Document DGxkdbpaL3Jeo8ggpYM76YD9n

(jjyj) H A ^ L S T O f M LABORATORIES AMERICA. INC. M O O lE I M u n S T U N IK C V I(N N A v ) e < N l* 8 8 1 O .U t.A PILOT RAT TERATOLOGY STUDY T-3352 FINAL REPORT Submitted to 3M Company St. Paul, Minnesota May 11, 1983 & HAZLETON n J ^ L A B O R A T O R IE S ArvtERlCA. INC. 92QCLEESBURG t^rnPKE VI6NNA VIRGINIA22' BO USA SUBJECT: P ilo t Rat Teratology Study Pro ject No. 154-159 We the undersigned, hereby declare that the work was performed under our supervision, according to the procedures herein described. Study D irector: a tu ii LAWRENCE T. WETZEL, Ph.D. Staff Scientist Department of Toxicology Laboratory Supervision: Teratology: GEORGE?A. BURDOCK, Ph.U. Rodent Toxicology I Department of Toxicology RUTH S. DURLOO, B.S . Research Associate Department of Toxicology Report Preparation: MARCIA A. MENSE, B.". Technical W riter S c ie n tif ic Resources Department blm ( i ^ ) H A Z L E T O N l a b o r a t o r i e s ,AM ERIC A INC. V^SP' .'.oouMaaumi tu-~--< v.o.~.'= TABLE OF CONTENTS Page SUMMARY INTRODUCTION CONTROL AND TEST MATERIALS TEST ANIMALS METHODS Mating Period Groups and Dosage Levels Compound Preparation and Administration Maternal Observations and Records Cesarean S a c rific e and Tissue Preservation S t a t is t ic a l Analyses Specimen, Raw Data, and Final Report Storage RESULTS - MATERNAL DATA C lin ic a l Signs M o rtality Body Weights Food Consumption Gross Pathology Uterine Weights RESULTS - CESAREAN DATA . Pregnancy Rates, Corpora Lutea, Uterine Implantations, and Implantation E ffic ie n c y Fetal V ia b ilit y , Weight, and Sex Fetal Development 1 3 3 4 4 4 5 6 6 7 8 9 1111110000 11 11 12 12 13 13 TABLES . Table 1 - C lin ic a l Observations During Gestation Table 2 - Mean Maternal Body Weights and Body Weight Change Table 3 - Mean Food Consumption Values Table 4 - Surrmary of Gross Pathology Findings Table 5 - Mean Terminal Body Weights, Gravid Uterine Weights, and . Terminal Body Weights Minus Gravid U terine Weights Key to Table 6 . Table 6 - Sumnary of Ovarian, U terin e, and L it t e r Data Table 7 - Mean Incidence Values for V isceral and Sk eletal Findings Key to Table 8 Table 8 - Mean Incidence of Visceral and Skeletal Findings per L it t e r 15 15 17 18 2210 22 23 25 25 1( ) H A Z ^ T P f \ l LABORATORIES AMERICA. INC. ' S* P ' MOO i M a u - o v,oi~ . ' o .u s - 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 D aily and Total Food Consumption Appendix 3 - Individual Body Wei ghts, Gravid Uterine Weights, and Terminal Body Wei ghts Minus Gravid Uterine Weights Appendix 4 - Individual and Mean Ovarian, U terine, and L it t e r Data Appendix 5 - Individual and Mean Live Fetal Data Appendix 6 - Individual Visceral Findings for Each L it t e r Appendix 7 - Individual Skeletal Findings fo r Each L it t e r Appendix 8 - References 27 28 31 34 37 40 43 44 46 HAZLETON LABORATORIES AMERICA, INC. s p o n s o r : 3M Company MATERIAL: T-3352 SUBJECT: FINAL REPORT P ilo t Rat Teratology Study Project No. 154-159 DATE: May 11, 1983 SUMMARY T-3352, suspended in corn o il at concentrations intended to d eliver 1, 10, 37.5, or 75 mg/kg/day (Groups 2, 3, 4, and 5, re s p e c tiv e ly ), was administered by oral intubation to four groups of mated female Sprague-Dawley S-D rats on gestation Days 6 through 15. A f i f t h 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. C lin ic a l 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 s a c r if ic e were noted in Groups 4 and 5 and these included incidences of l i v e r , adrenal, lung, and/or g astro in testinal tra c t e ffe c ts . (i)HAZLETOfM l a b o r a t o r ie s AMERICA. INC. M 0 0 . * . U - = T U . E - 2- 154-159 Evaluation of mean fe ta l weight data indicated that fe ta l weights of both sexes decreased with increasing dose le v e l. A sig n ific a n t decrease in mean fe ta l weight occurred in Group 4 males and females, although data for only two lit t e r s were a v a ila b le fo r that group. C le ft palate was found in two of six Group 4 fetuses (one fetus in each l i t t e r ) examined by W ilson's technique. Incompletely descended testes were noted in one fetus from each Group 4 l i t t e r evaluated for visceral defects. Incidences of skeletal variants were sim ila r fo r a ll groups. f jQ j) H A Z L E T O N LABORATORIES AMERICA. INC. 4b b o C L E E I B u P C ^ - ^ i V * N N * t f l O l N . i ' Q U * A -3- 154-159 INTRODUCTION This study was designed to determine the maternal and embryo/ fe ta l to x ic ity of T-3352 when administered by gavage to pregnant rats during the period of fe ta l organogenesis for the purpose of setting dose levels fo r 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 resu lts from this study. CONTROL AND TEST MATERIALS The vehicle and control m aterial, Duke's Corn Oil (C. F. Sauer Co., Richmond, V irg in ia ), lo t 80235, was received on January 24, 1983, and was stored at room temperature. The test m aterial, T-3352, an off-white s o lid , 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 s t a b ilit y , as well as data on composition or other ch a ra c te ris tic s which define the test m aterial, are on f i l e with the sponsor. H A Z L E T O N l a b o r a t o r ie s AMERICA, in c . M K L i(S .U S T U.N ! v.e~~A v.o.