Document QkLk7dEavY9M0yRQew8GR3k9L

6 "HAZLZMCNLABORATORIES Iw ............. AMERICA, INC. PILOT RAT TERATOLOGY STUOY T-3352 FINAL REPORT Submittedto 3M Company St.Paul,Minnesota May 11, 1983 HAZL=CN LABORATORIES AN4ERICA. 9200 I.EESSURG 'L@RNPIKE VieNNA K'4C- VORGINTA 22 180 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: 'LAW@RELWNf'@/T@T@E.EL, Ph.D. Staff Scientist Department of Toxicology Laboratory Supervision: Teratology: @/"GEORGEFA. BURDOCK, Ph.D. Rodent Toxicology I Department of Toxicology A-4 s. RUTH S. DURLOO, B.S. Research Associate Department of Toxicology Report Preparation: AL4e-MARCIA A. MENSE, b.A. Technical Writer Scientific Resources Department b".m HAZLL'ZIMNLABOFTATORIES ............... AMEFIICA-INC. TABLE OF CONTENTS Paoe 1 SUMMARY 3 INTRODUCTION 3 CONTROL AND TEST MATERIALS 4 TEST ANIMALS 4 METHODS 4 mating Period Groups and Dosage Levels 5 Compound Preparation and Administration 6 Maternal Observations and Records 6 Cesarean Sacrifice and Tissue Preservation 7 8 StatisticalAnalyses Specimen, Rcv Data, and Final Report Storage 9 RESULTS - MATERNAL DATA 10 Clinical Signs 10 Mortality 10 Body Weights 10 Food Consumption 11 Gross Pathology 11 Uterine Weights 11 RESULTS - CESAREAN DATA 12 Pregnancy Rates, Corpora Lutea, Uterine Implantations, and ImplantationEfficiency 12 Fetal Viability,Weight, and Sex 13 Fetal Development 13 TABLES Table 1 - Clinical Observations During Gestation 15 Table 2 - Mean Maternal Body Weights and Body Weight Change 16 Table 3 - Mean Food Consumption Values 17 Table 4 - Sunary of Gross Pathology Findings 18 Table 5 - Mean Terminal Body Weights, Gravid Uterine Weights, and Terminal Body Weights Minus Gravid Uterine Weights 20 Key to Table 6 21 Table 6 - Summary of Ovarian, Uterine, and Litter Data 22 Table 7 - Mean Incidence Values for Visceral and Skeletal Findings 23 Key to Table 8 25 Table 8 - Mean Incidenceof Visceral and Skeletal Findings per Litter 26 HAZLEMCDNLABORATOF;IES .... ........ ... WolWar V@@@A AMERICA. INC. .@.' @ - - TABLE OF CONTENTS (CONTINUED) Page APPENDICES 27 Key to Appendix 1 Appendix 1 - InWdeiivgihdtuaClhaanngdesMean Maternal Body Weights and 28 Appendix 2 - Individual and Mean Daily and Total Food Consumption 31 Appendix 3 - Individual Body Weights, Gravid Uterine Weights, and Terminal Body Weights Minus Gravid Uterine Weights 34 Appendix 4 - Individual and Mean Ovarian, Uterine, and Litter Data 37 Appendix 5 - Individual and Mean Live Fetal Data 40 Appendix 6 - Individual Visceral Findings for Each Litter 43 Appendix 7 - Individual Skeletal Findings for Each Litter 44 Appendix 8 - References 46 (EHAZLZZONLABORATORIES 9200 LEESBL)RG TURNPIKE, VIEN(-JA. VIRGINIA 22180. AMERICA, INC. U-S.A@ SPONSOR: 3M company DATE: May 11, 1983 MATERIAL: T-3352 SUBJEC:'r: 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 femaie Sprague-Dawley S-DO 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 wei ght 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 gastrointestinaltract effects. H AZ LL"tCDNLABORATORIES AMERICA.INC. 2 154-159 Evaluation