Development 12 12 13 13 TABLES Table 1 - Clinical Observations During Gestation Table 2 - Mean Maternal Body Weights and Body Weight Change Table 3 - Mean Food Consumption Values Table 4 - Summary of Gross Pathology Findings Table 5 - Mean Terminal Body Weights, Gravid Uterine Weights, and Terminal Body Weights Minus GravidUterine Weights Key to Table 6 Table 6 - Summary of Ovarian, Uterine, and Litter Data Table 7 - Mean Incidence Values for Visceral and Skeletal Findings Key to Table 8 Table 8 - Mean Incidence of Visceral and Skeletal Findings per Litter 15 16 17 18 20 21 22 23 25 26 0003^7 @ H A Z L E T O N LABORATORIES AMERICA, INC. > 8 0 0 L lla S U H O T u * N * IK |. V N N A V I ai* * A a * i o . u .* a TABLE OF CONTENTS (CONTINUED) Page APPENDICES Key to Appendix 1 Appendix 1 - Individual and Mean Maternal Body Weights and Weight Changes Appendix 2 - Individual and Mean Daily and Total Food Consumption Appendix 3 - Individual Body Weights, Gravid Uterine Weights, and Terminal Body Weights Minus Gravid Uterine Weights Appendix 4 - Individual and Mean Ovarian, Uterine, and Litter Data Appendix 5 - Individual and Mean Live Fetal Data Appendix 6 - Individual Visceral Findings for Each Litter Appendix 7 - Individual Skeletal Findings for Each Litter Appendix 8 - References 27 28 31 34 37 40 43 44 46 000328 HAZLETON LABORATORIES AMERICA, INC. a a o o LEESBURG TURNPIKE. VIENNA, VIRGINIA 2 S1BO. U.S.A. S P O N S O R : 3M Company D A T E : May 11, 1983 MATERIAL: J-3352 SUBJECT: FINAL REPORT Pilot Rat Teratology Study Project No. 154-159 SUMMARY T-3352, suspended in corn oil at concentrations intended to deliver 1, 10, 37.5, or 75 mg/kg/day (Groups 2, 3, 4, and 5, respectively), was administered by oral intubation to four groups of mated female Sprague-Dawley S-D* rats on gestation Days 6 through 15. A fifth group (Group 1) of mated females served as the control and received the vehicle only. Apparent compound-related deaths occurred in seven of seven Group 5 and three of seven Group 4 females on or before Day 17 of gestation. Clinical observations in Groups 4 and 5 included hunched, thin appearance, languid behavior, and apparent anorexia. Body weight gain during treatment was less in Group 3, 4, and 5 females compared to control values obtained during that time. The terminal body weight minus the gravid uterine weight was decreased in Group 4 females compared to control. A number of gross pathology findings at necropsy or sacrifice were noted in Groups 4 and 5 and these included incidences of liver, adrenal, lung, and/or gastrointestinal tract effects. f'r'f' S Z 9 [ffi H A Z L E T O N LABORATORIES AMERICA, INC. a8 s9n0 n0 Li i if t m8 9 uU m 0 nT Ul Aa MN Bi IKKCE . vViIiEr gNNN A . V lM O tA A 'A 8 8 1 8 0 . U .9 .A - 2- 154-159 Evaluation of mean fetal weight data indicated that fetal weights of both sexes decreased with increasing dose level. A significant decrease in mean fetal weight occurred in Group 4 males and females, although data for only two litters were available for that group. Cleft palate was found in two of six Group 4 fetuses (one fetus in each litter) examined by Wilson's technique. Incompletely descended testes were noted in one fetus from each Group 4 litter evaluated for visceral defects. Incidences of skeletal variants were similar for all groups. 000330 0jjj) H A Z L E T O N LABORATORIES AMERICA, INC. a a o o I.IE S B U A Q T l> A N * IK ( V ilfw M A . V lH O lN )* M 'l O , u l * -3 154-159 INTRODUCTION This study was designed to determine the maternal and embryo/ fetal toxicity of T-3352 when administered by gavage to pregnant rats during the period of fetal organogenesis for the purpose of setting dose levels for an expanded teratology study. The rats were placed in breeding on January 17, 1983, and cesarean sections were completed on February 18, 1983. This report presents the methods and results from this study. CONTROL AND TEST MATERIALS The vehicle and control material, Duke's Corn Oil (C. F. Sauer Co., Richmond, Virginia), lot 80235, was received on January 24, 1983, and was stored at room temperature. The test material, T-3352, an off-white solid, was received from the sponsor on January 5, 1983, and was stored at room temperature. The test material was assumed to be 100% active compound. Information on methods of synthesis and stability, as well as data on composition or other characteristics which define the test material, are on file with the sponsor. 000331 @ H A Z L E T O N LABORATORIES AMERICA. INC. 9 aaciiissuno v h n n a , v im q im ia a * i a o . u ..a -4- 154-159 TEST ANIMALS Sexually mature cesarean-derived Sprague-Dawley Crl:CD (SD)BR rats were chosen for this study because they are sensitive to a number of agents which are known to be embryotoxic and/or teratogenic. Rats have historically been used in safety evaluation studies of this type and are required by the regulatory agencies. Thirty-six twelve to fourteen-week old male rats and thirty-six eight to ten-week old female rats were received from Charles River Breeding Laboratories, Kingston, New York, on December 22, 1982. The rats were held in quarantine for three and one-half weeks, during which time a health status examination was performed by a staff veterinarian. The rats were housed one male and one female per cage during breeding. Following confirmation of mating, the females were housed individually in elevated wire-mesh cages with food (Purina Rodent Laboratory Chow 5001) and tap water available ad libitum. The females were uniquely identified by ear tag after mating was confirmed. Tempera tures in the study room ranged from 70 to 78F with a relative humidity between 44 and 695t. METHODS Mating Period During the mating period, one female was paired with one male until mating was confirmed or until two weeks had elapsed. Daily vaginal oooo @ H A Z L E T O N LABORATORIES AMERICA. INC. s a oai-cli- l. C E M u.Ra Sn ?t iuiDb un bb iivk *i . uv iicimn ini a . v i 0 **a i a a s in o u . I a -5 154-159 examinations of each female were performed to detect the presence and viability of sperm or the presence of a copulatory plug. The day of observation of sperm or copulatory plug was designated as "Day 0" of gestation. Groups and Dosage Levels Upon confirmation of mating, each female was assigned to one of the following groups. Group 1 2 3 4 5 Dose mg/kg/day 0 1 10 37.5 75 Number of Females 6 7 7 7 a Based on individual animal body weights at each weighing interval during the dosing period. b An additonal Group 2 female was confirmed to have mated on a day when no other matings were confirmed. Because this animal's dose level would have been known at cesarean sacrifice, she was removed from the study. The females were placed into the dose groups one at a time beginning with the high-dose group and continuing sequentially through the control group until all mated females were assigned to a group. All males were sacrificed via carbon dioxide asphyxiation and discarded without necropsy once a sufficient number of matings were confirmed. 