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Oral Teratology Study of FM-3422 in Rats Experiment No.: Conducted At: Inclusive Dosing Period: Study Director: 0680TROO10 Safety Evaluation Laboratory Riker Laboratories, Inc. St. Paul, Minnesota August 19 to September 4, 1980 E. G. Gortner E. G. Gortner Senior Research Technologist Animal Reproduction-Teratology Study Director Date E. G. Lamprecht, DVM, PhD Research Veterinary Pathologist Date &6t@. .1h 0r&4.c T. Case, DVM, PhD *,*/,tjjof/ Date Manager, Pathology-Toxicology Safety Evaluation Laboratory Summary oral administration of FM-3422 at 75, 37.5 and 25 mg/kg/day to pregnant Spraque-Dawley rats during days 6 through 15 of gestation (period of organogenesis) was teratogenic to rat fetuses. Teratogenic changes included a developmental eye abnormality, parenchyma and sternebrae malformations. cleft palate, blood in the kidney The developmental eye abnormality appeared to be an arrest in development of the primary lens fibers forming the embryonal lens nucleus followed by secondary aberrations secondary lens fibers of the fetal nucleus. The proportions of the of fetuses with the lens changes were significantly higher in all FM-3422 groups than in the control group. Cleft palates were produced in the 75 and 37.5 mg/kg/day groups. All three groups receiving compound had fetuses with blood in the kidney parenchyma. considered skeleton aberrations, The sternebrae changes, although were viewed as compound-related normally malformations because of their severity. FM-3422 also produced an increase in other fetal skeleton aberrations. FM-3422 was not embryotoxic and did not affect the ovaries tract contents of the dams. or reproductive FM-3422 was maternally toxic to the 75 and 37.5 mg/kq/day dose animals in reducing their group mean body weight gain during the dosing interval. Toxic clinical signs and deaths occurred in only the 75 mg/kg/day dose group. 2. Introduction This teratology study 1 in rats was conducted to evaluate the embryotoxic and teratooenic effects of orally administered FM-3422. The study was sponsored by 3M Commercial Chemical Division, St. Paul, )4innesota and was conducted by the Safety Evaluation Laboratory, Riker Laboratories, Inc., St. Paul, 14innesota. Two sets of compound administration groups were dosed between August 19 and September 4, 1980. The protocol and list of the principal participants and supervisory personnel can be found in Appendices I and II respectively. All portions of this study were conducted according to the Good Laboratory Practice (GLP) regulations and the Safety Evaluation Laboratory Standard Operating Procedures (see Appendix III for Quality Assurance Unit statment). The storage location for specimens, raw data and a copy of the final report is maintained in the Safety Evaluation Laboratory's record archives. Methods Time mated Sprague Dawley derived rats were obtained from Charles River Breeding Laboratory and assigned cages according to a computer-generated random numbers table. The rats were then divided into four groups of 22 animals weighing 140 to 240 grams. The rats were housed individually in hanging stainless steel cages with wire mesh fl ors and fronts in a 9 temperature and humidity controlled room. Food7- and water were available ad libitum. The lights were on a 12 hour light/dark cycle. The animals were observed daily from day 3 through day 20 of gestation for abnormal clinical signs. Body weights were recorded on days 3, 6, 9, 12, 15 and 20 of gestation and the rats dosed accordingly using a constant dose volume of 5 ml/kg of body weight. The four groups were dosed with FM-3422 (Lot 784) suspended daily in corn oil at 0, 75, 37.5 or 25 mo/kg/day. FM-3422 was administered daily by oral intubation with a syringe equipped with a ball-tipped intubation needle to the rats on days 6 through 15 gestation (day 0 indicated by sperm-positive vaginal smear). FM-3422 analytical characterization (see Appendix IV) was provided by 3M Commercial Chemical Division, St. Paul, Minnesota. All surviving animals were sacrificed on day 20 by cervical dislocation and the ovaries and uterus, including its contents, were