Document 0gEV8R2Rv019RbNkDGNvMgYob

HA!@LEOCON IASC)PATCD;;IES ANAEPICA. INr -FE Be @E zz, S PROTOCOL 1 . Study 2. Purpose 3. Test Material A. Identification B. Purity C. Characteristics D. Reserve Samples 4. Compliance 5. Quality Assurance Rat Teratology Study (Segment 11) To determine the embryo/fetal toxicity and teratogenicpotential of a test material administered by gavaqe to pregnant rats during the period of fetal organogenesis. T-3352 Assume 100% Information on the methods of synthesis and stability, as well as data on composition or other characteristicswhich define the test material, are on file with the sponsor. Reserve samples of the test and control articleswill be taken at initiationand retained. These samples, as well as any remaining test material,will be returned to the sponsor upon issuance of the final report. This study will be conducted in compliance with all Food and Drug Administration guidelines including the Good Laboratory Practice Regulations, Federal Registe December 22, 1979.- The protocol, in-life phase, and the final report will be audited by the Office of Quality Assurance in accordance with standard operating procedures at Hazleton Laboratories. HAZLEOCON ASOPATO=41ES ATAERICA. INC. -7 - - -==ze-mz: -@0%.--E @-RG:N-A 22,SC S 2 6. Experimental Design A. Animals (1) Spec ies (2) Strain/Source (3) Ingeat Receipt (4) Number/Sex (5) Identification (6) Husbandry (a) Housing (b) Food (c) Water Rat Sprague-Dawley CDe/Charies River Breeding Laboratories, Inc. Sexually mature, B-10 weeks old (females) and 12-14 weeks old (males). Rats having clinical signs and/or body weight extremes will be culled prior to mating. 100 females/100 males Individual eartags Individual housing, except during mating when one female is placed with one male, in hanging wire cages. Purina Rodent Laboratory ChowID5001, ad libitum. Sufficientquantities of food will be retained at study initiationto ensure that the same lot nunber of feed is available for the duration of the study. Feed is analyzed on a prospective basis for concentrations of specified heavy metals, antibiotics,aflatoxin, pesticides, and nitrosamines. Tap water, ad libitum. The water is routinely a-n-aTyz-oend a retrospective basis for specified microorganisms, pesticides, heavy metals, alkalinity,and halogens. HAZLEOCC)N LABOPATC)RIES AMEPICA. "C @.zz@C--EE se-@Z a;, _j 3 (d) Contaminants (e) Environment (f) Quarantine (7) Randomization Procedures The study director and/or the sponsor have considered possible interfering substances potentially present in animal feed and water, including the test material itself or possible structurally related materials as well as the items listed in (b) and (c) above. None of these contaminants are reasonably expected to be present in animal feed or water at levels sufficient to interferewith this study. Every attempt will be made to maintain temperatures at 72 + 60F with a relative humidity of 50 + 20%. The project coordinator -will be notified immediately if the room temperature exceeds a range of 50-900F. A 12-hour light-dark cycle will be maintained. Minimum of two weeks. Upon observation of sperm or vaginal plug, the females will be assigned to experimental groups using a table of random numbers. All animals will be placed on treatment within a two-week period. Prior to terminal sacrifice and cesarean section on day 20 of gestation, all surviving dams will be assigned random numbers so that all personnel performing cesarean sections and external, visceral, or skeletal examination of the fetuses will be unaware of the dose level from -which the animals were derived. The numbers -4illbe assigned by random card draw beginning with the first Group 1 animal and continuing numerically HAZLEOZON LAEBC)PAT(--)RIES AN4ERICA. INr-. 