0 0 0 3 AI-O H A Z LE T O N(jjjjj) LABORATORIES AMERICA. INC. aaoo u C E H u a s t u d n k e . v u n n * . v i iv ia aio. u * .a 6- - 154-159 Compound Preparation and Administration Suspensions of the test material in the corn oil vehicle were prepared on a weight per volume basis. The required amount of compound for levels 2, 3, or 4 was weighed into a homogenizer on an electronic Arbor 126 balance. Compound for Group 5 was weighed on powder paper on an Arbor 126 balance and rinsed into a Waring blender. Approximately 4 ml of corn oil was added and the test material was ground into a fine suspension. This suspension was rinsed from the homogenizer or the blender into a beaker and additional corn oil was added to produce the desired concentration. The suspension was mixed on a stirrer for approximately fifteen minutes. Prepared suspensions were mixed with a magnetic stirrer during dosing. Females were given the appropriate dosing suspension or vehicle by oral intubation on a daily basis beginning on Day 6 and continuing through Day 15 of gestation. The test material was administered orally because of the relative ease and accuracy of dosing. Fresh dosing suspensions were prepared weekly and stored under refrigeration (approximately 41F). Samples of each test mixture as well as the vehicle were sent to the sponsor for analysis. Maternal Observations and Records All animals were examined twice daily for mortality and mori- bundity (from day of receipt through study termination) and once daily for clinical signs of toxicity and pharmacologic effects (throughout 000334 @ H A Z L E T O N LABORATORIES AMERICA, INC. aa as no rc li feica sa9iui*n Sn Ttiul auMn Bv ii Kk il . vi/iiCeMa*Ka*a . vim o in ia 8 9 1 b d . u s a -7 154-159 gestation). Body weights and food consumption were recorded on Days 0, 6, 8, 12, 16, and 20 of gestation. Cesarean Sacrifice and Tissue Preservation A gross necropsy was performed on all animals (found deads, moribund sacrifices, and terminal sacrifices). The uterus and ovaries from found dead animals and animals sacrificed because of moribundity were removed and examined for the number of implantation sites and corpora lutea, respectively. Prior to gestation Day 20, surviving females were assigned random numbers and all personnel performing cesarean sections and/or external, visceral, or skeletal examinations of the fetuses were unaware of the dose level from which the animals were derived. On Day 20 of gestation, all surviving females were weighed and sacrificed by carbon dioxide asphyxiation. The uterus from each female was weighed and examined for the number and placement of uterine implantation sites, number of live and dead fetuses, early and late resorbing fetuses, and any abnormalities. The uterus of each animal was reweighed after the contents were removed. The ovaries were examined for the number of corpora lutea. Each live fetus was sexed, weighed, and examined for external abnormalities. Findings were recorded. Beginning at the ovarian end of the right uterine horn, the first six fetuses, regardless of sex, were selected for further evaluation. After the external examination was completed, the first, 000335 @ H A Z L E T O N LABORATORIES AMERICA, INC. M O O l l l M U H a ru A N M K i. V lIM M , V tm O lfiilA 0 0 * 0 0 . u.o^*. 8- - 154-159 third, and fifth fetuses were identified with a tag and fixed in Bouin's solution for soft tissue evaluation (Wilson, et al, 1965). The second, fourth, and sixth fetuses were eviscerated, tagged, and processed for skeletal examination using a technique modified from that reported by Staples (Staples and Schnell, 1964). Statistical Analyses Mean maternal body weight gains (Days 6-16 and 0-20), total food consumption, gravid uterine weight, terminal body weight minus gravid uterine weight, resorption incidence, percent males, and fetal viability were analyzed in the following order. Levene's test for homogeneity of variances (Levene, 1960; Draper and Hunter, 1969) was performed and if the variances proved to be homogeneous, the data were analyzed by one-way classification analysis of variance (ANOVA) (Winer, 1971). If the variances proved to be heterogeneous, a series of transformations was performed until variance homogeneity was achieved. These transformations were: log1Q, square (X2), square root (X^k1), reciprocal (1/X), angular (arcsine X1*), and rank, in that order. If rank transformation was ineffective in removing variance heterogeneity, ANOVA of ranked data was completed. If ANOVA of untransformed or transformed data was significant, Dunnett's t-test (Dunnett, 1955 and 1964) was used for control vs. compound-treated group mean comparisons. If ANOVA was not significant, the analysis was complete. 