~.Aa*'a. U.A -4- 154-159 test animals Sexually mature cesarean-derived Sprague-Dawley Crl:CD (SD)BR rats were chosen for th is study because they are sen sitive to a number of agents which are known to be embryotoxic and/or terato g enic. Rats have h is t o r ic a lly been used in safety evaluation studies of th is type and are required by the regulatory agencies. T h irty-six twelve to fourteen-week old male rats and th irty - s ix 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 s ta ff veterin arian . The rats were housed one male and one female per cage during breeding. Following confirmation of mating, the females were housed in d iv id u a lly in elevated wire-mesh cages with food (Purina Rodent Laboratory Chow 5001) and tap water availab le ad lib itu m . The females were uniquely id e n tifie d by ear tag after mating was confirmed. Tempera tures in the study room ranged from 70 to 78F with a r e la tiv e humidity between 44 and 69%. METHODS Mating Period During the mating period, one female was paired with one male u n til mating was confirmed or u n til two weeks had elapsed. D aily vaginal ( ^ H A Z L E T O N LABORATORIES AMERICA. INC 920C LeiU"GTuM"<i VI*MN - 5- 154-159 examinations of each female were performed to detect the presence and v ia b il i t y 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 Dosea mg/kg/day 0 1 10 37.5 75 Number of Females 6 6b 7 7 7 a Based on individual animal body weights at each weighing in terval 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 th is animal's dose level would have been known at cesarean s a c r ific e , 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 seq u en tially through the control group u n til a ll mated females were assigned to a group. A ll males were sa crifice d via carbon dioxide asphyxiation and discarded without necropsy once a s u ffic ie n t number of matings were confirmed. @ HAZLETON LABO RATO RIES AMERICA. INC. V^ S ? / m L i i x u a c T u -- M V.e~~- v . - o . . . . * a * * . o u . . -6 154-159 Compound Preparation and Administration Suspensions of the test m aterial in the corn o il veh icle 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 ele ctro n ic 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 o il was added and the test m aterial was ground into a fin e suspension. This suspension was rinsed from the homogenizer or the blender into a beaker and additional corn o il was added to produce the desired concentration. The suspension was mixed on a s t ir r e r for approximately fifte e n minutes. Prepared suspensions were mixed with a magnetic s t ir r e r during dosing. Females were given the appropriate dosing suspension or vehicle by oral intubation on a d a ily basis beginning on Day 6 and continuing through Day 15 of gestation. The test material was administered o r a lly because of the re la tiv e ease and accuracy of dosing. Fresh dosing suspensions were prepared weekly and stored under re frig e ra tio n (approximately 4 1 F). Samples of each te s t mixture as well as the vehicle were sent to the sponsor for an alysis. Maternal Observations and Records A ll animals were examined twice d a ily for m o rta lity and mori- bundity (from day of receipt through study term ination) and once d a ily fo r c lin ic a l signs of to x ic ity and pharmacologic e ffe c ts (throughout HA2 LETOIMr ^ 1 l a b o r a t o r i e s AMERICA. INC a a c c k .E E U B G TU W PIICE v . ~ ~ A < / oh m i* u m .j -7- 154-159 g estatio n). Body weights and food consumption were recorded on Days 0, 6, 8, 12, 16, and 20 of gestation. Cesarean S a c rific e and Tissue Preservation A gross necropsy was performed on a ll animals (found deads, moribund s a c r ific e s , and terminal s a c r if ic e s ). The uterus and ovaries from found dead animals and animals sa c rifice d because of moribundity were removed and examined for the number of implantation s ite s and corpora lutea, resp e ctive ly. P rio r to gestation Day 20, surviving females were assigned random numbers and a ll personnel performing cesarean sections and/or external, v is c e r a l, or sk eletal examinations of the fetuses were unaware of the dose level from which the animals were derived. On Day 20 of gestation, a ll surviving females were weighed and sa c rifice d by carbon dioxide asphyxiation. The uterus from each female was weighed and examined fo r the number and placement of uterine implantation s ite s , number of liv e and dead fetuses, e a rly and late resorbing fetuses, and any abnorm alities. The uterus of each animal was reweighed a fte r the contents were removed. The ovaries were examined for the number of corpora lu te a . Each liv e fetus was sexed, weighed, and examined for external abnorm alities. Findings were recorded. Beginning at the ovarian end of the rig h t uterine horn, the f i r s t six fetuses, regardless of sex, were selected fo r further evaluation. After the external examination was completed, the f i r s t , | i?S) ^ T Q f M l a b o r a t o r i e s A m e r i c a . INC. 0 - 0 0K< S P ' . ^ o L.. Tu~-- C V -~ ~ . V. .