examined immediately to determine the following: number of corpora lutea, number of viable fetuses, number of resorption sites, pup weights and sex, and any gross fetal abnormalities. Approximately one-third of the fetuses were fixed in Bouin's solution for subsequent free-hand sectioning by the Wilson technique to determine visceral abnormalities. The remaining fetuses were preserved in alcohol for clearing and staining of the skeleton with alizarin red to detect skeletal abnormalities. Selected free-hand sections were processed for histological evaluation. a Riker Experiment No. 0680TROO10 Purina Laboratory Chow, Ralston Purina Company, St. Louis, MO 3. Results and Discussion FM-3422 was maternally toxic to the high and mid dose groups (75 and 37.5 mq/kq/day) in reducing their group mean body weight gain during the dosing interval. All groups had lower mean weight gain than the controls at all weighinqs during the dosing interval of days 6 through 15 of gestation (Table 1). In the case of the high dose group at gestation days 9, 12 and 15 and in the case of the mid dose group at gestation days 9 and 15, the group mean weight gains were significantly lower than the mean weight gains of the control group (0 mg/kg/day). The lower mean weight gains of the high and mid dose groups during the dosing interval were responsible for their sicjnificantly lower mean body weights between the end of dosing and the termination of the study (Appendix V) - The mean body weights and mean weight gains of the low dose group (25 mg/kg/day) were not significantly different from the control. Abnormal clinical signs were observed and deaths occurred only in the high dose group. Three rats in the high dose group died. One rat died without clinical signs. Two of the rats that died plus one surviving rat had abnormal compound-related clinical signs which included some of the following: thin, lethargic, ataxic, blood in stool, urinary incontinance and bloody nares. The onset of abnormal clinical signs was on day 11 but the signs disappeared in the surviving rat by day 19 of gestation. The remaining 18 high dose rats and the mid and low dose rats did not have abnormal compound-related clinical signs. The compound was not embryotoxic and did not affect the ovaries or reproductive tract contents of the dams. The mean number of male, female, total and dead fetuses, the mean number of resorption sites, implantation sites and corpora lutea of the three FM-3422 dose groups were not significantly different from the control (Table 2, Appendix VI). FM-3422 was not fetal toxic. However, the combination of reduced maternal body weight gain (Table 1) plus higher numbers of fetuses in the treatment groups than the control group (Table 2 Appendix VI) resulted in mean fetus weights of all FM-3422 groups which were significantly lower than the control mean fetus weight. The reduced mean fetus weights were not associated with an increase in runting or other gross fetus findings (Table 3). FM-3422 administration resulted in malformations in fetal sternebrae. The changes, althouch normally considered skeleton aberrations, were interpreted as compound-related malformations because of their severity. The severity and often the incidence of sternebrae malformations were greater in the three treatment groups than the control group. These malformations included the followina: sternebrae asymmetrical, sternebrae bipartite, sternebrae scrambled, sternebrae enlarged, aternebrae missing and sternebrae misshapen (Table 4). All three FM-3422 dose groups had significantly higher proportions of fetuses with sternebrae asymmetrical than the control group. In addition, the high dose group had a 4. significantly higher proportion of fetuses with bipartite sternebrae than the control group. An increase in other skeleton aberrations also occurred as the result of FM-3422 administration. These skeleton aberrations included nonossification changes of the cranial bones and sternebrae plus other sternebrae and rib changes (Table 4). The high dose group had significantly higher proportions of fetuses with all of these skeleton changes than the control group. The