000336 H A ZLETO N|jg j > LABORATORIES AMERICA, INC. >S' MObllMUMB TUHNMKI.VlfNNA W*QINI*irO.U l.A -9- 154-159 In addition to the above data, analysis of covariance (ANCOVA) (Winer, 1971) was used to analyze mean fetal weights. The litter was used as the experimental unit. Levene's tests and ANOVA were evaluated at the 5.OX one-tailed probability level. Control vs. compound-treated group mean comparisons were evaluated at the 5.OX two-tailed probability level. Statistical references are appended to this report, and statisti cally significant differences, as indicated by the aforementioned tests, are designated throughout this report by the term "significant" and/or as follows: S+ * Significantly higher than the control value. S- = Significantly lower than the control value. Specimen, Raw Data, and Final Report Storage All specimens, raw data, and the final report are stored in the archives of Hazleton Laboratories America, Inc. 00033# ( g lH A S lg C O tV LABORATORIES AMERICA, INC. aaOn On H IM U>! Ai a AS TTiU MNaMfiiKf I.Vu iIBkWlk*i*A. VlAOlMIAISO. U.S-A. 10- - 154-159 RESULTS - MATERNAL DATA Clinical Signs Summary clinical signs are presented in Table 1. Treatment-related clinical observations were noted in the Groups 4 and 5 animals during treatment and in Group 4 animals posttreatment. No Group 5 rats survived the treatment phase. Clinical signs for Groups 4 and 5 animals included hunched and thin appearance, languid behavior, urine stains, and bloody crusted eyes, eyelids, nose, mouth, legs, paws, or genitals. Anorexia, ataxia, dyspnea, rough coat and pale appearance were also noted for several Group 4 or 5 rats. One Group 4 rat aborted thirteen fetuses on Day 20 prior to sacrifice. Alopecia was noted for some rats in each dose group and the control group. Mortality All Group 5 females were found dead on or before Day 16 of gestation and three Group 4 females were found dead on or before Day 17 of gestation. All other animals survived to cesarean sacrifice on Day 20. Body Weights Individual and mean body weights and body weight changes are presented in Appendix 1. Mean weight values and body weight changes are presented in Table 2. During gestation, lower than control mean body weight values were noted in Group 5 beginning on Day 8 and in Group 4 beginning on Day 12. Decreased body weight gain during gestation was noted in Groups 3 uOv3^8 djjj) H A Z L E T O N l a b o r a t o r ie s AMERICA, in c . *aOQUtittUAS T u A M A IK E . V lI N N A VIMO*f>JIA 991BO. U-SA - 11 154-159 and 4. The decrease in the Group 4 animals was significantly different than the control value. Because of high mortality, Group 5 data were not included in statistical evaluation. Food Consumption Individual and mean food consumption values are presented in Appendix 2. Mean food consumption values are presented in Table 3. Slightly decreased food consumption values were noted for Groups 4 and 5 compared to control on and after Day 8 of gestation. Mean total food consumption values for Groups 2, 3, and 4 were statistically comparable to control. Total food consumption for Group 5 was not determined because of mortality. Gross Pathology Summary gross pathology findings are presented in Table 4. No gross lesions were noted at necropsy for animals from the control group or dose Groups 2 and 3. Findings noted for Group 4 animals were discolored liver, kidney, and adrenals; enlarged adrenals; dilated renal blood vessels, and red vaginal discharge. Findings noted for Group 5 animals were reddened and/or distended lungs, enlarged and/or friable liver, enlarged adrenals, discolored and/or thin glandular gastric mucosa, fluid in stomach or intestines, and red vaginal discharge. Uterine Weights Individual and mean uterine weights are presented in Appendix 3. Summary uterine weights are presented in Table 5. 00033? @ H A Z L E T O N LABORATORIES AMERICA. INC. M O D l. E S a u B S T u O N B lK f V IEN N A VlHO*NIA 8 ff H O .U .IA - 12 - 154-159 Mean terminal body weights were comparable for Groups 1, 2, and 3, but the mean terminal body weight of the Group 4 females was decreased compared to control animal weights. There was an approximate 9% decrease in the mean gravid uterine weights of the Group 2, 3, and 4 animals com pared to the control mean weight. For Group 2, this may be attributable to one dam with eight early resorbing fetuses. Terminal body weight minus gravid uterine weights were comparable for Groups 1, 2, and 3, but that weight was decreased in the Group 4 animals compared to the control value. RESULTS - CESAREAN DATA Individual and mean ovarian, uterine, and litter data are presented in Appendix 4. Individual and mean live fetal data are presented in Appendix 5. Summary ovarian, uterine, and litter data are presented in Table 6. Pregnancy Rates, Corpora Ltea, Uterine Implantations, and Implantation Efficiency Pregnancy rates were 100 percent for Groups 1, 2, and 3, and 85.7 percent for Groups 4 and 5. The mean number of corpora ltea and the mean number of uterine implantations were comparable for all groups as were mean implantation efficiencies. 000340 H A Z L E T O N l a b o r a t o r ie s AMERICA, in c . 8 SO O L C E H U H G T u R N V iK f v iI N N A V lffO lfg iA 9 8 *0 0 . U -S - 13 - 154-159 Fetal Viability, Weight, and Sex Mean resorption incidences were higher for Groups 2 and 4 than for the control group, but these differences were not statistically significant. The increased incidence in Group 2 may be attributable to one dam with eight resorbing fetuses. Mean incidence of fetal viability and percent males were comparable for Groups 1-4. Mean live fetal weights of both sexes decreased with increasing dose level with Group 4 values significantly lower than control. Fetal Development Individual visceral findings are presented in Appendix 6; indivi dual skeletal findings are presented in Appendix 7. Mean incidence values for visceral and skeletal findings are presented in Table 7 (based on number of fetuses) and Table 8 (based on number of litters). Gross external examination of fetuses at cesarean section revealed one abnormal fetus from a Group 3 dam. This fetus had no tail and appeared to have no thoracic or lumbar vertebrae. However, no skeletal evaluation was done on this fetus so that finding could not be verified. Skeletal examination of the selected fetuses revealed that the incidence of skeletal variants was comparable for the control group and treated groups. No skeletal anomalies were noted. The incidence of visceral variants was markedly higher for Group 4 when compared to the 000341 iffi H A Z L E T O N LABORATORIES AMERICA. INC. o0a8n0n0 Li (E CI BunAo Cn Tt iUiABNi0u IB KitEr .t Vi/iIEEKN^NA. vMQMNIA 88100.U0A - 14 - 154-159 control group (50 percent vs. 5.56 percent). However, only two Group 4 litters were available for evaluation compared to six of the control group. Variants noted were dilated renal pelves (Group 2), dilated ureters (Groups 1 and 4), and incompletely descended testes (Group 4). Cleft palate was found in two of six Group 4 fetuses examined by Wilson sectioning. That anomaly was noted in one fetus from each of the two litters from which the fetuses selected for visceral examination were derived. No other visceral anomalies were observed. 