22'BC) LJS 4 (8) Justification through Group 4. The order of cesarean sections on any given day will be determined by the random number assignment, beginning with the lowest code number and continuing through the highest for that day. This method will result in cesarean sections being performed in a random manner without knowledge of actual animal identity or dose level. Copies of the coded dam numbers will be forwarded to the project coordinator and to the sponsor by the group leader of the study. Code numbers will be recorded on the prepartum examination records, fetaT-i-d-e-n-tc-ait7iion tags , visceral examination records, and skeletal examination records. Upon completion of all data collection and teratologic evaluations, the permanent animal identification and dose level assignments will be transcribed to all data records. Rats are sensitive to a number of agents which are kno-Nn to be embryotoxic and/or teratogenic. Rats historically have been used in safety evaluation studies of this type and are required by the appropriate regulatory agencies. In addition, background teratology control data are available for rats of the proposed strain and source. VHAZLEMCC)N LABORATORIES AMERIC:A. "C: S2@DC v.@r-:N,A z21e= - s- - 5- B. Group Designationg Dose Levels, and Dosing Schedule Group No. 1 (Control) Dose Level* mg7kg 0 Dosing Schedule Day of GestatlE-n 6-15 No. of Mated Female Rats 25 2 (1-ow) 1 6-15 25 3 k'Mid) 10 6-15 25 4 (High) 30 6-15 25 *Based on individual body weights at each weighing interval during the dosing period. Dose levels will be determined upon review of the pilot teratology study data by the sponsor. C. Dosing Procedures (1) Method of Administration The test material will be administered by oral intubation daily on Day 6 through Day 15 of gestation. The dose administered to each female will be based on the most recently recorded individual animal body weight during the dosing period and will be given at approximately the same time each day. (2) Reason for Dosing Route Oral intubation is used because of the relative ease of dosing and assurance of the dose amount. (3) Preparationof Dosing Solutions/ Suspensions Solutions/suspensions of each concentration will be prepared fresh weekly in Duke's(Dcorn oil. Vehicle and samples of each dosing solution should be shipped within 48 hours of preparation to: (eHAZLE':C:N LABC);;4Z7(--)RiES C.ME--41C:A. INC. .,Ecz 22,BZ@ SA 6- William C. McCormick, III Toxicologist Toxicology Services Medical Dept./3M 220-2E 3M Center St. Paul, MN 55101 Freezing is not required. (4) Analysis of Dosing Solutions/Suspensions (a) Stability (b) Homogeneity To be conducted by the sponsor. To be conducted by the sponsor. (,.) Routine Analyses To be conducted by the sponsor. D. Mating Procedures (1) Breeding (2) Vaginal Examinations One male and one female will be placed in a breeding cage until a sufficient number of mated females is obtained (maximum breeding period is two weeks). During mating, daily vaginal examinations will be made for the presence and viability of sperm or the presence of a copulatory plug. Day of observation of sperm or plug is Day 0 of gestation. (3) Disposition of Males The males will be anesthetized, sacrificed, and discarded without necropsy upon completion of the mating. E. Observation of Animals (Dams) (.1) Observations All animals will be examined twice daily for mortality and moribundity. HAZLEOZON LASC:)RATORIES AMEPIC:A. INC ENN- S 7 (2) Body Weights (3) Food Consumption F. Termination (1) Sick/Moribund/Found Dead Dams All animals will be observed once daily throughout gestation for clinical signs including time of onset, degree, and duration of effect. Cageside observations will include, but not be limited to, changes in skin and fur, eyes and mucous membranes, respiratory system, circulatory system, autonomic and central nervous system, somatomotor activity, and behavior. In addition, careful clinical observations will be made at each weighing interval. Signs will be recorded as they are observed. Animals will be weighed on Days 0, 6, 8, 12, 16, and 20 of gestation. A measured quantity of food will be provided on gestation day 0, and food consumption will be measured on gestation days 6, 8, 12, 16, and 20. Any food added during the gestation intervals will be recorded. Dams found dead or sacrificed because of sickness or moribundity will be examined for any grossly abnormal thoracic, abdominal, or pelvic viscera. No abnormal viscera will be retained unless requested by the sponsor. Uteri and ovaries will be examined for implantations and corpora lutea, respectively. Findings will be recorded. HAZLEZON LAE3C:)RATORiES AMEPIC:A. INC, s (2) Dams Showing Signs of Abortion or Premature Delivery Da,,nesxhibiting these signs will be sacrificedby carbon dioxide inhalation. Any grossly abnormal thoracic, abdominal, or pelvic viscera will be noted. No abnormal viscera will be retained unless requested by the sponsor. Uteri and ovaries will be examined for irnplantationsand corpora lutea, respectively. Findings will be recorded. (3) Terminal Sacrifice On Day 20 of gestation, all surviving dams will be weighed and sacrificed by carbon dioxide inhalation. The fetuses will be taken immediately by cesarean section, see Section G.