mid and low dose groups had significantly higher proportions of fetuses with some of these changes than the control group; notably nonossification of the cranial bones, sternebrae missing and 13 ribs spurred. The skeleton aberrations found are generally considered minor but they are of appreciable significance in this study with FM-3422 because of the high proportion of fetuses with the abnormalities. The control group had a higher proportion of fetuses with one or two bodies of the vertebrae bipartite than the three treatment groups (Table 4). This difference was significant in all instances except for the finding of one body of the vertebrae bipartite in the low dose group. FM-3422 administration produced the teratogenic effect of cleft palate in the high and mid dose groups and blood in the kidney parenchyma in all three dose groups. The proportions of fetuses with cleft palate and blood in the kidney parenchyma were sigificantly higher in the high dose group than in the control group (Table 5). No cleft palates were present in control and low dose fetuses examined. FM-3422 was teratoqenic to the eye of the rat at all dose levels administered in this study. The teratogenic effect was a developmental eye abnormality which appeared to be an arrest in development of the primary lens fibers forming the embryonal lens nucleus, followed by secondary aberrations of the secondary lens fibers of the fetal nucleus. All eye abnormalities were localized to the area of the embryonal lens nucleus although a variety of morphological appearances were present within that location. The range of morphological appearances as observed under the dissecting microscope varied from a slight discoloration running through the lens to a discoloration of part of the lens and the presence of a cleft beneath the lens epithelium (Table 5). Histologically the discolorations were due to the presence of lens vesicle remnants forming clefts or surrounding the lens nucleus. Also contributing to the discolorations were primary lens fibers which appeared to have not elongated and the possible presence of degenerated epitrichial cells. Secondary lens fiber development progressed normally except immediately surrounding the abnormal embryonal nucleus. Prominant secondary aberrations of secondary lens fibers include V-shaped clefts between the embryonal nucleus and lens epithelium and lens vesicle remnants surrounding the nucleus. The proportion of fetuses with the lens abnormality in one or both lenses was significantly higher in all groups than in the control group (Table 5). 5. No lens abnormalities occurred in the control group. A no-effect dose level for the teratogenic abnormality was not established in this study. Further Discussion on Lens Embryology Lens structural and functional requirements are met during embryonic development by the differentiation of highly specialized populations of cells from undifferentiated procursers and by the coordinated morphogenesis of the resulting tissues. Both processes are controlled to a remarkable extent by interactions which occur among emerging tissues. Each tissue of the eye is brought to its final state of differentiation, its cell population, size and its definitive geometry, not only by intrinsic process s, but also by extrinsic influences exerted by neighboring 7 tissue-ct--. The embryonal origin of the lens is undifferentiated ectoderm. The tip of the optic vesicle, presumably the neural retina, plays the final role in inducing lens from overlying ectoderm and in aligning the lens precisely with the rest of the eye. Additional action of tissues derived from endoderm (foregut) and mesoderm (heart) on the same target tissue decreases the probability that lens formation would be aborted by accidents durina the early phases of induction. While the nature of the inductive influence remains unknow?i, there are indications that substances may be transferred from the presumptive neural retina to the overlying ectoderm during induction. A prolonged period of inductive interaction not only increases the probability that lens induction will occur successfully in the face of interference, but provides