000342 Table 1 Clinical Observations During Gestation Pilot Rat Teratology Study of T-3352 OBSERVATION Number of animals observed Hunched rs-zo o^50 wq/ltq Ion '0-5 " TPT5-- "T T Z Group 2 - l ug bay of Gestation ~rn> 6-15 16*20 Group 4 - 37.5 mg/kg "0-5 ~5&:T5 16-20 0-5 6 6 6 6 6 6 7 i 7 6(1) 65((11)) 43((11)) 6(1) Thin Languid Anorexia 2 1 33(1) 1 Ataxia Dyspnea 2 Urine stains Alopecia (paws, legs, chest, neck, hips, side, abdomen) 2 Rough halrcoat Bloody crust (nose, mouth, paws, eyelids, legs, genitals, eyes) 4 4 12 2 3 1i 2 2 3 1 5(1) 3(1) Pale 1 Aborted fetuses/red vaginal discharge 1 151-159 5-ir 6666((((1111)))) 32(1) 4(1) en i 11 6(1) () * Nonpregnant female. O CO 0* CO Table 2 Mean Maternal Body Weights and Body Weight Change Pilot Rat Teratology Study of T-3352 Group and Dose Level 1 0 mg/kg 2 1 mg/kg 3 10 mg/kg 4 37.5 mg/kg 5 75 mg/kg Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N 0 231.60 6.935 6 239.38 18.051 6 240.93 10.348 7 250.25 17.535 6 238.90 18.020 6 6 263.57 6.342 6 259.25 29.056 6 271.81 12.006 7 263.63 21.958 6 259.47 13.286 6 Body Wei ght (grams) Day of 6estatlon 12 264.17 6.735 6 285.23 3.885 6 266.23 22.061 6 283.70 20.883 6 267.73 17.535 7 280.87 14.384 7 265.10 7.558 6 245.65 16.890 6 246.37 9.816 6 206.80 17.331 6 16 304.07 8.703 6 304.90 23.384 6 301.56 17.119 7 227.98 32.551 4 20 366.52 8.422 6 363.95 31.752 6 359.67 19.150 7 317.00 58.690 2 -- 00 a Statistical group comparisons performed on rank-transformed data 154-159 Body Weight Change (g) Days of Gestation fa^Tb 0^20^ +40.50 6.111 6 +134.920 7.161 6 +45.65 21.052 6 +124.57 20.838 6 +29.74 10.317 7 +118.74 18.776 7 -35.65$" 21.324 4 +60.70$" 32.527 2 -80.7 - 1 _ 0 Group and Dose Level " 7(in1gi/iK:1g)i 1 0 mg/kg 2 1 mg/kg 3 10 mg/kg 4 37.5 mg/kg 5 75 mg/kg Mean > S.D. N Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N 6 18.28 1.981 6 18.32 4.058 6 17.20 1.681 7 18.90 2.346 6 20.03 7.684 6 Table 3 Mean Food Consumption Values Pilot Rat Teratology Study of T-3352 Food Consumption (qm/day) Day of Gestation 8 12 16 ------ 2D 19.22 1.933 6 19.92 1.685 6 19.97 1.039 6 26.13 1.895 6 19.32 3.370 6 20.05 2.665 6 23.00 4.785 6 26.72 3.996 6 14.80 4.246 7 19.14 2.539 7 17.71 3.060 7 24.26 0.860 7 12.20 3.774 6 15.73 19.816 6 16.75 24.530 4 15.27 13.201 3 9.62 2.690 6 8.55 11.886 6 0 . 0 a Statistical group comparisons performed on rank-transformed data. 154-159 Total Food Consumption(q) Days 0-20a 430.23 17.578 6 445.45 58.101 6 394.17 31.539 7 411.43 156.013 3 0 CooO J wA 0003-56 Table 4 Sunmary of Gross Pathology Findings Pilot Rat Teratology Study of T-3352 154-159 ORGAN AND DESCRIPTION Group: 1 2 3 4 5 Dose Level (mg/kg): U ~T Iff i m r 75 Number of rats examined Number with no gross lesions LUNGS Reddened Distended 6 6 7 6(1) 6(1) 6 6 7 2(1) 0 2 1 LIVER Enlarged Yellow Friable consistency 2(1) 3 1 KIDNEYS Greenish Blood vessels - dilated ADRENALS Enlarged Brown 2( 1) STOMACH Contained dark red fluid Contained black fluid Glandular mucosa - reddened - thin - black focal areas 1 ( 1) 3(1) 1 1 () = Nonpregnant female. I 00 I Table 4 - Continued Summary of Gross Pathology Findings Pilot Rat Teratology Study of T-3352 ORGAN AND DESCRIPTION INTESTINES Filled with dark fluid material OVARIES Autolyzed VAGINA Red/bloody discharge/dark red fluid Group: 1 Dose Level (mg/kg): 2 ~T 000348 Table 5 Mean Terminal Body Weights, Gravid Uterine Weights, and Terminal Body Weights Minus Gravid Uterine Weights Pilot Rat Teratology Study of T-3352 154-159 Group and Dose Level mg/kg 1 0 mg/kg 2 1 mg/kg 3 10 mg/kg 4 37.5 mg/kg 20 mg/kg Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N Terminal tody Weight grams 366.52 8.422 6 363.95 31.752 6 359.67 19.150 7 317.00 58.690 2 Gravid Uterine Weight grams 77.30 7.925 6 68.33 21.509 6 68.34 11.081 7 67.40 19.233 2 Terminal Body Weight Minus Gravid Uterine Weight3 grams 289.22 2.378 6 295.62 22.459 6 291.33 22.396 7 249.60 39.457 2 00 0 a Statistical group comparisons performed on rank-transformed data. ro 0 1 - 21 - Key to Table 6 154-159 Pregnancy Rate (percent) = (number of pregnant rats/number of rats mated) x 100. Survival Rate (percent) * (number of rats surviving to Day 20/number of rats placed on study) x 100. Mean Implantation Efficiency (percent) * Group mean of ([ implantations per litter/corpora lutea per litter] x 100). Mean Resorption Incidence (percent) = Group mean of ([resorptions per litter/implantations per litter] x 100). Mean Incidence of Fetal Mortality (percent) * Group mean of ([dead fetuses per 1itter/implantations per litter] x 100). Mean Incidence of Fetal Viability (percent) * Group mean of ([live fetuses per litter/imp!antations per litter) x 100). 000349 ro a o Table 6 Sunmary of Ovarian, Uterine, and Litter Data Pilot Rat Teratology Study of T-3352 154-159 Group: 1 Dose Level (mg/kg): -- 0 Number of females with mating confirmed Number of females pregnant Pregnancy rate (X) Number of females surviving to Day 20 Survival rate (X) 6 6 100 6 100 Mean number of: Corpora lutea Implantatlons Resorptions Fetuses - dead - live 15.0 14.5 1.2 0.0 13.2 Indices calculated on a per Utter basis: Mean Implantation efficiency (X) Mean resorption Incidence (X) Mean Incidence of fetal mortality (X) Mean Incidence of fetal viability (X) 95.28 7.95 0.0 90.78 Live fetuses Mean body weight (g) Males - unadjusted - covariance adjusted Females - unadjusted - covariance adjusted Percent males * 3.42 3.40 3.25 3.25 55.03 2 r-- ~ 6 6 100 6 100 16.8 13.3 2.0 0.0 11.3 81.82 15.75 0.0 84.25 3.33 3.37 3.23 3.22 53.05 3 1 7 7 100 7 100 15.6 13.7 1.1 0.0 12.6 88.01 7.66 0.0 92.36 3.26 3.27 3.11 3.12 51.31 4 7 6 85.7 4 57.1 16.2 14.5 2.8 0.0 13.5 89.00 13.25 0.0 86.75 5 75 7 7 85.7 0 0 15.7 14.0 - 90.76 - 2.75 2.72S- 2.60 2.60S- 55.40 - An additional Group 2 female was confirmed to have mated on a day when no other matings were confirmed. in Because this animal's dose level would have been known at cesarean sacrifice, she was removed from the study. I rr\o> i Table 7 Mean Incidence Values for Visceral and Skeletal Findings Pilot Rat Teratology Study of T-3352 154-159 Group: Dose Level (mg/kg): 1 0 