of mean fetal weight data indicated that fetal wei ghts 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 availablefor 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. Incidencesof skeletal variantswere similar for all groups. -"-"HAZL=CNLABORATORIES AMERICA. INC. 3 154-159 INTRODUCTIDN 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, Dukels(TCorn 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 characteristicswhich define the test material, are on file with the sponsor. "HAZL=CDNLABORATORIES AMERICA, INC. 4 154-159 TEST ANIMALS Sexually mature cesarean-derived Sprague-Dawley Crl:CD'9 (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 historicallybeen used in safety evaluationstudies of this type and are required by the regulatory agencies. Thirty-six twelve to fourteen-week old male rats and thirty-sixeight 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 Chow4P5001) and tap water available ad libitum. The females were uniquely identifiedby ear tag after mating was confirmed. Temperatures in the study room ranged from 70 to 780F with a relative humidity between 44 and 69%. 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 HA,=L=CDNLABORATORIES AMERICA. INC. 5 154-159 examinations of each female were performed to detect the presence and viabilityof sperm or the presence of a copulatoryplug. 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. Grouo 1 2 3 4 5 Dose a 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 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. "HAZLEMCDNLABORATORIES AMERICA. INC. 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 410 F). 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 HAZL=:ZNLABORATORIES AMERICA, INC. 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 females lutea, respectively. were assigned random Prior to gestation Day 20, surviving 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, c"HAZLL'CCNLM30RATORIES AMEF;ICA.INC. 8 154-159 third, and fifth fetuseswere identifiedwith a tag and fixed in Bouin's solutionfor soft tissueevaluation(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 (Staplesand Schnell, 1964). Statistical AaLls@es Mean maternal body weight gains (Days 6-16 and 0-20), total food consumption,gravid uterine weight, terminal body weight minus gravid uterine weight, resorptionincidence,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 classificationanalysis of variance (ANOVA) (Winer, 1971). If the variances proved to be heterogeneous, a series of transformationswas performed until variance homogeneity was achieved. These transformations were - logi., square (x2 square root (Xk), reciprocal (1/X), angular (arcsineX2), 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 analysiswas complete. 