~. -8 154-159 th ird , and f if t h fetuses were id e n tifie d with a tag and fixed in Bouin's solution for soft tissue evaluation (Wilson, et a l , 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 S c h n e ll, 1964). S t a t is t ic a l Analyses Mean maternal body weight gains (Days 6-16 and 0-20), to ta l food consumption, gravid uterine weight, terminal body weight minus gravid uterine weight, resorption incidence, percent males, and fe ta l v i a b i l i t y were analyzed in the following order. Levene's te s t fo r homogeneity of variances (Levene, 1960; Draper and Hunter, 1969) was performed and i f the variances proved to be homogeneous, the data were analyzed by one-way c la s s ific a tio n analysis of variance (ANOVA) (Winer, 1971). I f the variances proved to be heterogeneous, a series of transformations was performed u n til variance homogeneity was achieved. These transformations were: log10, square (X2), square root (X%) , recip ro cal (1/X), angular (arcsine X1* ), and rank, in that order. I f rank transformation was in e ffe c tiv e in removing variance heterogeneity, ANOVA of ranked data was completed. I f ANOVA of untransformed or transformed data was s ig n ific a n t, Dunnett's t- te st (Dunnett, 1955 and 1964) was used for control vs. compound-treated group mean comparisons. I f ANOVA was not s ig n ific a n t, the analysis was complete. (9 ^HAZLETON LABO RATO RIES AMERICA. INC. '^SP' ;00 Tu-- !* v.0~.. '<=uMJ -9- 154-159 In addition to the above data, analysis of covariance (ANCOVA) (Winer, 1971) was used to analyze mean fe ta l weights. The l i t t e r was used as the experimental u n it. Levene's tests and ANOVA were evaluated at the 5.0% one-tailed p ro b a b ility le v e l. Control vs. compound-treated group mean comparisons were evaluated at the 5.0 two-tailed p ro b a b ility le v e l. S t a t is t ic a l references are appended to th is rep o rt, and s t a t i s t i c a lly sig n ific a n t d ifferen ces, as indicated by the aforementioned te s ts , are designated throughout th is report by the term "s ig n ific a n t" and/or as follows: 5+ = S ig n ific a n tly higher than the control value. S- = S ig n ific a n tly lower than the control value. Specimen, Raw Data, and Final Report Storage A ll specimens, raw data, and the fin a l report are stored in the archives of Hazleton Laboratories America, Inc. HAZLETOfMri^ l LABORATORIES AMERICA. INC. a a o c LlEIBU>a T u m i . v.oi~. *'o o.-< - 10 154-159 RESULTS - MATERNAL DATA C lin ic a l Signs Summary c lin ic a l signs are presented in Table 1. Treatment-related c lin ic a l 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. C lin ic a l signs fo r Groups 4 and 5 animals included hunched and thin appearance, languid behavior, urine sta in s, and bloody crusted eyes, e y e lid s, nose, mouth, legs, paws, or g e n ita ls. Anorexia, ataxia, dyspnea, rough coat and pale appearance were also noted for several Group 4 or 5 ra ts . One Group 4 ra t aborted th irteen fetuses on Day 20 p rior to s a c r if ic e . Alopecia was noted for some rats in each dose group and the control group. M ortality A ll 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. A ll other animals survived to cesarean s a c r ific e 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 HAZ LETON l a b o r a t o r ie s A, M E RICA, INC n o o L i( ia u B S T u N > w E v ta s iM i* a a '* o . u * - 11 - 154-159 and 4. The decrease in the Group 4 animals was s ig n ific a n tly d iffe re n t than the control value. Because of high m o rta lity , Group 5 data were not included in s t a t is t ic a l evaluation. Food Consumption Individual and mean food consumption values are presented in Appendix 2. Mean food consumption values are presented in Table 3. S lig h tly decreased food consumption values were noted for Groups 4 and 5 compared to control on and a fte r Day 8 of g estation. Mean to ta l food consumption values for Groups 2, 3, and 4 were s t a t i s t ic a l l y comparable to control. Total food consumption for Group 5 was not determined because of m o rta lity. Gross Pathology Surmiary 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 liv e r , kidney, and adrenals; enlarged adrenals; d ilated renal blood vessels, and red vaginal discharge. Findings noted fo r Group 5 animals were reddened and/or distended lungs, enlarged and/or f r ia b le liv e r , enlarged adrenals, discolored and/or thin glandular g astric mucosa, flu id in stomach or in te stin e s, and red vaginal discharge. Uterine Weights Individual and mean uterine weights are presented in Appendix 3. Summary uterine weights are presented in Table 5. LABORATORIES AMERICA. INC. - 12 - 154-159 Mean terminal body weights were comparable fo r 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 cornoared to the control mean weight. For Group 2, th is may be attrib u tab le to one dam with eight e a rly 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 l i t t e r data are presented in Appendix 4. Individual and mean liv e fe ta l data are presented in Appendix 5. Summary ovarian, uterine, and l i t t e r data are presented in Table 5. 