Visceral Number of fetuses examined Number of fetuses within normal limits 18 17 Number/percent of fetuses having one or more visceral variants Number/percent of fetuses with: Dilated renal pelvis Dilated ureter(s) Incompletely descended testis(es) 1/5.56 0 1/5.56 0 Number/percent of fetuses having one or more visceral anomalies 0 Number/percent of fetuses with: Cleft palate 0 Skeletal Number of fetuses examined Number of fetuses within normal limits Number/percent of fetuses having one or more skeletal variants Number/percent of fetuses with: Lagging Ossification Skull - Incomplete closure (less than 75%) O - Interparietal ossification incomplete - supraoccipital ossification Incomplete - supraoccipital nonoss1f1cat1on t4 - hyoid ossification Incomplete vj - hyoid - nonossifled ^ - parletals irregularly ossified 18 5 13/72.22 4/22.22 8/44.44 9/50.00 0 1/5.56 9/50.00 4/22.22 2 1 18 17 1/5.56 1/5.56 0 0 0 0 17 6 11/64.71 2/11.76 2/11.76 7/41.18 0 0 2/11.76 3/17.65 3 10 21 21 0 0 0 0 0 0 21 10 11/52.38 1/4.76 6/28.57 6/28.57 0 0 3/14.29 5/23.81 4 37.5 6 1 3/50.00 0 1/16.67 2/33.33 2/33.33 2/33.33 6 1 5/83.33 0 2/33.33 3/50.00 0 0 0 0 Table 7 - Continued Mean Incidence Values for Visceral and Skeletal Findings Pilot Rat Teratology Study of T-3352 Group: Dose Level (mg/kg): 1 Skeletal (Continued) Number of fetuses examined Number/percent having one or more skeletal variants Number/percent of fetuses with: Lagging Ossification Rib-cage - sternebrae bipartite - rib irregularly ossified Vertebral column - thoracic centra ossification 1ncomplete - thoracic centra nonfused - caudaIs less than three ossified Bone alignment Extra rlb(s) Angulated rubs 13 0 1/5.56 0 1/5.56 1/5.56 1/5.56 2/11.11 Number/percent having one or more skeletal anomal les 0 2 -- r~ 11 1/5.88 1/5.88 0 1/5.88 0 0 1/5.88 0 154-159 3 nr~ 4 37.5 11 2/9.52 1/4.76 1/4.76 1/4.76 1/4.76 0 0 5 4/66.67 0 0 1/16.67 1/16.67 0 0 0 00005 N - 25 - Key to Table 8 154-159 Mean Incidence of Visceral Anomalies (percent) = Group mean of ([number of fetuses with anomalies per litter/number of fetuses examined viscerally per litter] x 100). Mean Incidence of Visceral Variants (percent) * Group mean of ([ number of fetuses with variants per litter/number of fetuses examined viscerally per litter] x 100). Mean Incidence of Skeletal Anomalies (percent) * Group mean of ([number of fetuses with anomalies per litter/number of fetuses examined skeletally per litter] x 100). Mean Incidence of Skeletal Variants (percent) * Group mean of ([number of fetuses with variants per litter/number of fetuses examined skeletally per litter] x 100). 000353 Table 8 Mean Incidence of Visceral and Skeletal Findings per U t t e r Pilot Teratology Study of T-3352 In Rats 154-159 Group: 1 Dose Level (mg/kg): DH Visceral Examination Number of litters examined Number of litters with anomalous fetuses Percent of Utters with anomalous fetuses (X) Number of litters with variant fetuses Percent of litters with variant fetuses (X) 6 0 0 1 16.67 Mean values calculated on a per litter basis: Number of fetuses with variants Incidence of variants(X) {%)Number of fetuses with anomalies Incidence of anomalies 0.17 5.56 0 0 Skeletal Examination Number of litters examined Number of Utters with anomalous fetuses Percent of Utters with anomalous fetuses (X) Number of Utters with variant fetuses Percent of litters with variant fetuses (X) 6 0 0 6 100.00 Mean values calculated on a per Utter basis: Number of fetuses with var1ants Incidence of variants (X) Number of fetuses with anomalies Incidence of anomalies (X) 2.17 72.22 0 0 T2 3 4 I T " 3775 6 0 0 1 16.67 7 0 0 0 0 0.17 5.56 0 0 0 0 0 0 2 2 100.00 2 100.00 1.50 50.00 1.00 33.33 6 0 0 5 83.33 7 0 0 7 100.00 2 0 0 2 100.00 1.83 61.11 0 0 1.57 52.38 0 0 2.50 83.33 0 0 000354 - 27 - Key to Appendix 1 Individual and Mean Maternal Body Weights Pilot Rat Teratology Study of T-3352 154-159 P * Pregnant NP = Not pregnant C * Cesarean section performed (preceded by day of gestation) FD = Found dead (preceded by day of gestation A * Aborted pups (preceded by day of gestation) a Body weight on day of death. b Statistical group comparison performed on rank-transformed data. Note: Body weights in parentheses are excluded from mean calculations and statistical evaluation. 000355 M aternal Number 21606 21607 21608 21609 21610 21611 Mean S.O . 21613 21614 21615 21616 21617 21618 Mean S.O Date of Mating 1/18/83 1/18/83 1/19/83 1/19/83 1/19/83 1/20/83 1/18/83 1/18/83 1/19/83 1/19/83 1/19/83 1/20/83 D isp o sitio n P.20-C P.20-C P.20-C P.20-C P.20-C P.20-C P.20-C P.20-C P.20-C P.20-C P.20-C P.20-C Appendix 1 Individual and Mean Maternal Body Weights and Weight Changes Pilot Rat Teratology Study of T-3352 Body Weight (gram s) - Day of G e sta tio n 0 6 8 12 16 20 Group 1 - 0 mg/kg 229.2 2 2 8 .6 235.4 2 3 8 .5 237.6 220.3 271.1 263.4 265.7 262.4 266.6 252.2 272.3 256.2 2 6 9 .8 2 6 1 .9 2 6 7 .6 257.2 288.8 2 8 0 .6 289.7 2 8 5 .0 286.5 2 8 0 .8 309.9 2 9 3 .9 313.6 308.2 306.1 292.7 367.2 366.3 374.7 376.8 358.1 356.0 231.60 6 .9 3 5 263.57 6.342 264.17 6 .7 3 5 285.23 3.885 304.07 8 .7 0 3 366.52 8 .4 2 2 Group 2 - 1 mg/kg 2 3 8 .6 261.8 250.7 213.6 223.4 248.2 2 1 6 .6 2 9 7 .6 285.3 2 5 1 .8 245.2 2 5 9 .0 2 5 7 .6 2 9 4 .0 290.4 238.4 251.1 2 6 5 .9 2 8 4 .5 304.3 309.4 253.9 269.7 280.4 300.5 329.2 3 3 3 .8 274.7 2 8 5 .5 305.7 336.4 4 0 3 .8 4 0 0 .9 3 4 8 .6 3 3 0 .9 363.1 239.38 18.051 259.25 29.056 266.23 22.061 283.70 20.883 304.90 23.384 363.95 31.752 154-159 Weight Change (gram s) Days 6-16 Days 0-20b 3 8 .8 30.5 4 7 .9 4 5 .8 39.5 4 0 .5 4 0 .5 0 6.111 1 3 8 .0 1 3 7 .7 139.3 1 3 8 .3 120.5 1 3 5 .7 134.92 7.161 8 3 .9 3 1 .6 4 8 .5 22.9 4 0 .3 4 6 .7 4 5 .6 5 21.052 9 7 .8 1 4 2 .0 150.2 1 3 5 .0 1 0 7 .5 1 1 4 .9 124.57 20.838 ooo CO Ci 000357 M aternal Number Date o f H a tin g 21620 21621 21622 21623 21624 21625 21626 Mean S.D . 1/18/83 1/18/83 1/19/83 1/19/83 1/19/83 1/20/83 1/29/83 21627 21628 21629 21630 21631 21632 21633 Mean S.D . 