4a@HAZLEMCDNLABORATOARMEIRIECAS. INC. lw 9- 154-159 In addition to the above data, analysis of covariance (ANCOVA) (Winer, 1971) was used to analyze mean fetal weights. The 1itter was used as the experimental unit. Levene's tests and ANOVA were evaluated at the 5.0% one-tailed probabilitylevel. Control vs. compound-treated group mean comparisons were evaluated at the 5.0% two-tailedprobabilitylevel. Statisticalreferencesare appendedto this report, and statistically significantdifferences,as indicatedby the aforementionedtests, are designatedthroughoutthis report by the term "significant"and/or as follows: S+ = Significantlyhigher than the control value. S- = Significantlylower 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. "HA,ZLEMONLABORATORIES AMERICK INC. 10 - 154-159 RESULTS MATERNAL DATA Clinical Signs Summary clinical signs are presented in Table 1. Treatment-relatedclinical 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. Bo v Weiqhts 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 "HAZLZMCNLABORATORIES AMERICA. INC. 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. '"-"'HAZLETC)NLABORAATMEORIRCAI. EINSC. 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 comoared 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 Lutea, 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 lutea and the mean number of uterine implantations were comparable for all groups as were mean implantation efficiencies. H AZ LL"tCDNLABORATORIES AMERICA. INC. 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 differenceswere not statistically significant. The increased incidence in Group 2 may be attributable to one dam with eight resorbing fetuses. Mean incidenceof fetal viability and percent males were comparable for Groups 1-4. Mean live fetal weights of both sexes decreased with increasingdose level with Group 4 values significantlylower than control. Fetal Development Individualvisceralfindingsare 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 (basedon 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 evaluationwas done on this fetus so that finding could not be verified. Skeletal examinationof the selected fetuses revealed that the incidence of skeletal variants was comparablefor 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 '-""HAZLL'MCDNLABORAATMEORIRCAI. EINSC. - 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. G) w @. I z, 06 Ln u, tIj cL) cD 06 21, s_ cm c%j 0 w cu .0 an _u 0 cm E w c%j ev 06 CD cr CD Lm co aj mw c@ u C" Ec im o all C) v cn w 3r o E ow w cn L. c o-- -5j -'C ccL ol 9w z m SL 16 - U-D, ceno Ln co -W tr-o f-I r@ @0 CD 1-0 r@ LO a -w a; %o C6 r-- r@: m cz I cn -4 -4 + + + + 41 40 w tA :*I to tD Lo cc CL:n) .-4 -4 c:, "C; o 'cr + Lo Ln to CD L,; -M- m r@ mr -4 cn CA cr LO " cn " co + C.%j U") q r %o co to Lo Lo Ln cn I-- 14 _:Io %o M en r-_ LO %01-4 0; ko P, c@6 ci I I c:, ull r,- en CD CD co en C:) m U, cn #@o CD ul LO c@';-tr I I CD cn to cm to 0 co 'a to to (V Cj a 0 -to 41 MCD =C 0 >1 ec C, 10 co Lo m -.0 %o co r@ co 11 c; 10 co cli m C@ a! %o OJ to on co m 64, CO -4 Ln en m I-- Ln r@. .: ko.-q C"i to co Lr; UD -W 93 m CD to co CD LO Ln .: o ko en -.4 @ c@ wr m to r.-cr ulim m LO LO CD m c@ 1.0 C) co CD cz f@, r.