(l) below. G. Postmortem Procedures (1) Cesarean Section A cesarean section will be performed on those females sacrificed on Day 20 of gestation. Any grossly abnormal thoracic, abdominal,or pelvic viscera will be noted. No abnormal viscera will be saved unless requested by the sponsor. The uterus from each female will be excised, weighed, and examined for the number and placement of uterine implantation sites, number of live and dead fetuses, number of early and late resorbing fetuses, and any abnormalities. The uterus will not be reweighed after the contents have been removed. The ovaries will be examined for the number of corpora lutea. Findings will be recorded. (2) Fetal Evaluations Each fetus will be sexed, weighed, examined for external abnormalities, and identifiedwith a tag. HAZLE"CON 9- Approximately one-third of all live fetuses from each litter %..,ilbl-?selec@*edand processed for visceral examination by the Wilson Technique for assessing soft tissue developme-it. During visceral examination of the fetuses, particular att-?,ltio@ntiilble paid to the examination of the eyes (size, lens, and retina),palate, and brain (third and lateral ventricles). Stainlesssteel razor blades will be used for sec'L,ioninpgrocedures. Abnormalities will be described as ano,-qalieosr variants. The remaining fetuses will be eviscerated and processed for skeletal examination using the Alizarin Red 5 staining method. This evaluationwill include examination of the skull, long bones, vertebralcolumn, rib cage, extremities, and pectoral and pelvic girdles. Bone alignment and degree of ossificationwill be assessed. Abnormalitieswill be described as anomalies or variants. Fetuses will be selectedfor visceral or skeletal exar,linatioinn the following manner: Beginning at the ovarian end of the left uterine horn and continuing past the cervix into the right uterine horn, the first availablefetus will be fixed in Bouin's solutionfor visceral evaluation. The next two fetuses will be processed for skeletal evaluation. This sequence will be repeated until all availablefetuses have been selected. Dead fetuses will be discarded .qithoutexamination. (3) Tissue Preservation The ilollo,..itiinsgsues from each dam will be preserved in 10% neutral bul'ilerefdormalin: ovaries uterus All fetuses will be retainedin alcohol,Bouin's fixative, or glycerine. HAZLEOCON LAe(:)PATOFRANIIEEPSICA. TL---'-r@ v,E INC -mZ,N-A 22' 6Z: Lj S A 10 7. Final Report At terminationof the study,a finalreport which includesthe following informationwill be prepared and submitted: A. Experimental Design and Methods B. Results (1) Maternal Data - Arranged by test group and supplied in tabular form. (a) Data for each animal showing: identificationnumber disposition (pregnancy status and day of cesarean section or sacrifice) food consumption at each interval body weight at each weighing interval daily clinicalsigns (b) Data for each dose level showing: number of animals placed on study mean body weight change for gestationdays 0-6, 6-8, number and percent 6-16, 16-20, 0-20, and 6-20 pregnant number and percent surviving to cesarean mean food consumption at each interval mean terminal body weight section on gestation (individualterminalbody day 20 number and percent weights minus gravid uterineweights) that aborted (including mean uterine weights premature delivery) summary incidence of mean body weight at each clinicalsigns weighing interval summary incidence of gross pathology (2) Fetal Data - Arranged by test group and supplied in tabular form. (a) Data for each littershowing: identificationnumber number and percent of live fetuses number of each sex number