1/18/83 1/18/83 1/19/83 1/19/83 1/19/83 1/19/83 1/29/83 D isp o sitio n P.20-C P.20-C P.20-C P.20-C P.20-C P.20-C P.20-C P.20-C P.15-FD P .2 0 -A P .1 6 -F D P .1 7 -F 0 P.20-C N P .20 -C Appendix 1 - Continued Individual and Mean Maternal Body Heights and Height Changes Pilot Rat Teratology Study of T-3352 ____________________ Body H e ig h t (gram s) - Day o f G e s ta tio n _______________________ 0 6 8 12 16 20 Group 3 - 1 0 mg/kg 241.5 242.1 2 2 6 .9 237.4 231.9 2 5 7 .9 248.8 2 6 4 .3 2 7 1 .6 2 5 6 .9 2 8 0 .5 2 5 9 .8 2 8 1 .0 288.6 2 5 7 .6 2 6 5 .9 245.2 2 6 8 .7 255.1 2 8 9 .0 2 9 2 .6 2 7 4 .9 278.1 267.3 282.1 2 6 3 .9 300.1 299.7 2 8 0 .9 301.5 300.5 301.3 282.2 315.2 329.3 344.7 3 5 9 .3 360.5 3 7 0 .6 3 3 3 .8 3 5 5 .0 3 9 3 .8 240.93 10.348 271.81 12.006 267.73 17.535 280.87 14.384 301.56 17.119 359.67 19.150 Group 4 - 3 7 .5 mg/kg 2 7 4 .8 2 6 9 .3 2 4 0 .6 2 4 6 .3 2 3 2 .7 2 3 7 .8 (2 8 1 .2 ) 2 9 8 .3 300.3 2 6 4 .8 2 7 4 .8 2 5 9 .3 2 6 4 .5 (2 9 9 .6 ) 272.5 2 7 5 .6 264.1 2 6 3 .5 2 5 7 .5 2 5 7 .4 (2 8 0 .0 ) 273.9 241.2 244.7 2 5 5 .0 2 2 8 .6 2 3 0 .5 (2 4 6 .6 ) 2 8 4 .6 (2 1 9 .0 ) 214.1 2 2 5 .2 * 2 0 3 .9 212.1 (2 1 5 .4 ) 358.5 (1 6 1 .9 ) (1 9 6 .3 ) 2 7 5 .5 (2 5 1 .8 ) 250.25 17.535 263.63 21.958 265.10 7 .5 5 8 245.65 16.890 227.98 32.551 317.00 5 8 .6 9 0 154-159 Height Change (gram s) Days 6-16 Days 0-20" 1 6 .6 2 9 .9 4 3 .6 2 0 .8 2 2 .4 3 4 .2 40.7 29.74 10.317 1 0 3 .2 1 1 7 .2 133.6 133.2 1 0 1 .9 9 7 .1 1 4 5 .0 118.74 18.776 -1 3 .7 (-5 0.7 ) - 2 1 .1 -5 5 .4 -5 2 .4 -3 5 .6 5 S 21.324 8 3 .7 (-7 8.7 ) i to 3 7 .7 6 0 .7 0 S ` 32.527 la te rn a l Number 21634 21635 21636 21637 21638 21639 21640 Mean S.O . Date of Mating 1/18/83 1/18/83 1/18/83 1/19/83 1/19/83 1/19/83 1/21/83 D isp o sitio n P , 16-fO P. 14-FD P. 13-FD NP, 15-FD P, 14-FD P. 15-FD P. 13-FD Appendix 1 - Continued Individual and Mean Maternal Body Weights and Weight Changes Pilot Rat Teratology Study of T-3352 Body Weiqht (gram s) - Day o f G e sta tio n 0 6 B 12 16 Group 5 - 7 5 mg/kg 2 5 1 .7 2 1 5 .6 258.1 (2 4 9 .4 ) 222.7 2 3 1 .3 2 5 4 .0 273.4 2 4 9 .6 251.0 (2 5 3 .6 ) 2 4 2 .9 266.4 2 7 3 .5 258.2 233.4 2 5 4 .0 (2 4 6 .1 ) 2 3 7 .7 243.1 2 5 1 .8 226.3 1 8 2 .6 2 1 2 .9 (2 1 4 .3 ) 189.3 209.4 220.3 192.7* (1 7 1 .6 )* (203.4 * (1 9 0 .1 )* 1 7 1 .2 )* (1 8 7 .6 )* ( 1 9 8 .3 ) 238.90 18.020 259.47 13.286 246.37 9 .8 1 6 206.80 17.331 154-159 Weiqht Chanqe (grams) Days 5-16 Pays 0-20 - 8 0 .7 u> o I Appendix 2 Individual and Mean Dally and Total Food Consumption Pilot Rat Teratology Study of T-3352 154-159 lacerna i Number 21606 21607 21608 21609 21610 21611 Mean S.D. 21613 21614 21615 21616 21617 21618 Mean S.D. 6 16.5 20.4 19.8 15.3 18.6 19.1 18.28 1.981 12.9 24.8 20.5 17.8 18.0 15.9 18.32 4.058 Food Consumption (gm/day) ay of Gestation 8 12 16 ---- 20" Group 1 - 0 mg/kg 20.0 17.4 17.5 22.6 19.2 18.6 19.3 18.3 19.0 22.0 18.8 22.1 20.7 19.1 18.6 19.7 20.3 21.4 25.0 25.4 25.1 25.2 26.2 29.9 19.22 1.933 19.92 1.685 19.97 1.039 26.13 1.895 Group 2 - 1 mg/kg 19.5 24.9 20.7 16.8 18.2 15.3 20.7 24.3 21.7 17.9 17.8 17.9 20.7 22.4 22.1 32.4 18.8 21.6 26.1 31.7 27.6 28.5 19.6 26.8 19.23 3.370 20.05 2.665 23.00 4.784 26.72 3.996 a Statistical group comparisons performed on rank transformed data Total Food Consumption Days 0-20 (grams}a 415.4 428.5 424.0 419.5 429.6 464.4 430.23 17.578 399.2 537.3 470.1 472.7 386.7 406.7 445.45 58.101 I I 000 O f1 o Appendix 2 - Continued Individual and Mean Dally and Total Food Consumption Pilot Rat Teratology Study of T-3352 154-159 Maternal Number 21620 21621 21622 21623 21624 21625 21626 Mean S.D. 21627 21628 21629 21630 21631 21632 21633 Mean S.D. 6 16.6 17.8 19.2 18.8 17.8 14.6 15.6 17.20 1.681 23.2 18.8 16.6 19.5 17.3 18.0 (17.8) 18.90 2.346 Food Consumption (gm/day) Day of Gestation 8 12 16 20 Total Food Consumption Days 0-20 (grams)d Group 3 - 1 0 mg/kg 9.0 14.1 12.1 14.5 15.3 22.9 15.7 15.8 17.2 21.8 22.7 17.9 20.4 18.2 12.0 15.6 20.8 19.7 17.6 20.0 18.3 23.4 24.2 25.6 24.3 23.0 24.7 24.6 339.0 380.5 430.7 427.1 389.1 408.3 384.5 14.80 4.246 19.14 2.539 17.71 3.060 24.26 0.860 394.17 31.539 Group 4l - 37.5 mg/kg 7.3 9.4 17.9 11.1 13.1 14.4 (6.2) 9.2 2.0 55.7 9.8 7.3 10.4 (2.8) 12.1 24.8 Found dead Day 15 52.7 0.2 Found dead Day 16 0.3 Found dead Day 17 1.9 20.8 (0.7) (13.2) 361.1 (157.8) 586.4 (198.0) (177.5) 286.8 (203.6) 12.20 3.774 15.73 19.816 16.75 24.530 15.27 13.201 411.43 156.013 oscooo a Statistical group comparisons performed on rank transformed data. NOTE: Numbers in parentheses are excluded from mean calculation and statistical evaluation. Appendix 2 - Continued Individual and Mean Daily and Total Food Consumption Pilot Rat Teratology Study of T-3352 154-159 Maternal Number 21634 21635 21636 21637 21638 21639 21640 Mean S.D. Food Consumption (gm/day) WOay of Gestat ion 6 8 12 IB Group 5 - 7 5 mg/kg 17.5 14.7 17.4 (H.7) 14.1 21.8 34.7 10.3 12.4 7.9 (14.9) 13.0 6.6 7.5 10.5 1.6 1.9 (6.4) 1.7 31.8 3.8 Found dead Day 16 Found dead Day 14 Found dead Day 13 Found dead Day 15, NP Found dead Day 14 Found dead Day 15 Found dead Day 13 20.03 7.684 9.62 2.690 8.55 11.886 Total Food Consumption Days 0-20 (grams) (185.0) (134.0) (145.1) (137.3) (131.6) (293.2) (273.1) NOTE: Numbers in parentheses are excluded from mean calculation and statistical evaluation. 000361 23COOO Maternal Number 21606 21607 21608 21609 21610 21611 Mean S.O. 21613 21614 21615 21616 21617 21618 Mean S.O. Appendix 3 Individual Body Weights, Gravid Uterine Weights, and Terminal Body Weights Minus Gravid Uterine Weights Pilot Rat Teratology Study of T-3352 154-159 Terminal Body Weight (g) 367.2 366.3 374.7 376.8 358.1 356.0 366.52 8.422 Gravid Uterine Weight (g) Group 1 - 0 mg/kg 79.3 74.1 87.5 84.9 68.7 69.3 77.30 7.925 Terminal Body Weight Minus Gravid Uterine Weight (g) 287.9 292.2 287.2 291.9 289.4 286.7 289.22 2.378 336.4 403.8 400.9 348.6 330.9 363.1 363.95 31.752 Group 2 - 1 mg/kg 36.4 90.7 90.0 68.3 73.5 51.1 68.33 21.509 300.0 313.1 310.9 280.3 257.4 312.0 295.62 22.459 Appendix 3 - Continued Individual Body Weights, Gravid Uterine Weights, and Terminal Body Weights Minus Gravid Uterine Weights Pilot Rat Teratology Study of T-3352 154-159 Maternal Number 21620 21621 21622 21623 21624 21625 21626 Mean S.D. 21627 21628 21629 21630 21631 21632 21633 Mean S.D. Terminal Body Weight (g) Gravid Uterine Weight (g) Group 3 - 1 0 mg/kg 344.7 359.3 360.5 370.6 333.8 355.0 399.8 65.7 67.7 77.9 75.5 65.7 46.8 79.1 360.53 20.979 68.34 11.081 Group 4 - 37.5 mg/kg 358.5 Found dead Day 15 (161.9) Aborted pups Day 20 Found dead Day 16 Found dead Day 17 275.5 (251.8) Not pregnant 81.0 53.8 317.00 58.690 67.40 19.233 Terminal Body Weight Minus Gravid Uterine Weight (g) 279.0 291.6 282.6 295.1 268.1 308.2 314.7 291.33 16.396 277.5 221.7 249.60 39.457 000363 NOTE: Numbers in parentheses are excluded iron mean calculation and statistical evaluation. 