a mechanism for continuously adjusting 2the size, shape, position and orientation of the lens to that of the retina - During the early stages of the inductive process, the ectodermal cells immediately overlying the tip of the optic vesicle elongate perpendicularly to the body surface to form a thickened disc (lens placode). The change in cell shape is accomplished without change in cell volume. The number of cells, however, continues to increase during this period. Toward the end of lens placode formation, acidophilic fibrils appear in the apices of the lens placode cells. At about this time, the placode invacrinates to form the lens cup. This invagination is independent of the concomitant invagination of the underlying optic vesicle, and is probably due to forces operating within the lens ectoderm. As the lens cup deepens, its opening (lens pore) becomes progressively constricted until its lips meet and fuse, cutting off the lens vesicle internally and re-establishing continuity in the overlying ectoderm. Closure of the lens pore is attended by, and possibly accomplished by, a local and temporary restricted wave of cell death. Followina closure of the lens pore, the cells at the back of the lens vesicle continue to elongate, under the influence of the neural retina, to form the lens fibers. As the fibers grow the cavity of the lens vesicle is obliterated. The lens cells to ard the ectoderm, which do not I elongate further, form the lens epithelium-. The cuboidal lens epithelial cells which face the cornea continue to grow 6. after the lens vesicle forms. As the cells rotate through the equator region, they take their places on the surface of the crrowing fiber mass. These cells differentiate into secondary lens fibers at the equator and elongate rapidly toward the poles of the lens where they meet with other fibers in planes of junction called sutures. As secondary fibers grow their nuclei become positioned at about the center of the fibers and form a convex lens bow outward. Since the newer fibers are always deposited superficially, the oldest fibers in the lens come to lie centrally and are referred to collectively as the lens nucleus. With time the lens cell nuclei in this region become pycnotic and finally disappear. The cell fibers, however, are not broken down and removed but remain in place. Thus the size and shape of the lens are contr5lled by factors which control the number, size and shape of the lens cells-. The teratogenic lens effect of FM-3422 probably occurred durina the portion of organogenesis between differentiation of lens tissue from ectoderm and the formation of secondary lens fibers surroundina the embryonal lens nucleus. The exact time of the teratogenic insult and the morphogenesis of the abnormality were not determined in the study. The developmental lens abnormality appears to be unique because it has not been described as a compound-related abnormality - A similar-appearing structural lens abnormality has been reported to ccur spontaneously in rat fetuses but with a very low incidence of 1.2% - The abnormality resembles the Fraser developmental lens abnormality ?f a mutant mouse strain which results from degenerative primary lens cell&--. 7. References 1. Coulombre AJ, Couloirbre JL: Abnormal Organogenesis of the Eye, in Wilson J, Fraser FC (eds): Handbook of Teratology 2:Mechanisms and Pathogenesis. New York, Plenum Press, 1977, pp 329-341. 2. Couloubre AJ: The Eye, in DeHaan RL, Ursprung H (eds): Organogenesis. New York, Holt Rinehart and Winston, 1965, pp 227-232. 3. Mann I: Development Abnormalities of the Eye, 2nd ed. Philadelphia, JB Lippincott Co., 1957. 4. Weisse I, Niggeschulze A, Stotzer H: Spontaneous congenital cataracts in rats, mice and rabbits. Archiv Puer Toxikologie 32: pp 199-207, 1974. 5. Hamai Y, Kuwabara T: Early cytologic changes of Fraser cataract. An electron microscopic study. Investigative Ophthalmology 14 (7): pp 517-527, 1975. 8. Table 1 Mean Body Oral Teratology Study of FM-3422 in Rats Weight Gains of Pregnant Rats Between weighings with Standard Deviations Dose Group 0 mg/kg/day 75 mg/kg/day 37.5 mg/kg/day 25 mg/kg/day Gestation Day 6 9 12 15 20 :17 2- 2 4. 