- co Ln ko m 11; m tD r@ co -01 Ln -4 %o co Ln m CD en -W Ln Ln en CIIJLn c@ Cli cn CD c@ c@ 'D c@ m c@ cn m 1.4 -tr -4 Ul) 1.4 en -4 ko to Ln (V c'm w In cn cm cn > NC .19 _u to 0) -i tm tm CL -4 E C%i E w %A CD L. 0 CD 9= Le) IC en E Ln im LO r= cu to S- E So Ln (A go E CL Sto u vi 41 to 4-) to 17 - LO LO CL to cl@j Ln m ewn -4 cn cn r.. 01 Ln wr -4 -4 Ln CD ti C:or-4l- %a qUr-)CU,O5 to 0% cn -.44 %UlA; c:o fa mr en cr 0 U- LO cn m to 0 CD La co C, i c, 4 Lc; t-; m --4 m en Ln co c:l C:) Ln Cn r.:C4 to CD a cn 0 o 4- tm c 0 4..) Ln LO ko to m co LO %o m m Ln a to m to CD %o -A LO f%.co LO 4--; CL CD C;, 4 o 6 c@ o 0; c@ c@ 4- tn 0 4-) to m 0 a- to en U- co cn (n 14 %o cv cn m c@ I.; CD ff;to -4 a; cl;to co .-4 C, c@ co M LO en C:, co cv .119 r- -4 -4 -W en co f@ CD.1.9 0 " tm r go = cu . = a) - tn Ln Ln V) CL en .= 0 S- cn 41 LM .19 be be AC 41 en E m cn E en tm qt:r Ul Ln r= cm In -4 CD -4 Ull) rl_ 18 In t.0 CD C%i cn CL"j en m 4>1 >1 to tm CM tm 00 S- LD In 41 ra v, en -4 -4 C%J -.4 en tA S- u LA cu W 4-- L. E CA to 0 4- fe 4- x S- gA ui w cm In 4A cn 4A (V -u u 0 &1 s- u a to = = r= tn S- u (A a) a) LC6D- CA CL -c:c 3: CD r- & 4w S- E.-O C/)-r- a 0 S- S- -0 41 41 (V (L) Le)-0 LA 0 .0.0 C.5 (D LLJ S- LLi C S- =I- >- LL. m CM m Ewm u C-)CD vi 19 U') S- LLJ CD LLJ LL. MCC =I. C:) CL) CD - 20 - cLrn% Ln =C ai =C "C 4A cc ,a cc f@, cli en c-@ %a co m to to to C@ Lo a% C%i to m 0% c@ ai C%i C) Ln c@J CD SCl.) Ln en 4- S- 4j o 0 tb- 4A (A Ln s- 3c r= CD C-i en CD > cn go go S- tn S- CD = cm CD r-_ LO r C) -W co cn CD m oi UD -4 LM tm cu 04- CNi LC) Lr) V) a ci E Ln Mr 4D -4 3c fa fa r= S- to en r@. r@. co cj VI -4 to S- cn en m en S-. CL) r-L I-- e 0 Li to a) Ln Ln to (1) cu V) im tm > tm cm tm cm a) bc cm -Ibf m -. 0) tm t7) C"i t7) m E qr Ln Ln 01 E- IV 4-3 Ln tA CD CD 0 en 21 - Key to Table 6 154-159 Pre nancy Rate (percent) x 00. (number of pregnant rats/number of rats mated) Survival Rate (percent) (number of rats surviving to Day 20/number of rats piaceo on study) x 100. Mean implantation Efficiency (percent) = Group mean of (I implantations per litter/corpora lutea per litter] x 100). Mean Resorption Incidence (percent) = Group mean of ([resorptions per ri-t-te-rFIiamnptati-o-ns--P-t---tre-r7]ix 100). Mean Incidence of Fetal-M-ortality (percent) = Group mean of ([dead fetuses per litterrimplantations per litter] x luu). Mean Incidence of Fetal Viability (percent) = Group mean of fetuses per litter/implantations per litterl-x 100). live 22 - LO f--ui C) Ulq)r Ln Ln %0 Uc'o@-W rl- U') %o qcr r,- c@m c:,o 00-4 co c@ C:) en S.-Ul) cu cn r_ 4w -W Ln tm >1 -0 S- CD to ko CD %o LO tm E > cu C) Lo '9 -@ -@ . . LO M C) m CO r-%0 C-,i c@ m Ln Ln CO r,- m -4 U-) uc c U-)-cr-ICDen -4 M Lo W m 0% C:) r@. ul f@.CD CD m -cr cz cz cllc;z u; LO tm r@, -I m -4 c@ m m m ff;"@ Ul L4 S- to Ew m r-- m m Ln mmmmc> c@; C.; Ln C) 4- c (ru- 100 m C) Lo Ln M -1*1-:r cv r;el;u; LO m ei > > SM CL E Ln LM w E >1 -0W >1 4j--- ISW-o7D 0- 4w 41 LM fa u 3: CA to > tv LA cm CA CA vt vt 4-4- S- Sw a) .0.0 w to -w z w go 41 1-4- #0 S>.,4- uo r_ to S- > r- WM.0 > w e s- S- = = A 4.- (U C tu > W o -w o oo 0 - r- S..- -A-)0 1 cu M- .0 ee 41 44 0 s- r- a) 0 'a CA r- =@- = s- CL CA -I..) c o e (L)(L) 0w LL. (U to- ra 4&I.