000364 Maternal Number 21634 21635 21636 21637 21638 21639 21640 Appendix 3 - Continued Individual Body Weights, Gravid Uterine Weights, and Terminal Body Weights Minus Gravid Uterine Weights Pilot Rat Teratology Study of T -3352 154-159 Terminal Body Weight (g) Gravid Uterine Weight (g) Group 5 - 7 5 mg/kg Found dead Day 16 Found dead Day 14 Found dead Day 13 Found dead Day 15, Not pregnant Found dead Day 14 Found dead Day 15 Found dead Day 13 Terminal Body Weight Minus Gravid Uterine Weight (g) 8 000365 M aternal Humber O v arian Corpora Ltea U terine im plantations 21606 2160; 21608 21609 21610 21611 Mean S.D . 14 13 (15)* 17 14 17 1 5 .0 1.87 14 13 16 15 14 15 14.5 1.05 21613 21614 21615 21616 21617 21618 Mean S.D . 24 17 15 13 15 17 16.8 3.82 13 17 15 11 14 10 1 3 .3 2 .5 8 O vary c u t - e x clu d e d from mean c a l c u l a t i o n . Appendix 4 Individual and Mean Ovarian, Uterine, and Litter Data Pilot Rat Teratology Study of T-3352 Im plantation E ffic ie n c y (X) 100.0 100.0 a 88.2 100.0 88.2 9 5 .2 8 6 .4 6 3 54.2 100 100 8 4 .6 9 3 .3 5 8 .8 81.82 20.461 E a rly R e so rp tio n s Resorpt ion Incidences (X) Group 1 - mg/kg 00 o0 1 6.3 00 3 21.4 3 20.0 1 .2 1.47 7.95 10.183 Group 2 - mg/kg 8 61.5 t 5.9 o0 o0 i 7.1 2 20.0 2 .0 3 .0 3 15.75 23.576 Nunber of F e tu se s Dead A live Fetal M o rtality (X) 0 14 0 12 0 15 0 15 0 11 0 12 0 0 0 0 0 0 0 13.2 0 0 1 .7 2 0 05 0 16 0 15 0 11 0 13 08 0 0 0 0 0 0 0 11.3 0 0 4 .2 3 0 154-159 Fetal V ia b ility (X) 100 9 2 .3 9 3 .8 too 78.6 8 0 .0 9 0 .7 8 9 .4 4 5 3 8 .5 94.1 100 100 9 2 .9 8 0 .0 8 4 .2 5 23.576 ' Co ^ i 000366 M aternal Nueber 21620 21621 21622 21623 21624 21625 21626 Mean S.O . 21627 21628 21629 21630 21631 21632 21633 Mean S.O . O v a r1an Corpora Lutea 14 16 15 18 15 15 16 15.6 1.27 19 14 14 17 14 19 1 6 .2 2 .4 8 U terine Im plantatlons 12 16 15 15 14 9 15 13.7 2 .4 3 18 13 13 17 13 13 1 4 .5 2 .3 5 Appendix 4 - Continued In d iv id u a l and Mean O v a r ia n . U t e r in e , id L i t t e r O ata P ilo t Rat Teratology Study o f T-3352 Im plantation E ffic ie n c y (X) E a rly R e so rp tio n s R e so rp tio n Incidences (X) Group 3 - 10 mg/kg Number of F e tu s e s Dead A live 85.7 100 100 8 3 .3 93.3 6 0 .0 9 3 .8 88.01 13.911 0 0 0 12 3 18.8 0 13 1 6 .7 0 14 1 6 .7 0 14 3 21.4 0 11 0 0 09 0 0 0 15 1.1 1.35 7.66 0 12.6 9.044 0 2 .0 7 Group 4 - 3 7 .5 mg/kg 94.7 2 11.1 0 Found dead Day 15 13 F e tu se s aborted p r io r to s a c r i f i c e Day 20 100 5 Found dead Day 16 9 2 .9 2 Found dead Day 17 68.4 2 1 5 .4 0 Not p regnant 16 11 8 9 .0 0 14.060 2 .8 1 .5 0 13.25 0 13.5 3.041 0 3.54 Fetal M o rtality (X) 0 0 0 0 0 0 0 0 0 0 0 0 0 154-159 Fetal V ia b ility (X) too 8 1 .3 9 3 .3 93.3 7 8 .6 100 100 92.36 9 .0 2 4 8 8.9 8 4.6 86.75 3.041 1 Ui 00 ' M aternal Humber O v a ria n Corpora Lutea U terine Im plantations 21634 21635 21636 21637 21638 21639 21640 Mean S.O . 15 a 16 20 11 17 15 15.7 2 .9 4 11 17 15 11 17 13 14.0 2 .7 6 a Ovary too autolyzed to count corpora lu t e a . Appendix 4 - Continued Individual and Mean Ovarian, Uterine, and Litter Data Pilot Rat Teratology Study of T-3352 Im plantation E f f ic ie n c y (>) 73.3 a 93.8 Hot pregnant 100 100 8 6 .7 90.76 11.195 R e so rp tio n s R e s o rp tio n In cid en ces (<) Group 5 - 7 5 mg/kg Day 16 Day 14 Day 13 Found dead Day 15 Day 14 Oay 15 Day 14 Humber of F e tu s e s Dead XTfve Fetal M o rtality (X) 154-159 Fetal V ia b ility ) co co i 000368 M aternal Nuaber 21606 21607 2161 21609 21610 21611 Mean S.O . 21613 21614 21615 21616 21617 21618 Mean S.O . Ntfuber o f Males Sex____________ Nuaber of Females 7 7 6 9 7 7 7 .2 0 .9 6 7 5 9 6 4 5 6 .0 1 .7 9 2 4 9 9 8 4 6 .0 3.03 3 12 6 2 5 4 5 .3 3 .5 6 Appendix S Individual and Mean Live Fetal Data Pilot Rat Teratology Study of T-3352 154-159 Percent M ales ______________________________________ Mean W eight (g r a n s ) H ales Fem ales U n ad ju sted A d ju ste d U n a d ju ste d Group 1 - 0 mg/kg 5 0 .0 5 8 .3 4 0 .0 6 0 .0 6 3 .6 58.3 3 .3 3 .6 3 .5 3 .5 3 .4 3 .2 3 .1 3 .4 3 .2 3 .4 3 .2 3 .2 55.03 8.611 3.42 0 .1 4 7 3 .4 0 0 .1 7 6 3 .2 5 0 .t2 2 6roup 2 - 1 mg/kg 4 0 .0 2 5 .0 6 0 .0 8 1 .8 6 1 .5 5 0 .0 2 .9 3 .3 3 .3 3 .6 3 .4 3 .5 3 .3 3 .2 3 .2 3 .3 3.1 3 .3 53.05 19.559 3 .3 3 0 .2 4 2 3.37 0 .1 7 9 3 .2 3 0 .0 8 2 A d ju ste d 3 .2 5 0 .1 5 7 3.22 0 .1 5 8 1 * O 1 000369 M aternal Niaaber 21620 21621 21622 21623 21624 21625 21626 Mean S.D . 21627 21628 21629 21630 21631 21632 21633 Mean S.D . Appendix 5 - Continued Individual and Mean Live Fetal Data Pilot Rat Teratology Study of T-3352 154-159 Number of Males Sex Number of Females 7 3 6 9 7 3 11 6 .6 2.94 5 10 8 5 4 6 4 6 .0 2.24 Percent M ales M ales Unadjusted Group 3 - 10 mg/kg 5 8 .3 23.1 42.9 64.3 63.6 3 3 .3 73.7 3.1 3 .0 3 .3 3 .3 3 .6 3 .2 3 .3 51.31 18.533 3 .2 6 0 .1 9 0 Group 4 - 3 7 .5 mg/kg 9 Found Dead Day 15 Pups absorted p r io r Found Dead Day 16 Found Dead Day 17 6 Not pregnant 7 to s a c r if ic e 5 5 6 .3 Oay 20 5 4 .5 2 .9 2 .6 7 .5 2.12 6 .0 1.41 5 5 .4 0 1.273 2 .7 5 0.212 Mean U e lq h t (g r ams) Fem ales A d ju ste d Unadjusted A d ju ste d 3 .2 7 0 .1 7 6 3 .0 2 .8 3 .2 2 .9 3 .4 3.1 3 .4 3.11 0 .2 3 4 3.12 0 .1 5 7 2 .7 2 .7 2 S 0 .1 7 6 2 .5 2 .6 0 0.141 2 .6 0 S 0 .1 5 7 M aternal Number 21634 21635 21636 21637 21638 21639 21640 _ ____ ______________ Sex Number of Males Number of Females Found Dead Day 16 Found Dead Day 14 Found Dead Day 13 Found Day 15, Not pregnant Found Dead Day 14 Found Dead Day 15 Found Dead Day 14 Appendix S - Continued Individual and Mean Live Fetal Data Pilot Rat Teratology Study of T-3352 154-159 Percent M ales U n ad ju sted ________________Mean H eig h t (gram s)_______________________________________ M ales Fem ales A d ju ste d U n a d ju ste d A d ju ste d Group 5 - 7 5 mg/kg i l\3 0003 o'i  Appendix 6 Individual Visceral Findings Tor Each Litter Pilot Rat Teratology Study of T-3352 154-159 M aternal Number: Number o f f e t u s e s examined by W ils o n 's te ch n iq u e * Number o f f e t u s e s w it h in normal l i m i t s Ntaaber o f f e t