1 :1 t-FI-fl -.1Ht.l. :1il. t-E1 H t-4 T Fit-., 4 cia :14. E..2@ :1' C :1 j- L-. 1 r-.i.9 J_7 :14A 1;7'::' -q. I Ci. 4 :15. i-I 2 !f..4 11. E, a Significantly lower than the control (Dunnett's t test p -c,0.05) Table 2 Oral Teratology Study of FM-3422 in Rats Mean Litter Data with Fetus Weights and Standard Deviations Dose Group 0 mg/kg/day 75 mg/kg/day 37. 5 mg/kg/day 25 mg/kg/day No. of Animals V I FiE:L-E F E E M F TCII FIL 18 C. 5. 4 -E,-. E. 1. 8 2. 17 5. 1 4. 7 1 2. 3 2. 1 20 4. 4 5. 4 1 2. 1 1. 1? 21 4. 3 5. 8 10. 1 I E. I . 9 :1. :4 [:-EFiL,@ F*E 1 F,E,::.i")RTFI,f-it-4 I IIF'LFit-4*TAITIit-4 C:'-'P 1 ES 'EI@ TES LI-IT (."I C-1 1 c Cl. ci ci. -0 Fi. cl f.,i -7 1 I..ri Cl. 5 c . IF. Cl. 9 0. 5 Cl. !@-I q. F 2. 5 10. 4 1. 9 10. 4 1 ci.7 2. 0 2. 10. 1 li. 1. Significantly lower than the control (Dunnett's t test p 0.05) 10. Table 3 Oral Teratology Study of FM-3422 in Rats Number of Fetuses with Gross Findings@@ Finding Total Fetuses Examined Runted Umbilical hernia Total Normal Fetuses Total Abnormal Fetuses 0 mgAg/day 161 --- 1 160 1 75 mgAg/day 167 2 --165 2 37.5 mg/kg/day 195 ----195 0 25 mg/kg/day 213 2 2 209 4 Treatmnt groups were not significantly different from control (Chi-square p -@0.05) Table 4 Oral Teratology Study of FM-3422 in Rats Number and Percent of Fetuses with Skeleton Findings Skeleton Finding 0 mg/kg/day Fontanelle not closed 27 Holes in parietal 1 Parietal scalloped 1 Frontal nonossified 21 Parietal nonossified 21 Interparietal nonossified 14 occipital nonossified Sternebrae nonossified 80 Sternebrae asymmetrical 10 Sternebrae bipartite 2 Sternebrae scrambled Sternebrae enlarged Sternebrae misshapen one sternebrae missing 23 Two sternebrae missing 2 Three sternebrae missing one body vertebrae missing 13 ribs 1 13 ribs spurred 3 Wavy ribs 5 Protrusion on ribs 8 one body of the vertebrae 29 bipartite Two bodies of the vertebrae 17 bipartite Three bodies of the vertebrae bipartite Four bodies of the vertebrae bipartite Five bodies of the vertebrae bipartite Total Normal Fetuses 9 Total Abnormal Fetuses 104 Total Fetuses Examined 113 (24) (1) (1) (19) (19) (12) (71) (9) (2) (20) (2) (1) (3) (4) (7) (26) (15) (8) (92) 75 mg/kg/day 37.5 mg/kg/day 25 mg/kg/day 26 (22) 1 25 (18) 28 (19) 62 (53)a 62 (53)A 70 (51)@l 70 (51)A 75 (50)a 74 (50)a 54 (47)a 4r, (33)A 59 (40)a 1 (1) 100 (86)a 102 (74) 42 (36)@L 34 (25)a 37 (32).@l 6 (4) 111 (75) 36 (24)2@ 5 (3) 1 (1) 1 (1) 1 (1) 32 (@-.'8) 16 (14)@l 1 (1) 31 (22) 9 (7) 33 (22) 16 (11)a 1 (1) 1 (1) 3 (3) 32 (28)2@ 8 (7) 12 (10) 15 (13)11 3 (2) 28 (20)a 4 (3) 5 (4) 21 (15)!! 5 (3) 9 (6) 2 (1) 7 (5) 30 (20) 4 (3)b 5 (4)11 3 (2)b 1 (1) 2 (1) 1 (1) 1 2 114 (2) (98) 116 6 132 138 (4) (96) 7 (5) 142 (95) 149 a Significantly higher than the control (Chi-square p <,0.05) b Significantly lower than the control (Chi-square p < 0.05) percent of total examined 12. Table 5 Oral Teratology Study of FM-3422 in Rats Number and Percent of Fetuses with Internal Findings Internal Finding 0 mg/kg/day 75 mg/kg/day 37.5 mg/kg/day 25 mg/kg/day Fetuses with eye abnormalities 0 Discoloration the lens of Discoloration the lens of Discoloration 3/4 through running through one eye running through both eyes running 1/2 to the lens of one eye Discoloration 3/4 through running 1/2 to the lens of both eyes Discoloration in back of lens Bubble on outside of lens and discoloration running through the lens of one eye Cleft in the lens and discoloration running through the lens of one eye Cleft in the lens and discoloration running through the lens of both eyes Bubble on outside of lens cleft in the lens of one eye Cleft in the lens of one eye open space in the rear of the lens of one eye Small eyes Cleft palate Enlarged atrium Enlarged renal pelvis area in 5 (10) the kidney Blood in the kidney parenchyma Abdominal cavity full of blood 1 (2) 35 (69)@S 7 (1,3) a 16 (31) 5 (10) 1 (2) 5 (10) 1 (2) 1 (2) a 7 (14)1 (2) a 11 (22)- a 29 (51)@@ 2 (4) 13 (23)@S 1 (2) a 7 (12)1 (2) 1 (2) 5 (9) 3 (5) 3 (5) Total Normal Fetuses Total Abnormal Fetuses Total Fetuses Examined 42 (87.5) 8 (16) 6 (12.5) 43 (84) 48 51 25 (44) 32 (56) 57 -SSignificantly different from the