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 fo r a ll groups as were mean implantation e ffic ie n c ie s . @) HAZLETON LABORATORIES AMERICA. INC. 49 8 0 0 L i l l B u B 5 T u * N ' * E v i f MIM(*SA VlSQINIA aB'SO. L - 13 - 154-159 Fetal V ia b ilit y , Weight, and Sex Mean resorption incidences were higher fo r Groups 2 and 4 than for the control group, but these differences werenot s t a t i s t ic a l l y s ig n ific a n t. The increased incidence in Group 2 may be a ttrib u ta b le to one dam with eight resorbing fetuses. Mean incidence of f e t a l v ia b il i t y and percent males were comparable for Groups 1-4. Mean liv e fe ta l weights of both sexes decreased with increasing dose level with Group 4 values s ig n ific a n tly lower than control. Fetal Development Individual visceral findings are presented in Appendix 6; in d iv i dual skeletal findings are presented in Appendix 7. Mean incidence values for visceral andskeletal findings are presented inTable7 (based on number of fetuses) and Table 8 (based on number of l i t t e r s ) . Gross external examination of fetuses at cesarean section revealed one abnormal fetus from a Group 3 dam. This fetus had no t a i l and appeared to have no thoracic or lumbarvertebrae. However, no skeletal evaluation was done on th is fetus so that finding could not be v e rifie d . 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 HAZLETON|f^ ) LABO RATO RIES AMERICA. INC. s a o o L. . M U = r u -- . v .e ~ ~ * v . - o . ~ . a * i o . urn - - 14 - 154-159 control group (50 percent vs. 5.56 percent). However, only two Group 4 l i t t e r s were availab le for evaluation compared to six of the control group. Variants noted were dilated renal pelves (Group 2), d ilated ureters (Groups 1 and 4 ), and incompletely descended testes (Group 4 ). C le ft 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 l i t t e r s from which the fetuses selected for v isc e ra l examination were derived. No other visceral anomalies were observed. Table 1 C lin ic a l Observations During Gestation P ilo t Rat Teratology Study of T-3352 154-159 OBSERVATIOH Group 1 - 0 mq/kg Day of G estation ~IT5 5-I5~ T^TO Group 2 - 1 mg Day of Gestation 0-5 6 -1 5 ------- Number of animals observed G Hunched Thin Languid Anorexia Ataxia Dyspnea Urine stains Alopecia (paws, le g s, chest, neck, h ip s, sid e , abdomen) 2 Rough halrcoat Bloody c ru st (nose, mouth, paws, e ye lid s, legs, ge n ita ls, eyes) Pale Aborted fetuses/red vaginal discharge 6 4 6 66 412 6 2 Group 3 - 1 0 mq/kg Pay of Oesta tlo n 0 ^ --- 6-15-- 15-70 // 12 Group 4 - 3 7 . 5 mg/kg Day of Gestation T J - 5 -----E - I5 I5 -7 Group 5 - 7 5 mg/kg bay of Gestation TT-TT-- 575--15-JO 6(1) 6(1) 5(1) 2 1 3 12 4(1) 6(1) 3(1) 3 3(1) 1 2 3 1 6(1) 6(1) 6(1) 6(1) 3 2(1) 4(1) 1 1 0 i ui i 5(1) 3(1) 1 6(1) 1 () Nonpregnant female. Table 2 Mean Maternal Body Weights and Body Weight Change P ilo t 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 06 231.60 6.935 6 263.57 6.342 6 239.38 18.051 6 259.25 29.056 6 240.93 10.348 7 271.81 12.006 7 250.25 17.535 6 263.63 21.958 6 238.90 18.020 6 259.47 13.286 6 Body Wei qht (grams) Day of Gestation 8 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 S t a t i s t i c a l group comparisons performed on rank-transform ed data. 154-159 Body Weight Change (g) Days of 1Testation 6-16 0-20d +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 S- 21.324 4 +60.70S- 32.527 2 -80.7 - 1 - 0 Group and Dose Level (mg/kg) 1 0 mg/kg 2 1 mg/kg 3 10 mg/kg 4 37.5 mg/kg 5 75 mg/kg Mean i 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 P ilo t Rat Teratology Study of T-3352 Food Consumption (gm/day)______ Day of Gestation 8 12 T6 20 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 9.62 2.690 6 15.73 19.816 6 8.55 11.886 6 16.75 24.530 4 - 0 15.27 13.201 3 ' - 0 Total Food Consunptlon(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 a S t a t i s t i c a l group comparisons performed on rank-transform ed data. Table 4 Suirmary of Gross Pathology F in d in gs P ilo t Rat Teratology Study of T-3352 154-159 ORGAN AND DESCRIPTION Group: 1 2 3 4 5 Dose Level (mg/kg): T J ~ r TIT 1 7 3 71 Number of rats examined Number with no gross lesions LUNGS Reddened Distended 66 66 7 6(1) 6(1) 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 flu id Contained black flu id Glandular mucosa - reddened - thin - black focal areas 1 (D 3(1) 1 1 () * Nonpregnant female. I 00 I Table 4 - Continued Sutmiary of G ross Pathology F indings P ilo t Rat Teratology Study of T-3352 ORGAN AND DESCRIPTION INTESTINES F ille d with dark flu id material OVARIES Autolyzed VAGINA Red/bloody discharge/dark red flu id Group: 1 Dose Level (mg/kg): I T Table 5 Mean Terminal Body Weights, Gravid Uterine Weights, and Terminal Body Weights Minus Gravid Uterine Weights P ilo t 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 5 20 mg/kg Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N Mean S.D. N Terminal Body Weight grams 366.52 8.422 6 363.95 31.752 6 359.67 19.150 7 317.00 58.690 2 - 0 Gravid Uterine Weight grams 77.30 7.925 6 68.33 21.509 6 68.34 11.081 7 67.40 19.233 2 - 0 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 - 0 a S t a t is t ic a l group comparisons performed on rank-transformed data. - 21 - Key to Table 6 154-159 p ,.n n,n rv 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 E ffic ie n c y (percent) -G roup mean of ([ implantations per litte r/co rp o ra lutea per li t t e r j x 100). Mean Resorption Incidence (percent) = Group mean of ([reso rp tio n s per litter/im p lan tatio n s per l i t t e r j x 100). mTM incidence of Fetal M o rta lity (percent) 'G r o u p mean of ([dead fetuses per litte r/im p lan tatio n s per 1it t e r j x 100). Mean I ncidence of Fetal V ia b ilit y (percent) 'G r o u p mean of ( [ l i v e fetuses per litter/im p lan tatio n s per l i t t e r ] x 100). Table 6 Surtmary o f O varian, U te rin e , and L it t e r Data P ilo t Rat Teratology Study o f T-3352 Group: 1 Dose Level (mg/kg): -- <5~ 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 Implantations Resorptions Fetuses - dead - live 15.0 14.5 1.2 0.0 13.2 Indices calculated on a per l i t t e r basis: Mean Implantation e ffic ie n c y (X) Mean resorption Incidence (X) Mean Incidence of fe ta l m o rta lity (X) Mean Incidence of fe ta l v ia b il i t y (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 --- j-- 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 10 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 37.5 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 2.72S2.60 2.60S55.40 a An additional 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 s a c r if ic e , she was removed from the study. Table 7 Mean Incidence Values fo r V isce ra l and S k e le ta l F in d in gs P ilo t Rat T eratology Study of T-3352 154-159 Group: Dose Level (mg/kg): 1 Visceral Number of fetuses examined Number of fetuses within normal lim its 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 te s tis (e s ) 1/5.56 0 1/5.56 0 Number/percent of fetuses having one or more visceral anomal1es 0 Number/percent of fetuses with: Cleft palate 0 Skeletal Number of fetuses examined Number of fetuses within normal lim its Number/percent of fetuses having one or more skeletal vari ants Number/percent of fetuses with: Lagging O ssificatio n Skull - Incomplete closure (less than 75%) - In te rp a rie ta l o s s ific a tio n Incomplete - supraoccipital o s s ific a tio n Incomplete - supraoccipital nonossification - hyoid o s s ific a tio n Incomplete - hyoid - nonosslfled - parietals 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 f o r V isc e ra l and S k e le ta l F in d in gs P ilo t Rat T e rato logy Study of T-3352 Group: Dose Level (mg/kg): 1 0 Skeletal (Continued) Number of fetuses examined Number/percent having one or more skeletal variants Number/percent of fetuses with: Lagging O ssificatio n Rib-cage - strnebrae b ip a rtite - rib irregularly ossified Vertebral column - thoracic centra o ssifica tio n incomplete - thoracic centra nonfused - caudals less than three o ssified 13 0 1/5.56 0 1/5.56 1/5.56 Bone alignment Extra rlb (s ) Angulated rubs 1/5.56 2/11.11 Number/percent having one or more skeletal anomalies 0 ---2j _ 11 1/5.88 1/5.88 0 1/5.88 0 0 1/5.88 0 154-159 34 10 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 - 25 - Key to Table 8 154-159 Mean incid ence of V isceral Anomalies (percent) = Group mean of ([number of -- f etuses With anomalies per litter/number or fetuses examined v is c e r a lly per l i t t e r ] x 100). Mean in c idence of V isceral Variants (percent) = Group mean of ([ number of fetuses with variants per litter/number of fetuses examined v is c e r a lly per l i t t e r ] x 100). Mean incidence of Skeletal Anomalies (percent) = Group mean of ([number of -- fetuses with anomalies per 1itter/number or fetuses examined s k e le ta lly per l i t t e r ] x 100). Mean Incidence of Skeletal Variants (percent) = Group mean of ([ number of fetuses with variants per ntter/number ot fetuses examined s k e le ta lly per l i t t e r ] x 100). Table 8 Mean Incidence o f V isc e ra l and Ske le tal F in d in gs per L it t e r P ilo t Teratology Study o f T-3352 in Rats 154-159 Group: Dose Level (mg/kg): Visceral Examination 1 2 ~T ~ 3 "IT T 4 37.5 Number of lit t e r s examined Number of lit t e r s with anomalous fetuses Percent of U tt e r s with anomalous fetuses (X) Number of lit t e r s with variant fetuses Percent of lit t e r s with variant fetuses (X) 6 0 0 1 16.67 6 0 0 1 16.67 7 0 0 0 0 2 2 100.00 2 100.00 Mean values calculated on a per l i t t e r basis: Number of fetuses with variants Incidence of varlants(X) Number of fetuses with anomalies Incidence of anomalies (X) 0.17 5.56 0 0 0.17 5.56 0 0 0 0 0 0 1.50 50.00 1.00 33.33 Skeletal Examination Number of lit t e r s examined Number of lit t e r s with anomalous fetuses Percent of lit t e r s with anomalous fetuses (%) Number of U t t e r s with variant fetuses Percent of lit t e r s with variant fetuses (X) 6 0 0 6 100.00 6 0 0 5 83.33 7 0 0 7 100.00 2 0 0 2 100.00 Mean values calculated on a per U t t e r Number of fetuses with variants Incidence of variants (X) Number of fetuses with anomalies Incidence of anomalies (X) basis: 2.17 72.22 0 0 1.83 61.11 0 0 1.57 52.38 0 0 2.50 83.33 0 0 - 27 - Key to Appendix 1 Individual and Mean Maternal Body Weights P ilo t 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) 2 Body weight on day of death. b s t a t is t ic a l group comparison performed on rank-transformed data. Note: Body weights in parentheses are excluded from mean calculatio ns and s t a t is t ic a l evaluation. Maternal Number 21606 21607 21608 21609 21610 21611 Mean S.D. 21613 21614 21615 