u s e s h a vin g one o r more v i s c e r a l v a r ia n t s P e rcen t o f f e t u s e s h avin g one o r more v is c e r a l v a ria n ts (X) Number o f f e t u s e s w it h : D ilated renal p e lv is D ilated u re te r(s) Nusber o f f e t u s e s h avin g one o r more v is c e r a l anom alies P ercen t of f e t u s e s having one o r more v is c e r a l anom alies (X) 2 1 6 0 6 3 3 0 0 .0 0 0 .0 Group 1 - 0 mg/kg ___________ 2 ------- ~ 1-- 2 11111 66666 000 11 7B90 1 33333 32 333 0 1000 0 .0 33.33 0 .0 0 .0 0 .0 1 00 0 00 0 .0 0 .0 0 .0 0 .0 0 .0 __________ Group 2 - 1 mo/kg 1 --------- 2---------- 1 ----- 2------- ~ T ~ 1 1 1 11 1 66666 6 111111 3456 78 33333 3 323 3 3 3 0 10 0 0 0 0 .0 33.33 0 .0 0 .0 0 .0 0 .0 1 000000 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 M aternal Number: ~ 1 ------1 6 2 0 2------ -- 1 6 2 1 Group r^ - 1 6 2 2 3-10 7 \ 6 2 3 mg/kg 2 l 6 2 4 i l 6 2 5 2 1 6 2 6 Ntnber of fe tu se s examined by W ils o n 's tech niq u e* Number o f f e t u s e s w it h in normal l i m i t s 333 3333 3 33 333 3 Number o f f e t u s e s h a vin g one o r more v i s c e r a l v a r ia n t s 0 0 0 0 0 0 0 P e rcen t o f f e t u s e s having one o r more v is c e r a l v a ria n ts (X) 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 Number o f f e t u s e s w it h : D ilated renal p e lv is D ilated u re ter(s) In c o m p le te ly descended t e s t 1s ( e s } Nimber o f f e t u s e s h a v in g one o r more v i s c e r a l an o m alies 0 0 0 0 0 0 0 P ercen t o f fe tu se s having one o r more v is c e r a l anom alies (X) 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 Number o f f e t u s e s w it h : C le ft p alate Group 4 3 7 .5 mg/kg 2 11 66 23 72 3 2 1 33.33 3 1 2 6 6 .6 7 1 1 33.33 1 1 1 1 33.33 1 * Three fe tu se s from each l i t t e r . 000371 Appendix 7 Individual Skeletal Findings for Each Litter Pilot Rat Teratology Study of T-3352 154-159 M aternal Number : 2 1 6 0 6 Number o f f e t u s e s examined Number o f f e t u s e s w it h in normal l i m i t s Nimber o f f e t u s e s h a vin g one o r more s k e le t a l varian ts P e rcen t o f f e tu s e s having one or more s k e le t a l v a ria n ts (X) Number o f f e t u s e s w it h : Lagging O s s if ic a t io n S k u ll - Incom plete c lo s u r e ( l e s s than 75X) - In te r p a r ie ta l o s s if ic a t io n Incomplete - su p ra o c c lp ita ! o s s if ic a t io n Incomplete - hyoid o s s if ic a t io n incom plete - hyoid n o n o ssified - p a rleta ls irre g u la rly o ssified Rib cage - sternebrae b ip a rtite - rib s irre g u la rly o ssifie d V e rte b ra l column - th o r a c ic c e n tra o s s if ic a t io n in c o a p le te - th o ra c ic ce n tra nonfused - caudal le s s than three o ssifie d Bone Alignment " Extra rib (s ) Angulated rib s 3 0 3 100.00 2 3 2 1 1 Number o f f e t u s e s h a vin g one o r more s k e le t a l an o m alies Percen t o f fe tu se s having one or more s k e le t a l anomal te s (X) 0 0 .0 Group 1 -- P .M / k g 222 111 666 000 78 9 344 211 122 33.33 66.67 66.67 2 2 12 1 121 1 1 1 1 000 0 .0 0 .0 0 .0 22 11 66 l1 01 44 10 23 66.67 100.00 2 22 2` 1 21 2 1 1 1 00 0 .0 0 .0 Group 2 - 1 mg/kg 2 21 ? 22 111111 66666 6 l l 1111 3456 78 2* 3 3 3 3 3 2 10 20 1 02 3132 0 .0 66.67 100.00 33.33 100.00 66.67 2 1 22 2 3 1 1 3 1 1 1 000000 0 .0 0 .0 0 .0 0 .0 0 .0 0 .0 One f e t u s was damaged (h irin g p r e p a r a t io n so number o f m e t a t a r s a ls and p halang es co u ld not be d ete rm in e d . 000373 Appendix 7 - Continued Individual Skeletal Findings for Each Litter Pilot Rat Teratology Study of T-3352 154-159 M aternal Humber: ~T 1 6 2 0 Number o f f e t u s e s examined Number o f f e t u s e s w it h in normal l i m i t s Number o f f e t u s e s h a v in g one o r more s k e l e t a l varian ts P e rc e n t of fe t u s e s h avin g one or more s k e le t a l v a ria n ts (X) Number o f f e t u s e s w it h : la gging O s s if ic a t io n S k u ll - Incom plete c lo s u r e ( l e s s than 75X) - In t e r p a r ie t a l o s s if ic a t io n Incom plete - su p ra o cctp ita l o s s if ic a t io n Incomplete - hyoid n o n o sslfied - p arletals irre g u la rly o ssifie d Rib cage - sternebrae b ip a rtite V e rte b ra l column - th o r a c ic c e n tra o s s if ic a t io n incom plete - th o racic cen tra nonfused - caudal le s s than three o ssified Bone A lig nment x tra rlb (s) Angulated r ib s 3 2 1 33.33 1 Number o f f e t u s e s h a v in g one o r more s k e l e t a l an o m alies P e rcen t o f f e t u s e s havin g one o r more s k e le t a l anomalies (X) 0 0 .0 1 1 6 2 1 3 0 3 30.00 Group 1 6 2 2 3-10 2 1 6 2 3 mg/kg_________ __________ ~T 111 666 222 456 33 3 3 3 2 22 20 11113 3 3.33 33. 33 3 3.3 3 3 3 .3 3 1 00.00 1 31 2 31 2 ll 1 1 11 2 1 1 1 1 1 000000 0.0 0.0 0.0 0.0 0.0 0.0 Group 4 - 3 7 .5 mg/kq ------ ------------ ------ 11 66 23 72 33 10 23 6 6 .6 7 100.00 1 l1 12 13 t 00 0.0 0.0 i cn i - 46 - i Appendix 8 References Pilot Rat Teratology Study of T-3352 STATISTICAL METHODS/LABORATORY PROCEDURES 154-159 Bartlett, M. S., "Some Examples of Statistical Methods of Research in Agriculture and Applied Biology." J. Royal Statist. Soc. Suppl., IV:137-183, 1937. Draper, N. R. and W. G. Hunter (1969), "Transformations: Some Examples Revisited", Technometrics, 11:23-40. Dunnett, C. W., "A Multiple Comparison Procedure for Comparing Several Treatments with a Control." J. Am. Stat. Assoc., 50:1096-1121, 1955. Dunnett, C. W., "New Tables for Multiple Comparisons with a Control." Biometrics, 20:482-491, 1964. Games, P. A., and Howell, J. F., "Pairwise Multiple Comparison Procedures with Unequal N's and/or Variances: A Monte Carlo Study." J. Ed. Statist., 1:113-125, 1976. Levene, H., "Robust Tests for Equality of Variances," in I. Olkin edited, Contributions to Probability and Statistics, Stanford University Press. Palo Alto, 1960.------- Staples, R. E. and Schnell, V. L., "Refinements in Rapid Clearing Technic in the KOH-Alizarin Red S Method for Fetal Bone." Stain Technoloqy, 39:61-63, 1964. ---------------- Wilson, J. G. and Warkany, J., editors. Teratoloqy: Principies and Techniques, The Univ. of Chicago Press, Chicago, 1^65, pp. 251-27/. Winer, B. J., Statistical Principles in Experimental Desiqn. 2nd Ed.. McGraw-Hill, N.Y7,.Chs. 3-10, 1371. 000374