- aWu 4- 0 &Jw fa to m u UU -W c m w S-'Olo 0-- un u u W c r- w u m to to CU (U (U 'a QJ "D W U Wu -4j r- AJ a tn 4A oe in - =- = fe to to > to > 0 aa U U 4w X CA = >1 &.; "C Wa ev tn r= cu - > to U- s- cu 0- = cm r_ CA :2 >1 to c A 23 - C; CD to m C.; C) c@CD c:, co LO cv co P, r-@ a% Ln Ln CO CD CD CD c:l CD CD cz c@ CD CD 4 m cv -4 U") -tr CQ cli 4-@ Ln wm M vi fa D., S.. ow Sto ra cu 0 ,a m c:l CD LO Ln Ul) Lo ko CD v; CD CD CD CO Ln to CO P-- to U-) tO CD CD cv ko CCD:>m Ln cz 4 c:, c:, - 6 cz -crLO Ln Ln CT CO ON -4 0% qrr cm IV S- -W ei > to to LM to 41 cu Ln > CL Ln %- S- o S0 0 tn tm ol CA (A a r= to Xw 3: CA CA o) 4) (A = 4-14-; a) 4) 4-4- 4-400 &- I4) (U .0.0 %A (U vi (U cn 4-- a cu > (U 4- 11.-- - U o cu CA %A 4- 06- (V 0 @ 41 to c c 4) S- cu u 4) L. L- CA CL 4) 4.3@-"o r-Lr- s- w #a (D 41 S- "C 0) 41 >1 W@ I- w 4-3 (D @ (u CL A-) r= S- .0 to 0 (U S- .0 #0 > CA cu cu 4- 4-J 4w U CA L. cu d)- ea s- E (D o CA (V 4.j a) c -i..) L- 40 w 0. CL -- 41 S- 4a) w =- L.) S(A 0 E co 0 0LA LA W u 0 tn CA a 4-J S- CA tA 0 0 = tn 0 r- :: > I- to 0o cm CA (U O.C CA -- 0 S- (U UM 4.J 0 4j cv = cw w C)..@: 0 tn 4J = W - W &- tA r- Ln uo - w 4- A.) - M U 0 1M a) m = CL 0 4- U E s- M'0'0 0- a W S- s- - - CA CA 4- U AJ r-L 0 0 s- a) w 0 cn (A == a C tn u CD I I >1 U, 4) L- u 4) s- &A 0. r- 4- (D 4J 0 0 mc - Z3 M.3e s- Sw w a) L. .0 .0.0 to > IM" _ir 24 - CD CD CD Ln co co co co tA c@ c@ C9 cn U-) Ln LO U-) to 41 cu @ w _be Ln ulli m (n cu go S-.W 0 to 03 4- S- w Ln ko tm Ln CD Lr! CD V) Ln Ln 1-4 tm vi LA 0 _le to > 41 to to S- S- &i Ln (A tA - Lm 41 41 E o LA w tn to E- C.- 0 S- to a)- 0- == to C" .61 a to ei S- S- E (v- = - to cn &J S- E x r_ ic (U go to to be vi S(V LA ul 41 (V ta ev a U CD CM S(V w m S- (M@ - a) cn= I_ s- to cm S- to (D to S- cop 25 Key to Table 8 154-159 Mean Incidenceof Visceral Anomalies (percent)= Group mean of ([number of fetuses ii-i'atnhomat-iesper litter/numberof fetuses examined viscerally per litter] x 100). Mean Incidenceof Visceral-Variants (percent) Group mean of ([number of fetuses ;ti-ih -varia@ts-per 1itter/numDer ot fetuses examined viscerally per litter] x 100). Mean Incidenceof Skeletal Anomalies (percent)= Group mean of ([number of fetuses -w-l-t-h-a-nomaTielsi-tpteerr/numberot tetuses examined skeletally per litter] x 100). Mean Incidenceof Skeletal Variants (percent)r-Group mean of ([number of fetuses witK---Variantpser litter/numberot'fetuses examined skeletally per litter] x 100). 26 - C) C:l 9 U@ fi c, c@ c, c@ ci CD CD M CD CD LO C:) C) Ln cn co U*) en CD CD C@ CD CD 0 CD C> CD 9 f-I C> CD 0 CD r@ CO L@ Cl! -4 M 00 Ln ko -I LO u@ CD CD cn m %o C) m u, co rn .4 CO CD C> to a CD Ln LI; CD CD CD %a CD CD '0 C) m c,@ c; c:o U- Ln m cn co 0 w ->01 S.. 0 In cu 0- c.13CM E (V LU Ln LM u tn cn (A tn W tn LM cm = Ln w 4-) In tn = =0 0- 4-1 W (V 4- I+- 41 4a w CA (A .19 (D in 0 LM In = cu S- -W cu tn tn to.- = 4- 4.@ - CA w LA In 4- = L. C> r_ w do E CZ.- S- 0- 97 m to >- > 0=-= tA 0 a > a a > 0 41 CA -i.-iW - 4.