control (Chi-square p-, 0.05) 27 (42)a 1 (2) 1 (2) 1D (16)a 5 (8) 2 (3) 4 (6) 1 (2) 3 (5) 1 (2) 2 (3) 3 (5) 1 (2) 32 (50) 32 (50) 64 13. Appendix I Oral Teratology Study of FM-3422 in Rats Protocol objective A teratology study will be used to evaluate the embryotoxic and teratogenic effects of orally administered FM-3422 to pregnant rats during the period of organogenesis. The procedure complies with the general recommendations of the FDA issued in January, 1966 ("Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Use"). The study will be conducted according to the 1978 Good Laboratory Practice regulations and Safety Evaluation Laboratory's Standard Operating Procedures. Sponsor 3M Commercial Chemical Division, St. Paul, Minnesota. Testing Facility Safety Evaluation Laboratory, Riker Laboratories, Inc., St. Paul, Minnesota. Study Director E. G. Gortner Start of Dosing Mid August, 1980. Test System Eighty-eight sexually mature, time mated Sprague-Dawley derived female rats from Charles River Breeding Laboratory will be housed in hanging stainless steel cages with wire mesh floors and fronts in a temperature and humidity controlled room. This strain of rats will be used because of historical control data and time mated females are readily avilable. Purina Laboratory Chow and water will be available ad litibum. The lights will be on a 12 hour light/dark cycle. Test System IdeLtification Each animal will be ear tagged and that number will be indicated on the outside of the cage. Randomization The animals will be assigned cages according to a computer-generated numbers table. random 14. Appendix I (Concluded) Control Article Corn oil. Test Article FM-3422. Analytical Specifications The test article, composition and purity will be determined by the Sponsor (3M Commercial Chemical group) prior to the start of the study and at the end of dosing. Dosage Levels and Experiment Design The test article will be suspended in corn oil daily. The test article suspension and control article will be administered by oral intubation to the rats on days 6 through 15 of gestation according to the following: Dose Group High Mid Low Control Dose Level 75 mg/kg/day 37.5 mg/kg/day 25 mg/kg/day 0 mg/kg/day Group Size 22 @ 22 * 22 @ 22 @ The oral route of administration will be used because toxicity has been defined by this route in a rangefinder study. No dietary contaminants are known to interfere with the test article. The animals will be observed daily from day 3 through day 20 of gestation for abnormal clinical signs. Body weights will be recorded on days 3, 6, 9, 12, 15 and 20 or pregnancy and the rats dosed accordingly using a constant dose volume of 5 ml/kg of body weight. The females will be killed on day 20 and the ovaries, uterus and its contents will be examined to determine: number of corpora lutea, number of fetuses (live and dead), number of resorption sites, number of implantation sites, pup weight and gross abnormalities. Approximately one-third of the pups will be fixed in Bouin's solution for subsequent free-hand sectioning by the Wilson technique to determine any visceral abnormalities using a dissecting microscope. The remaining approximately two-thirds of the pups will be fixed in ethyl alcohol for subsequent skeletal examination after clearing and staining with alizarin red. Data Analysis and Final Report The proposed statistical methods to be used for analysis of the data are: Dunnett's t test for dam and pup weights, number of fetuses, number of resorption sites, number of implantation sites and number of corpora lutea; chi-square for percent abnormalities. The proposed date for the final report is 2-3 months after detailed pup examinations have been completed (approximately first quarter, 1981). 