21616 21617 21618 Mean S.D 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 l Individual and Mean Maternal Body Heights and Height Changes Pilo t Rat Teratology Study of T-33S2 Body Height (grams) - Day of Gestation 0 ___6___ 8 12 16 20 Group 1 - 0 mg/kg 229.2 228.6 235.4 238.5 237.6 220.3 271.1 263.4 265.7 262.4 266.6 252.2 272.3 256.2 269.8 261.9 267.6 257.2 288.8 280.6 289.7 285.0 2B6.5 200.8 309.9 293.9 313.6 308.2 306.1 292.7 367.2 366.3 374.7 376.8 358.1 356.0 231.60 6.935 263.57 6.342 264.17 6.735 285.23 3.805 304.07 8.703 366.52 8.422 Group 2 - 1 mg/kg 238.6 261.8 250.7 213.6 223.4 248.2 216.6 297.6 285.3 251.8 245.2 259.0 257.6 294.0 290.4 238.4 251.1 265.9 284.5 304.3 309.4 253.9 269.7 280.4 300.5 329.2 333.8 274.7 285.5 305.7 336.4 403.8 400.9 348.6 330.9 363.1 239.38 18.051 259.25 29.056 266.23 22.061 283. 70 20.883 304.90 23.384 363.95 31.752 154-159 Height Change (grains) Days 6-16 Days 0-20 38.8 30.5 47.9 45.8 39.5 40.5 40.50 6. I l l 138.0 137.7 139.3 138.3 120.5 135.7 134.92 7.161 83.9 31.6 48.5 22.9 40.3 46.7 45.65 21.052 97.8 142.0 150.2 135.0 107.5 114.9 rcoo , 124.57 20.838 Number 21620 21621 21622 21623 21624 21625 21626 Mean S.D. 21627 21628 21629 21630 21631 21632 21633 Mean S.D. Date of Hating 1/18/83 1/1S/83 1/19/83 1/19/83 1/19/83 1/20/83 1/29/83 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.20-A P.16-FD P.17-FD P.20-C NP.20-C Appendix 1 - Continued Individual and Mean Maternal Body Weights and Weight Changes P ilo t Rat Teratology Study of T-3352 Body Welqht (qrams) - Day of G estation 0 6 8 12 16 20 Group 3 - 1 0 mg/kg 241.5 242.1 226.9 237.4 231.9 257.9 248.8 240.93 10.348 264.3 271.6 256.9 280.5 259.8 281.0 288.6 271.81 12.006 257.6 265.9 245.2 268.7 255.1 289.0 292.6 267.73 17.535 274.9 278.1 267.3 282.1 263.9 300.1 299.7 280.87 14.384 280.9 301.5 300.5 301.3 282.2 315.2 329.3 301.56 17.119 344.7 359.3 360.5 370.6 333.8 355.0 393.8 359.67 19.150 Group 4 - 37.5 mg/kg 274.8 269.3 240.6 246.3 232.7 237.8 (281.2) 298.3 300.3 264.8 274.8 259.3 264.5 (299.6) 272.5 275.6 264.1 263.5 257.5 257.4 (280.0) 273.9 241.2 244.7 255.0 228.6 230.5 (246.6) 284.6 (219.0) 214.1 225.2 203.9 212.1 (215.4) 358.5 (161.9) (196.3) 275.5 (251.8) 250.25 17.535 263.63 21.958 265.10 7.558 245.65 16.890 227.98 32.551 317.00 58.690 151-159 Weight Change (grams) Days 6-16 Days 0-20 16.6 29.9 43.6 20.8 22.4 34.2 40.7 29.74 10.317 1i10|1397..201 133.6 11A3m903l71...19At 145.0 118.74 18.776 -13.7 (-50.7) -21.1 -55.4 -52.4 -35.65S21.324 83.7 (-78.7) *3177. 77 i vo i 60.70S" 32.527 ' Maternai Number 21634 21635 21636 21637 2163B 21639 21640 Mean S.D. Date of Mattnq 1/18/83 1/18/83 1/18/63 1/19/83 1/19/83 1/19/83 1/21/83 Dlsposltlon P, 16-FD 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 Heights and Weight Changes P ilo t Rat Teratology Study of T-3352 _______ Body Helght (g rains) - Day o f G estation ---- --------- HB 12 16 Group 5 - 7 5 mg/kg 251.7 215.6 258.1 (249.4) 222.7 231.3 254.0 273.4 249.6 251.0 (253.6) 242.9 266.4 273.5 258.2 233.4 254.0 (246.1) 237.7 243.1 251.8 226.3 182.6 212.9 (214.3) 189.3 209.4 220.3 192.7 (171.6) (203.4) 190.1) (171.2 (187.6) (198.3) 238.90 18.020 259.47 13.286 246.37 9.816 206.80 17.331 154-159 Welqht Chanqe Bays 6-16 -80.7 i uO I Appendix 2 In d iv id u a l and Mean D a lly and T otal Food Consumption P ilo t Rat T eratology Study o f T-3352 154-159 Maternal Nimber 21606 21607 21608 21609 21610 21611 Mean S.D. 21613 21614 21615 21616 21617 21618 Mean S.D. 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)___________ Day of Gestation 8 U 16 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 Total Food Consumption Days 0-20 (grams)3. 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 a S t a t i s t i c a l group comparisons performed on rank transformed data. Appendix 2 - Continued In d iv id u a l and Mean D a ily and T otal Food Consumption P ilo t 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 R rOo Total Food Consumption Days 0-20 (grams)3 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 4 - 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 a S t a t is t ic a l group comparisons performed on rank transformed data. NOTE: Numbers in parentheses are excluded from mean calculatio n and s t a t is t ic a l evaluation. Appendix 2 - Continued In d iv id u a l and Mean D a lly and Total Food Consumption P ilo t Rat Teratology Study o f T-3352 154-159 Maternal Number 21634 21635 21636 21637 21638 21639 21640 Mean S.D. ___________ Food Consumption (gm/day)___________ Day of Gestation 8 --- 12--- -- re------ oCSJ Group 5 - 7 5 mg/kg 17.5 14.7 17.4 (11.7) 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 ConsunptIon Days 0-20 (qrams) (185.0) (134.0) (145.1) (137.3 (131.6) (293.2) (273.1) NOTE: Numbers in parentheses are excluded from mean ca lcu la tio n and s t a t is t ic a l evaluation. 21606 21607 21608 21609 21610 21611 Mean S.D. 21613 21614 21615 21616 21617 21618 Mean S.D. Appendix 3 Individual Body Weights, Gravid Uterine Weights, and Terminal Body Weights Minus Gravid Uterine Weights P ilo t 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 P ilo t 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 NOTE: Numbers in parentheses are excluded from mean calculation and s t a t is t ic a l evaluation. 