@ cn 1^ S- (A L- L. w Scu W i..) W 4-) .6.3 4-@ 4J w r- 3: 4-1 ra to M CM tn S -W W = n n3 Ln a: u => to - 4.J (C W 4.- W u 4.. 0 4- 0 4-) 41 x w In 'n CA LA 'A S- Ss- W s- (W W &.) W 4-1 .1-i" .4-3-W 4-3 A.) 4.J r- 3: cc -z tn tv LA a = to 4.J 4-) W ti.- W 4- U 4- 0 4- 0 4- 4-4- 04- 0 4-J c wwwwai .0 .0 u -0 u 96 S- E S- w lw CL. CL Ln 4- CD @ s- a M w 4-- 4- 4- 044-- 0 cc 0 4-; &1 s- s- r- s- a wwwww -0.0 U.M u 9 9 S- S- IV w cn 4- (V 4W0 - s- s- #0 (v cu > .0 -.0 EU u a to 27 - Key to Appendix I Individual and Mean Maternal Body Weights Pilot Rat TeratologyStudy of T-3352 154-159 P = Pregnant NP = Not pregnant C = Cesareansectionperformed (precededby day of gestation) FD = Found dead (precededby day of gestation A = Abortedpups (precededby day of gestation) Body weight on day of death. b Statisticalgroup comparisonperformedon rank-transformeddata. Note: Body weights in parentheses are excluded from mean calculations and statisticalevaluation. 28 - Ln U, cIcn c@-: Cc0 LMCP@ 09 Icn.":* -*r' w'= - - -0-%C--OC--:l Ln - - -w ---CD - - am m CD tn V) tn tn O@ C" U.) Ln C9 g c@ c; c@ c@ cc w cu cg co 10 u@ Ln 10 c@ c@ 10 en c@ en U, Ln c@ f" orl L.; wm 3c'n m en c@ cc o w GO cr, CD Ln CO mc'-9 t7, col CL- 0 cm CL cu 0 t- co mc cr 'a co C@ c@ o C, 47, n %o @o %o n C, cv WW In m %o m Ln C- co mf ev Ln r m =cvzms@m@a"kC%n%o%ja co cri E @o C:)qr 0' Ln tn eq clicv c@ C9 cocl, Cl!9 ko C%i %D cv CNJ CD UD 19 0 c@ CY@ CO U! C@ 0,:C2,,@n m@ 0" w 1c@9o;C,z9 u0@ 1;9z ".wvC@c co @e!r,CID f"- CL. a co coco 41 coco w CO Lf L? 0 m CD CD 0 0 co cmco co co2 cn CQ" clim C%i Ln ;z LA 29 Ln M- C6C6 c@ en C%o>mm qr LM %@ cn @ = " Cwl!101: In cw -w cw ew ctum 3r w 0 Ln r! Lr! C@ C@ c; c@ 16 w ul %a LUm, mr,@ mm@ .%o Le; C6 Ln Lm C%:U; m ev en m W Ln Ln CO cv r4 CV UI,t! -" M fn -W ul 0, c@ c@ C-9 . .. .. C9 a% fn:; w %n Cc 0 %c %n %o -cvw%n t"v@qr -eyr- Cu 0 co m m c,@"@ Ln%o L@n ILnn CY," WM cwrj" CWCQ" Ir! m I %0- r ul 40 ul 61 Wa ev.u,. . . . . . . . .. ... L%nn ual . CD -D to 10 10 10 cw ew C-, C; cz m @,mwwmul%o C6', wolqrm no C', el ew m cl,co C, 10- _;_: 06 C, M@ m Ln r ar; Cv C-i Um, C971,97 co C. -W a% CD to r@ to C-i a. CL. 12.CI- cc !Zqrmnfccn 0. c@. 0. c@. Cl-c@. 95- cc go Cc fCnO MCc fCc"fC-OI cw rn o ew ew r4 cli 10 - Cc a- 0 "r4 "f" !! 10 CW en 10 c; ZC C^ 30 Cc WW 02 7toCl9y, co C@ c@ cz co* Ln n qr f-I6n cL.g@.c@ cL.cl. cc cc cc cc cc cc L. GO ch 0 c@j cNj c%j c@%ac%j r4 tn 31 - Ln ra LO 4C-A 10 Sc-n m rc@o Lo CD w Lr)CD LO $@D-92' Ln Lc;C6 c; If.n:rc-;.crz-.'0g0;CD L4w@ CL@n to m -4 cn Ul) -cr -cr cn qr -cr -tr -w -cr to r_ C"i 0 LO m m U- 4.- Ln c@ c,@ ci Ln LnLnLo%o4m tD -4 %o -4 C"i al ro CO fl- to m V) cu 4w -W M: m > CD cm 0 m -:r cn CO 0) C@ M 0% CD cn .0, CO %D C.@;cz c@l;C6 _: m -0 C%i C:) co CD c-; S- co a% to 0c r- M I--0% CO ON Ln to CD ko 0 4dj ) -4 cm 0. cm cn .cn .CD .C@ .CO . . cn a) ON CO M -4 cm . . . . . . CD C*- -4 r@ r@ r-, CD ew I -1-4 -4 ..4 -4 -4 -4 -4 qcr LO ko " to CD r.: I.: cz c@ c@ cli CIJ -1 -4 en M c@ -4 LI@ I'@ C@ f@ %9 tm CD ON Lf)CO (M co co 00-0 U-) cn r@ CO cv cn a; I'@ C6 Le; Cl@j -4 -$ -@ en r-_ C%i en a; C4 -.4 O@ LI@ C@ c@Jq:l.C:)r-,co Ln cli co LO C@ co -cr to Sa) P, co m CD CD CD CD C:l -4 --4 a io %o to %o tD %o ro .q -4 -# -4-4 --f 4) cli C%i C%i C%i cli m qa* LC) t.0 rl_ co @-4 -4 -4 -4 --4 -4 Lo to tD %o %o %o @@ 1-4 cv C%i 1.4 @@ @@ C"i to a) 'A S&i Sc 0 "C a) S(D 0. CA r_ 0 V) E 0 u 0. 