15. Appendix Il Oral Teratology Study of FM-3422 in Rats List of Principal Participating Personnel NAME Edwin G. Gortner Elden G. Lamprecht Cathy E. Ludemann Gary C. Pecore Loren 0. Wiseth FUNCTION Study Director Veterinary Pathologist Coordinator-Histology Supervisor-Animal Care Technician Appendix III STATEMENT OF QUALITY ASSURANCE 16. STUDY NUMBER: TITLE: 0680TROO10 oral Teratology Study of FM-3422 in Rats Auditsand/orinspectiownesreperformebdy theRikerQualitAyssurance Unit for the abovetitledstudyand reportedto the studydirectorand to management as follows: Date Performed 20 August 1980 2 September 1980 20 and 21 January 1981 22 January 1981 Date Reported 21 August 1980 4 September 1980 22 January 1981 22 Janaury 1981 .E. Orters trom LaboratoryQuality Assurance Riker Laboratories,Inc. January-22, 1981 Date Appendix IV 17. Test and/or Control Article Characterization for ;5 AA -3 14.1c).--L-o-T 14 1. The identity strength, uniformity, composition, purity or other per- tinent characterizations of the test andzor control substances have been determined and documented as of 1?74AI& 2. The method of synthesis or origin of the test and control substances, including their amount and the method of bioassay (if applicable) is documented. yes no 3. The stability of the test and/or control 9 stancel have been determined or will be determined as of The above information and documentation are located in the sponsor's records. Alece.f.C44 7 Sponsor Date' form 19793 PWO 18. Appendix V Oral Teratology Study of FM-3422 in Rats Individual and Mean Body Weights of Rats With Standard Deviations Dose Group and Rat No. Study Day 3 6 9 12 15 20 r.4Cil1-4@, 14 t4 C.-i 14 2:: 1:=.,l 2 Cl 2 2 'U-. 4 2,'.'-' 21'-4 f--l 2'1@ 1 Cl Ci --;.14 2'-:4 4--,t@. -i f.4i7lF*15 -':'--:':4 r-4C.F--i" :1 4 @-1 14 r-41-7F1' 1540:-=: tACiF, 15 4 0,-; 1 1,@::4 2 1,-:; l'-@4 1 2 1. 2---.,:-: 2E,4 2f@l 2 1--1 24C-1 2 Lz. 2- .'-:''27 8 21 240 171 2 h-E@,l 2 t- -4 21 2,ClE. 2 4 2 4 1 401 297 7-,:97 2 5 @--i-@'I:'@7' [-IEHt4 2 C-iLi "-1: 245 21:71 L, I ':7THt-4. E.IE'-.7,@''l. :-@: 4 2 Ci. Ci 2,---1,. F'F'Ei--It-4At-F4lTt-4IrIFIL-C.' r.jCiF:14 5,,:- 21 44 I.4C,F: 14@@,:-4--. "7,IC-1 2,--,-- NCIF* 14'471:"_:4 1!@44 t.JCiF, I!E, 1,:-24 2 5 2 4 2'17-@:- 268 2 24:@ 25t,- 244 2 5 2'-; @2 -4 250 280 19. Appendix V (continued) Oral Teratology Study of FM-3422 Individual and Mean Body Weights With Standard Deviations in Rats of Rats Dose Group and Rat No. Study Day 3 6 9 12 15 20 OCIF, :14 1 0 Ci1_.,:14 7@E.', 14 @4 :1 14 -C F.: 14, CIOF, 14 C,CiF.:11'* cicip, 14 OCIF: 15'-:; C, l@I-ICIF: I t 1 Cl 1-.Fi' I 0 CFi.1* 15 44 OOF;.' 15 4 1 @--i 0 CiF. 15411 C[CIF; I'-,cll4 0 @--Fi' It -4 14 185 140 1'-:@t- 4 24 11 2 C.i::-:: 2 ---Ci-:1 %4 C' 2'z:.' 4 c C, @i 21CI 2t-z 24 2el,;, 211--1 '5 2-:41 a 2 --.11 211 2 *c71-, *-'4CI --%.I- 215 1 5 182 211 216 237 2 t-.:l. 25- 4 "7,4'-. 2 E. @-1 317-. 4b b CiC.iF;::14 j- :,Ci,-- 4 Ci 1:-: :1 0 Ci R-ICIF, 14 1 Cii--iF,I!E.4 1 1 1'-:44 1 -@4 245 2'--":j 1 4 :1 @4 @;4 Li cicip: I!f4l 1 2clj- el a Rat died b Significantly lower than the control (Dunnett's t test p < 0.05) 20. Appendix V (Continued) Oral Teratology Study of FM-3422 in Rats Individual and Mean Body Weights of Rats With Standard Deviations Dose Group and Rat No. Study Day 3 6 9 12 15 20 37.5 r-1I:3. '--3.--1":1 I @1 :1':: 1 .1 2 1;:-@: 2 254 nj 14 2 Cl '---'C4-1 2@-:.'-:4 'c7E--'I F, r-,F-, 14 14 2 2--,.4 2 C-45 2'-;4 C-- F, c.-'P,: 14 C-i 1 24-:, Ci P' 149 F'@-1:F, 14 2C'4 1 FICIF,14 L, I F'C'F' 14 15 1:--: C@ 4 F,CLP, I t@ i,iii F,LIF1, 24' F,CIF, I F'OR 157!?--:4 189 217 POR 15415 PCIF* I -@4, 1,@. 205 I C-1 239 24... PCIF' It-41 F'CIF' I!f,4:1:'--1'-;E. F'C'F" I'f-4 l'-@ 24,1 2-71 E, F'CIF' 154 2 Cl,-:@ 245 21 ..,4 @'4 41 26 225 24 24 24 @7'5 Ci 2 237 245 2E.9 2 9 a-1@@7L,-1' 5 2 @-1--l-@ 2t-.-7 2:::., 2 Ci C.i 30@7-,:74 --,.4 C, 4 C, 2 C' 2 2 4b- 2,i.: TF :1 9 :1 -7. '7 22. F,F'E'3t-4FIr-4FTit-41t-lFiL'-= F'@-FI' :14 E. a,Ci 2j-@: 2:14 2.-:-:.-Z-2 b Significantly lower than the control (Dunnett's t test p < 0.05) 21. Appendix V (Concluded) Oral Teratology Study of FM-3422 in Rats Individual and Mean Body Weights of Rats With Standard Deviationsc Dose Group and Rat No. Study Day -3 6 9 12 15 20 H'T' !CiF, 1 4.@ 1 2 @-1: 2 t-5. 2 2 I ;i4, C, 2-:4.@ 2 2 7'@-,,. C!CIF, 14 i'C-iPi, 14 f@74 C!CIF' I 4'-ri@@ 1'-:4;"--' 2'r*. 2 1 2 ClC.L- 2 21!f. 21717-1 2 2!f,.E. 2,,!_:-:4 4,1 C!CIF, 14 C!C'F' 14 C! ILIF, 14-l' 2 f---1I.: 2E 2':41 5 2 2-.',"j- 1 2@-14 2!@.IJ. 2::-::l CiC, 7!