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 P ilo t 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) Appendix 4 Individual and Mean Ovarian, Uterine, and Litte r Data P ilo t Rat Teratology Study of T-3352 Maternal Number 21606 21607 21608 21609 21610 21611 Mean S.D. 21613 21614 21615 21616 21617 21618 Mean S.D. Ovar 1an Corpora Lutea 14 13 (15) 17 14 17 15.0 1.87 24 17 15 13 15 17 16.8 3.82 Uterine Implantations 14 13 16 15 14 15 14.5 1.05 13 17 15 11 14 10 13.3 2.58 Implantation E ffic ie n c y (X) 100.0 100.0 a 88.2 100.0 88.2 95.28 6.463 54.2 100 100 84.6 93.3 58.8 81.82 20.461 Early Resorptions Resorption Incidences (X) Group 1 - 0 mg/kg 00 00 1 6.3 00 3 21.4 3 20.0 1.2 1.47 7.95 10.183 Group 2 - 1 mg/kg 8 61.5 1 5.9 00 00 1 7.1 2 20.0 2.0 3.03 15.75 23.576 Number of Fetuses Dead A1 lve Fetal M ortality (X) 0 14 0 12 0 15 0 15 0 11 0 12 0 0 0 0 0 0 0 13.2 0 0 1.72 0 05 0 16 0 15 0 11 0 13 08 0 0 0 0 0 0 0 11.3 0 0 4.23 0 Ovary cut - excluded from mean c a lc u la tio n . 154-159 Fetal Viability (X) 100 92.3 93.8 100 70.6 BO.O 90.78 9.445 38.5 94.1 100 luo 92.9 80.0 84.25 23.576 Maternal Number 21620 21621 21622 21623 21624 21625 21626 Mean S.O. 21627 21628 21629 21630 21631 21632 21633 Mean S.D. Ovarlan Corpora Lutea 14 16 15 18 15 15 16 15.6 1.27 19 14 14 17 14 19 16.2 2.48 Uterine Implant at Ions 12 16 15 15 14 9 15 13.7 2.43 18 13 13 17 13 13 14.5 2.35 Appendix 4 Continued Individual and Moan Ovarian, Uterine, and L itte r Data P ilo t Rat Teratology Study of T-3352 Implantation Efficie n cy (X) Early Resorptions Resorption Incidences (X) Number of Fetuses bead Al t_ve Group 3 - 1 0 mg/kg 85.7 100 100 83.3 93.3 60.0 93.8 0 0 0 12 3 18.8 0 13 1 6.7 0 14 1 6.7 0 14 3 21.4 0 ll 0 0 09 0 0 0 15 88.01 13.911 l.l 1.35 7.66 0 12.6 9.044 0 2.07 Group 4 - 37.5 mg/kg 94.7 2 11.1 0 Found dead Day 15 13 Fetuses aborted p rio r to s a c r if ic e Day 20 100 5 Found dead Day 16 92.9 2 Found dead Day 17 68.4 2 15.4 0 Not pregnant 16 11 89.00 14.060 2.8 1.50 13.25 0 13.5 3.041 0 3.54 Fetal M o rta lity (X) 0 0 0 0 0 0 0 0 0 0 0 0 0 154-159 Fetal V iability (X) 100 81.3 93. 3 93.3 78.6 100 100 92.36 9.024 88.9 84.6 86.75 3.041 Maternal Number Ovarian Corpora Lutea Uterine Im p la n ta tlon s 21634 21635 21636 21637 21638 21639 21640 Mean S.D. 15 a 16 20 11 17 15 15.7 2.94 11 17 15 11 17 13 14.0 2.76 a ovary too autolyzed to count corpora lutea. Appendix 1 - Continued Individual and Mean Ovarian, Uterine, and L itte r Data P ilo t Rat Teratology Study of T-3352 Im p la n ta tio n E fficie n cy (X) 73.3 a 93.8 Not pregnant 100 100 B6.7 90.76 11.195 Resorptions Resorption Incidences (t) Group 5 - 7 5 mg/kg Day 16 Day 14 Oay 13 Found dead Day 15 Day 14 Day 15 Oay 14 Humber of Fetuses elfl AUve Fetal M ortality (X) 151-159 Fetal Viability (<) CCOD I Maternal Number 21606 21607 21608 21609 21610 21611 Mean S.O. 21613 21611 21615 21616 21617 21618 Mean S.O. Number of Hales Sex Number of Femali 7 7 6 9 7 7 7.2 0.98 7 5 9 6 1 5 6.0 1.79 2 4 9 9 8 4 6.0 3.03 3 12 6 2 5 4 5.3 3.56 Appendix 5 In divid u a l and Mean L ive Fetal Data P ilo t Rat Teratology Study of T-3352 151-159 Percent Males _________ __ ______________ Mean Hei ght (grains) Hales ~ ~ ~ ~ ___________ females Unadjusted Adjusted Unadjusted Group 1 - 0 mg/kg 50.0 58.3 40.0 60.0 63.6 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 0.147 3.40 0.176 3.25 0.122 Group 2 - 1 mg/kg 40.0 25.0 60.0 81.8 61.5 50.0 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 3.33 0.242 3.37 0.179 3.23 0.082 3.25 0.157 3.22 0.158 i o Maternal Number 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 P ilo t Rat Teratology Study of T-3352 154-159 Number of Hales Sex Number of Females Percent Males __________________________ Mean Weight (grams)_______ Hales Females ~ Unadjusted Adjusted UnadjustedAdjusted Group 3 - 1 0 mg/kg 7 3 6 9 7 3 11 6.6 2.94 5 10 8 5 4 6 4 6.0 2.24 58.3 23.1 42.9 64.3 63.6 33.3 73.7 51.31 18.533 3.1 3.0 3.3 3.3 3.6 3.2 3.3 3.26 0.190 3.27 0.176 3.0 2.8 3.2 2.9 3.4 3.1 3.4 3.11 0.234 3.12 0.157 Group 4 - 37.5 mg/kg 9 7 56.3 Found Dead Day 15 Pups absorted p rio r to s a c r if ic e Day 20 Found Dead Day 16 Found Dead Day 17 6 5 54.5 Not pregnant 7.5 2.12 6.0 1.41 55.40 1.273 2.9 2.6 2.75 0.212 2.72S0.176 2.7 2.5 2.60 0.141 2.60S0.157 i 4=. I Maternal Number 21634 21635 21636 21637 21638 21639 21640 Sex Number ' Number of Hales 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 5 - Continued Individual and Mean Live Fetal Data P ilo t Rat Teratology Study of T-3352 154-159 Percent Males Hales Unadjusted- Group 5 - 75 mg/kg Mean Weight (grains) ~ females Adjusted Unadjusted ~ Adjusted Appendix 6 Individual Viscera] Findings for Each lit t e r P ilo t Rat Teratology Study of T-3352 Maternal Number : 2l 6 0 6 Number of fetuses examined by U l ls o n 's technique* Number of fetuses w ithin normal lim it s Nunber of fetuses having one or more visc e ra l varian ts Percent of fe tu se s having one or more v isc e ra l variants (X) Number of fetuses with: Dilated renal pelvis Dilated ureter(s) Number of fetuses having one or more visc e ra l anomalies Percent of fetuses having one or more v isc e ra l anomalies (X) 3 3 0 0.0 0 0.0 2 ~ 7 -------- 7 ll1 666 000 789 3 3 0 23l 3 3 0 0.0 33.33 0.0 l 000 0.0 0.0 0.0 7 tl 66 11 01 33 33 00 0.0 0.0 00 0.0 0.0 "7 l 6 l 3 3 3 0 0.