0 S... m u to 4-@ vi 32 ulli LO cn M -w C) CD U-) f,- M Ln -4 Ln co CD LO %0 mr C:) 0 C-) C:l cli c;, c@ m CO cn c*,iCO CD CO m m -cr -C' M -cr M 4,4 cn en r.: C6 rl: 'g; t@; %0 U-) co cn - CO C:) m -I Ln -4 -4 -4 Iz -4 Ln am 0 Ln m en @.L u- w 0 >1 41 c @-- tm 0 C\i ,c w r_ 0-to W f-- -w to to CL > >1 c:, to -4 --- a 0.0 - CA 4-1 cu 0. CD 94- CD is C"! ",:,19 M -:r Ln -rz-m -*- -rr m cli . . . . c@j c@ qr C:o tm -IC tm E CD 0 tm m r- to C) m C) to Ln -4 c; u@ c@ P.: C4 m I @ ..4 Ci -.4 -4 -4 cn 0 S.CD mm .. Ln -4 co r-_ .. cli m m 113*m .. P., C:l CO -4 a, -W 1-4 Ln 0 .. L. a$ m cm ..4 LO %D co oo q:r CD cz 0 CD m L" Cli u@ C4 m r,-a m tm rl- o ci1 =o c; c; U- LO L.L. Ln m r@.Ln It; m 4D r@. co m -a, 00 . .. .. . . 0% Ln cn f,- CD csi Ln r@ co vi C3 a Li- L9 7 r@ ai q:r m -cr Ln M Ln --4 --4 --4 --1 C"J -4 C:) -cr c@ C@ -cr -cr -4 O@, I'@ C"! cn r@ -4 c) -cr to C) C@ r,: m cn 19 9 C"! 9 r@ m m C9 @9 t9 r- qr Ln --4 @4 -4 co C% %9 r@ -.4 C@ C9 1.9 LC! el! 9 C9 m to m rl- m r-4 -4 -4 --1 -4 -4 C=) mr a@ 7 co C%i a ei to 2: m qr Ln to cli M C%j Cli C%j C%i CNJ r_ to 1.0 to %0 to UD UD to --1 -4 -$ --I o C%i C%i csi C\i C%i r- M CA C) "M"Mmmm MM %0 MD "-4 C%J C'i to %0 tm to --4 @@ --4 -4 c-,i C-,i C',i C-.i rcz 'A 4A tA r_ to ,o CL4.- CA r- U to S- c E ra E 4S- Sfa (V CL 41 E r0 GJ u C) r_ Stm U.0 -E 6-1 :3 fa 33 cn Ln ,r Ln r- m e4 M Ch r*- io > to u m to -1**en Lr; -cr LM M "D Uci >, >-i >I to co to to m to to to -be wwwwwww to 0 cli u ci -0 ko aa cC aC a a >., -4 tr-nI == == == = r_ 1 0000000 u- U- u- u- u- U- m0 Ln a to 0. to Ww to &A I- W cm cm 4.. ul u, -4 to Cl- to cm 1= x --C*(-71 (7% C:) UD Ln C%j %D to LL> CD -4 -4 M mD 1-4 -4 C%i CL) L r-% -.4 00 f@- 1=) tz 4 --4 -4 -d S- m m ff) rn en -cr %0 to tD --4 4 -4 tz %o fa C"i C"i 'cr U, 'o co a,,=, ILI C) 34 LO LO cu =C =C cu r_ co Cri m c%j M mr r@ P,: c; r- cy, to co m co 0% co CO co C%i r, "M co SC) cm r: C%; GO Ln @@ M C%i LO Ln m m cm cu :m LO a cn cu 4C) 4 S- Ai - - -Y-. Ln -NC >c CD tn cm ff) -4 u-@ m m c7i M m 4A S- cm 4-) CD . m . r@.-W. CO. M. to w CM c@. 0 cu C%i = V) -W 4-) 4A to = Ai L. al-C a) 0 CU 4-2 c_n 0 co 0 0 CID - r.. CD M Ul c@ c@ 4 -4 T-@ LO ON m CD LO c@tm C%i >E tm S- 01 C%i m C%i CO CD LC) r.: C6 SCL-)mc -I:rco ON %D m LC)U-) c; 4 8 c@ 6 m CD CD R:rM 4.0 %D M to S- a) .0 9 co (n CD CD CD CD CD --4 -4 4D %o to tD tD tD to -4 -4 --4 --4 @-4 -I (D m co " c,..l04 C%J CO -.4 -4 a C%i C%i C%i C"i C%l C"i 35 - Ul) LO M 4-J LA 40 = =C tm W Ln a en =C en cu w cu S.