and percent of late resorbingfetuses number and percent of total resorbingfetuses (9[IAZLEIZON LABC)PATOPIES AMEMICA. INr mean live fetal weight by sex number and percent of dead fetuses number and percent of early resorbing fetuses number of corpora lutea number of implantations implantationefficiency (implantations X 100 corpora lutea) (b) Abnormalitydata for each litter showing: identification number number of fetuses examined for external abnormalities; number of fetuses within normal limits number of fetuses with a particular external variant number of fetuses with a particular external anomaly number of fetuses examined for skeletal abnormalities; number of fetuses within normal limits number of fetuses with a particularskeletal variant number of fetuses with a particular skeletal anomaly number of fetuses examined for visceral abnormalities; number of fetuses within normal limits number of fetuses with a particularvisceral variant number of fetuses with a particular visceral anomaly incidence and description of each type of abnormality (c) Cumulative data for each dose level showing: identificationof each group number of litters examined mean number and percent of live fetuses mean Neight of live fetuses by sex mean fetal viability (live fetuses X100 implantations) mean number and percent of dead fetuses mean number and percent of early resorbing fetuses mean number and percent of late resorbing fetuses mean number and percent of total resorbing fetuses mean number of corpora lutea mean number of implantations mean implantationefficiency number and percent of litterswith any external variant number and percent of litters with any external anomaly number and percent of litterswith a particular external variant number and percent of litterswith a particular external anomaly HAAZLEIZON LAsoRATORIES t-MEPIC:A. INC. -EEse @E VIQG-@,- 2Z IEZ: @ S - 12 - number and percent of litterswith any skeletal variant nu,-iberand percent of litterswith any skeletal anorraly nu.-,lbearnd percent of litterswith a particular skeletal variant number and percent of litterswith a particular skeletal anomaly number and percent of litterswith any visceral variant number and percent of litterswith any visceral anomaly nur,lberand percent of litterswith a particular visceral variant number and percent of litterswith a particular visceral anomaly nu-,iberand percent of litterswith any variant number and percent of litterswith any anomaly C. Statistical Analyses The significance of suggestive intergroupdifference will be tested using appropriatestatisticaltests. Body weight changes and total food consumption of pregnant animals during the treatment and post-treatment phases will be analyzed by one-way ANOVA and fetal weights will be analyzed by one-way ANCOVA. If appropriate,Dunnett's t-test will be performed to identifygroups with significantlydifferent means from the control. Heterogeneous data, as determined by Levene's test, will undergo a series of transformationsto achieve homogeneity of variances prior to performing the above analyses. Kruskal-Wallis, nonparametric one-way ANOVA or a modified Fisher-Erwin Exact Test, depending on the nature of the data, will be performed on incidence data. In addition, a non Dara,,ietritcest for trend will be performed on these data. Inc*idence data to be analyzed include the following: (1) Percent males/litter (2) Percent fetal viability (3) Percent resorptions (4) Percent of total fetuses examined with a particular external, visceral,or skeletal anomaly or variant rA"H-AAZ)Lr;EC(=N LASOPATO=IIES AMEPIC:A. INC 92CC @EESB@=-- -@c'4P"E V,Er4NA VIQG'R@jr2-Z IE:; WE A - 13 - (5) Percent of total littersexamined with a particular anomaly or variant (external,visceral,or skeletal) 'o) Percent of total fetuses examined with any anomaly or variant (7) Percent of total littersexamined with any anomaly or variant All tests for significanceare made at the 5.0% probability level, two-tailedfor the group comparisons and one-tailed for I-evene'sF-tests and trend analysis. The final report, or a draft copy of such, will be reviewed by the sponsor within three months of receipt. Any revisions or comments will be submitted to Hazleton within this period. 5089B 6/2/83