< 0 0. Maternal Number: 7-- 1 026 ? -------Group 3 -21 0 mg/kg Z l 11l 62 1 622 62 3 62 4 2 1 62 5 2 1 62 6 Number of fetuses examined by W ils o n 's technique* 333 3333 Number of fetuses w ithin normal lim it s 33 3 33 3 3 Number of fetuses having one or more v isc e ra l va ria n ts 0 0 0 0 0 0 0 Percent of fetuses having one or more visc e ra l variants (X) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Number of fetuses with: Dilated renal pelvis Dilated ureter(s) Incompletely descended te stls(e s) Nimber of fetuses having one or more visc e ra l anomalies 0 0 0 000 0 Percent of fe tu se s having one or more visc e ra l anomalies (X) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Number of fetuses with: Cleft palate 154-159 Group 2 - 1 mq/kq 77 f 111 666 111 456 23 3 3 3 3 100 33.33 0.0 0.0 1 Z l 6 1 7 3 3 0 0.0 0000 0.0 0.0 0.0 0.0 3 3 0 0.0 0 0.0 Group 4 - 37. 5 mg/kg rz 11 62 7 6 23 33 21 12 33.33 66.67 1 l 33.33 I 1 1 1 33.33 1 -UpJ* i * Three fetuses from each lit t e r . Appendix 7 Individual Skeletal Findings for Each lit t e r P ilo t Rat Teratology Study of T-3352 154-159 "7 1 6 Maternal 0 Number : 6 Number of fe tu se s examined Number of fe tu se s w ithin normal lim its Nimber of fe tu se s having one or more sk e le ta l variants Percent of fetuses having one or more ske le ta l va ria n ts (X) Number of fe tu se s with: Lagging O ssifica tio n sk u ll - Incomplete closure (le ss than 75X) - In te rp a rie ta l o ssific a tio n Incomplete - supraocclplta! o s s ific a tio n Incomplete - hyoid o s sific a tio n Incomplete - hyoid nonosslfled - parletals Irregularly ossified Rib cage - sternebrae bipartite - ribs Irregularly ossified Ve rte bral column - thoracic centra o s s if ic a t io n Incomplete - thoracic centra nonfused - caudal le ss than three o ssifie d Bone Alignment Extra rlb (s ) Angulated ribs 3 0 3 100.00 2 3 2 1 1 Number o f fetuses having one or more sk e le ta l anomal le s Percent o f fe tu se s having one or more ske le ta l anomalies (X)* 0 0.0 2l Group 1 2 - 0 m2q/kg 11 66 6 000 78 9 344 211 122 33.33 66.67 66.67 2 2 12 1 121 1 1 1 l 000 0.0 0.0 0.0 22 11 66 11 01 44 l0 23 66.67 100.00 2 22 21 1 21 2 1 1 1 00 0.0 0.0 Group 2 - 1 wg/kg 2 2 2 2 2T 11l 11 l 6 6 666 6 1l 111 1 3 4 56 7 8 2 3 3 3 3 3 2 1020 1 0 23132 0.0 66. 67 100.00 33.33 100.00 66.67 2 1 22 2 3 1 l 3 1 1 l 000000 0.0 0.0 0.0 0.0 0.0 0.0 * One fe tu s was damaged during preparation so number of m etatarsals and phalanges could not be determined. Appendix 7 - Continued Individual Skeletal Findings for Each U t t e r P ilo t Rat Teratology Study of T-3352 Maternal Number: Number of fetuses examined Number of fetuses w ithin normal lim its Number of fetuses having one or more ske le ta l variants Percent of fetuses having one or more ske le ta l v a ria n ts (X) Number of fetuses with: Lagging O ssification S k u ll - Incomplete closure (le s s than 75X) - In te rp a rie ta l o s s if ic a t io n Incomplete - supraocclpttal o s s ific a t io n Incomplete - hyoid nonosslfled - pari eta Is Irre gu la rly o ssifie d Rib cage - sternebrae b ip a rtite Vertebral column - thoracic centra o s s if ic a t io n Incomplete - thoracic centra nonfused - caudal le s s than three ossified Bone Alignment Extra rlb (s) Angulated ribs Number of fetuses having one or more ske le ta l anomalies Percent of fetuses having one or more ske le ta l anomalies (X) Group 3 - 10 mg/kg 7 2222 l 1111 6 66 66 2 22 22 0 123 4 3 33 33 20 222 13 111 33.33 100.00 33.33 33.33 33.33 3 3 l 1 1 l 1 1 1 l 1l 1 00000 0.0 0.0 0.0 0.0 0.0 _______ 7 7* 1l 66 22 56 33 20 13 33.33 100.00 Group 4 37.5 mo/kg " 7 ------------- 2 11 66 23 72 33 10 23 66.67 100.00 1 2 l1 2 12 1 2 1 13 1 1l 00 0.0 0.0 00 0.0 0.0 - 46 - Appendix 8 References P ilo t Rat Teratology Study of T-3352 STATISTICAL METHODS/LABORATORY PROCEDURES 154-159 B a r t le t t , M. S ., "Some Examples of S t a t is t ic a l Methods of Research in Agriculture and Applied Bio lo g y." J . Royal S t a t is t . Soc. Suppl IV:137-183, 1937. Draper, N. R. and W. G. Hunter (1969), "Transformations: Some Examples R e v is ite d ", Technometrics, 11:23-40. Dunnett, C. W., "A M ultiple Comparison Procedure for Comparing Several Treatments with a C ontrol." J . Am. S ta t. Assoc., 50:1096-1121, 1955. Dunnett, C. W., "New Tables for M ultip le Comparisons with a C o ntro l." Biom etrics, 20:482-491, 1964. Games, P. A., and Howell, J . F ., "Pairw ise M ultiple Comparison Procedures with Unequal N's and/or Variances: A Monte Carlo Study." J . Ed. S ta tis t^ , 1:113-125, 1976. Levene, H., "Robust Tests for Eq u a lity of Variances," in I . Olkin edited, Contributions to P ro b a b ility and S t a t is t ic s , Stanford U n ive rsity Press, Paio A i t o r r a r o : Stap les, R. E. and Sch n e ll, V. L ., "Refinements in Rapid Clearing Technic in the K0H-A1izarin Red S Method fo r Fetal Bone." Stain Technology, 39:61-63, 1964. Wilson, J . G. and Warkany, J . , e d ito rs. Ter at o1oqy: Pr i ncip1es and Techniques, The Univ. of Chicago Press, Chicago, 1965, pp. 251-27?. Winer, B. J . , S t a t is tic a l Prin cip le s in Experimental Design, 2nd Ed., McGraw-Hill, N .Y., Chs. 3-10, 1971.