- a L. 0 CD CD 4-J < >1 ar co 0 Co - -- E S- cc Ai CDtoto-4-4 UD M Ln _; C,; Lr; 0; m -r cri co cn %D C) --q CA cv c%i ci en cn to LA 8 L-Otr a, c@ -rr en CQ a m > cu u Ul to tm cm a)- E C:) P--Cn Ln r- Cc -0 u*) LO %D M 00 m CD CO UD -4 tm -be E Ul) co cn en CD m co -crcli en P, m LO ko,-f Scm cm CD CL E >1 to E 0 L. Ln cm >,,= >1 (D L. CL u x cm 4-1 -C tm E S- :c m c:@CD en Ln ON -crLn %D r@ M Ln M rn m m en m cn m cn r- Ln Cn CD CD to m Ln M -0 m CD CD m CD tD co Lr; c@ (V Ln o 0 0 f,-Ln LO CA M LA- U- U- w C=ll S- to S- M -tr Ln UD C%J IV-.i c%i" " " to UD %D to %D r_ to CD CA Scu -0 F.q M C@ -4 M """Mmmm MD to %o tD %o %o c to Cm 36 Ul) mc mc 0) S- SCD 0.t-m Ai CL) a) MC LC) CM 0 u> go(A >., I sCD en x &A .- Ln 'D 4-) S- o tm= CU a) - a) I-- 0. a) - CL CD 0-0 7Z '3 a, =D 4-) LCI to Ln SCL 0 L. tm 4) SCa. 4-1 CD -:rCn Ln -crLn C" to S- mc ca 0000000 U- U- U- LA- LL. U- LA- LO to r@ co (7% CD rnff) m m m en -W 41 to 37 cc .0t CD CD 01 CO 'CO,@ com rmLnu@-ei cu U, Cc qr c:,CD,0 4=0 Q CD fe 0 79 el cu w CU 0 CD Ln Ir CY- qr CL en 6 Ln cli ev 10 10 -i C:) C6 cc %a w-M cr, CC)@ac,o Ln U@ C! tokn-r Ln C9 co 10 1w0 E c@ C@ .0 qr U-)",Ul- %a fn G) 4D %oto m clicv;Z;Z tA ell CIO r4;Z;Z;z lea, x LI) CD n, 4u 0, 38 - ulz cccc cc kgf" U, In UD V! Lc,u tu z.c r- CL U tm m @Ln CD ON =ww-Ln cm Lall E w cm dCr CL 3.4 u E wi r, co CD u; @.4 cc c-0;CY4% ew U',ci M cz..; r -0 r qj 6. a, @D 0 co ol -- --- - cc ev C-if" ul to cv"ecw "M "M . . . CY m v@ CO m 0;9 1-1m f-i tv z 39 un C: w t7l E ,a ai cz CL CL -w en qr Ln r c a C) CC) t,, C:o L'i CL 1c0u 0 CL ci -W Ln D 06 10 cm - 40 CO men#" fn cv en*f"' on'f"* to IV @rnmLmI!@LIm! c@ cn cn E cL n Lm Cc Ln w Ln Ln w %o Ln w6on c:lul r ew to A@n cu LA L. w Ln C7, %c; m m %a ew ul w ul m C:l ev qr cn m Cc qr co c,, 4w to cj c%o c%o c%j ew LA zzzz;zz 41 e4c; :E.2 - w 0 ca_ 0! c@ M." en 0 cz CLnofc"vcw@ CD LO cz c@ 6 LM 6n In Ell. It L.n Ln CO Ln -w %D r o c@ %o en In LM.E %0 3..4 r =,-=-g - 1IV0 0 1" ol e@ f-)-W Lewn -Z M" CV" CO 0, C:@ m " CY f" 1-0 ev"CV "M m C; ICU, 42 Ln 01 m IV cn fn 99 Egg 99 C4 &n.10 0% 0 r_w mmmm mw cv cliC. m 43 co Mtn c; c; C) tne@ CL %*fn ew rn@- CD em ci cu me.- %o CD Cc M C. 9%., @o ew wt f-tt"C5 c; CD rw -%a ew r f"M 0 c@ em CL C5 rv %a @@O c; CY- f" CD, CD CW- %a 0 'a mmo 0 41 a SI- cu w w cm 01.5 2-. gi a) @ -@ 00 0 6 0 JODI cn C asA CLW %A W 41 ci w 0 oz cvo fn"-10 0 to s. v E cu w E cw im CA 4u IA V%4 w a) Gi - -0 -0 z 0- ol 0- -0 w .0 sw-cm Gi 'D co -cm 44 CD CD CD CD C, 0 cn cvM- a a, u -.00 co -W- m Iz to rw %c 0 rQ m m cu CD o ci CD c; CD 0 0 0 0. C, Ln IV 0 ol G, o w 'A c 2-.v cL u cmo Iwo so-I, ol ej be t.. a, 8 E E I- CL t- 0 im cr. c 7i:; o jm 9=' =0 , 4) ou Gj a) = lo ol 0o c o s- s- I-7: w L.co 6w C cn 4u @x c a- -6 $- a = E 'm 4LL,'o 45 CD C%i%0 rn en CV C%i C%i %o evqr fn CD cu I-0 en, ca r@ %o CD Cl.w ,cc ci fn e@ tD r 0 cu w c9w CL WTA- cc ra re C:l L. 1. : -: t w .0.401 IuV 0.0 2: a: ;c CL. 0ow-cwc- u CLU iz; 9 7; 01 11 $u_-10 0 u c G) 00 4) ol cm im r_ CL IV m 0 iz ai r ol CM -C 1- :1 -I=C 0 OL) .0 c u c ar t,6 46 - 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 Technology, 39:61-63, 1964. Wilson, J. G. and Warkany, J., editors. Teratology: Principles and Techniques, The Univ. of Chicago Press, Chicago, 1965, pp. 251-277. Winer, B. J., Statistical Principles in Experimental Design, 2nd Ed., McGraw-Hill, N.Y., Chs. 3-10, 1971.