@, C!CIF, 1 -4 4 i@ '-.'4:=: E.4 1 -7::11 14 C!CIF* 141--0 C'F-' :1.54C'Ci 15 C!r-F-'! I!t,4 C'1, 1'-::14 1..!C'F*1 4 Cl.:-@.' '@7'C'6 Q 0 F..'15 4 C' 179 OOR 15404 192 2 11 212 2 2'-;1 -4'C-i c.@ I=-. @--'C1l. 'Z:-4'l 24F-l c.-7.L.--, 220 228 254 274 2@ :-:'4 2 4. @l 4 E. 247 11 --<0 ---: 9 F!C'F' 15 4 "@--'C' 214 4 1 25C.1 2E.2 2,-;1, I C,F.' I F-14 1: C-L,Ci,7 2 1,-; 274 5 C!@jF: 15@l I 'E: 2 1 t-. 2-@-I 2 t-C.l 2:E.'C-I I C,F-, 15 4 2 a-:, 251@ 2 *,*:c:7-, I-1 0 C'F.' :15. 4 r_g-l* 2 2--117 '1:@-. '5'-;'a- C, C'2 L:-E@.@ *--'--@c 24C-i '--5';4 281 5'-- 2 c-.C, 9 2 1. NOt-4 F,F*.Ei--)*r.4FFiitt..44TItiFiL'=-, FA@-IF'ltl4 :1'-E:.@ 2 -@-K:-7. 1 27-.4 2 t-z 271 Means not significantly different from control (Dunnett's t test p -,0.05) 22. Appendix VI Oral Teratology Study of FM-3422 in Rats Individual Litter Data With Mean Fetus Weights Dose Group and Rat No. 0 mg/kg/day 1.1 F TOT FiL. FETLI'-=ES PT ICit-4T H"f'l1-t1-4 LUI-ER S I T E'-='. 5 1 T E'-:-. t-c4.P-,'i:15@-.::- 4 7 :11 ci I.4C'P: 15 t.4Cil F,F,Ei3t.4Ftt.4l 4 C-i f-4171 1 7: 4 ci 1 4 Ci'-, I!f.4 ci ci r-4@i f-: 15 4 iii t-4C,F' 1 4 Ci: 4 5 ci N P, 15 4 1,--4$ 7 1 ci t-_i 7..: 4 1-4F' 14 4 11 ci t-4k7-iF:I cl t@ E. 0 t-417-1 F, 14 5:':: 1-41--IT F'RE,3t-JHt-4T t-4cip,14 PFEt3t-4Fir-41' 14C'F' :14 7 E.Cl I Nt--'Fl14 7 7 io 0 1-4C,f-, 14 7 E. I ci t-4'Lif@'14," -7 4 11 ci t-4C,@' 14 1.4 -1 F,F,Ei:it.4Fit-41 NOR 14 7 a 4 4 E: r-C4.f-@I' 14 5 4 i_:i ci 4 4 1 t-I 11 0 q C-1 lc-i C-1 7 ci 11 1 9 :1 4 0 io 1 14 0 11 1 9 7 4 OE. 1 ci :li 1 I ci 11 Iz-1 4, 4 4. 1 4. !f4. 1 4. S' 4. gI. 4- 4 4. 4. 4. zl 1. .4. 4. Ci 4. 4. el gl. 4 4. 4 4. !f, 4. 1 4. 4 4. 4. C, 4. -:1 4. 4. t- 4. 12 4. 2 4. .a. 4. :1 14 4. 0 4. C, i:l t,. :1 ti. 2 4. 11 5. 5. 5. 11 23. Appendix VI (Continued) Oral Teratology Study of FM-3422 in Rats Individual Litter Data With Meaji Fetus Weights Dose Group and Rat No. 1%,'IFiE.LF-FETI-I-cE--:-. I.-EHL@t-1 F Ti-ilFIL FETLISE@-@-. F.E'-=OF* It-IF'LAt-4C,.CIF:F,F.H tIEHt-4 FEIU--'. L-JT(.1@3.,f F,IIF-11.T4HTli--it.4LUTEFI r.,l F I TES S I TEE. 75 mg/kg/day cicif: 4 0 CiCiF, :15 CiOF;., :15 C4 ci CICIF, I CICIF' 15 4 4 5 1:@ CiCiF,, :1541Ci 5 :E: CiCiF, :154:11 4 7 :1:1 0 0 @-1F' :15412 [@EHL:, CiC.iF-.,15 4 1 Ni--iT F'F.E 131-F4lt--4F OC.,F: 15414 C iC-.F',i 15 4,--.E,. ti 7 1 ci CIC,F: 14 1 E: :1 0 147 CiC.iF, 14 4 12 C-i 1 17i ci Ci17F-1,*14 7 4 5 4 0 OOR 14765 7 4 11 ci OOR 14781 DEFi[:- OCIF, 147.,E:2 E. 5 11 0 OCIF. 14 7 E. 1 5 E. 0 OC.IF, :L4784 t-4i--ITF,F:Ei:it.4F-it-41 C,F' 14 5 4 11 ci CiCiF., :14'@":47 5 7 C-i :I :Ici :Ici c 8 0 :1:1 5 -7. E' - c 4 14 j-4 12 12 0 1 C-i 0 1 11 11 0 :1 12 0 11 11 :1 7 1 4. 1 4. 2 4. 4 4 4. 1 4. 1 4. :1 4. 1 4. :1 4. 4. 4 5 5. 4. 1 4. C. 4, 24. Appendix VI (Continued) Oral Teratology Study of FM-3422 in Rats Individual Litter Data With Mecwi Fetus Weiglits Dose Group and Rat No. VIABLE FETUSES t-1 F TOTAL DERE@, FETUSES RES-CIF, ItIF'LFit-4C:OF'PF'Fi PTIOt-4 TFiTIOt-4 LLITEFI SITES SITE'-=, tIEFit-4FEII-]---,@,41."-3.1, FiIlfe-,i t-I F 37.5 mg/kg/day PCIF' 15---t95 4 5 9 ci PCIF' 15--:,9t. 71: 5 8 C-1 POP 157-:-ci7 5 E. ll C-i F,L-iP, 15 8 11 0 POF: 15 5 8 0 POP 15415 6 E. :1 0 PC-IF' :15416@ :12 0 POF' 15411@ a 1: POF' 15 4 1 2 8 :Lc-i POP 15 4 1'-:@ 6 8 14 C-i F'OF' 14 7 5 8 0 F,CIF, 14 4 2 el 0 F'OF'. 14 7 11 ci POP 1476q 5 4 9 0 F,CIF: 1477Ci 5 4 9 ci POP 1478G. NOT PRE!-it-4Fit-41 POF' 14787 POP 14788 4 5 E4 0 4 7 li 0 POF: 14 7 8'-; 1 8 9 0 PCIF' :1479C, 1 7 E: 0 P C-iF-., 1479:-= 7 2 9 0 0 9 0 8 11 2 10 1 1 ci :12 1 11 0 :14 11 2 8 0 11 q I I C-i 0 9 1 12 1 io 1 9 0 9 9 1:@ 1 C-i :11 e. :1 :li 14 1 E: 11 9 10 10 :L2 11 :11 7 E. 4. --,: 4. 1 4. Ci E: 4. 4. 7 4. Cl 4. C, 4, 1 4. ti 4. 4 4. 4. ri -8 4. 5. 0 4. 1 4. ;1 4. 4. 4. :L' 4. :1 4. E. -7..I--. 71 4. 1 4.4 E. 4. 4 4. 4. 'C7-1 4.--,: 4. 1 4. 4. 1 4.4 5 4. 5 4. 0 25. Appendix VI (Concluded) Oral Teratology study of FM-3422 in Rats Individual Litter Data With Mean Fetus Weights Dose Group and Rat No. VlFiE:LE FETLISES, ti F TOTAL I'@EFi[:- PEE-OF-.'.Ir-lPLAt-4 t--OF.F'F-..*MFElFit-4 F*ETI-I':--. FETUI---.EF. PIIOI.4 TATIOt-4 LUTER Fl'-oG 1.1 F SITES SITE'-=. 25 mg/kg/day OOR 154CiCi E. 4 1 ci k--l QC-IF, lt;4CII E. C-i c@ ciF. 15 4 Ci - E: 11 ci QCIF 15 4 Ci 2 7 C4 0 OCIF 15404 5 7 Clc!iR 15420 5 1,-, ci QCIF, 15421 C4 t--F4: 15 4 2 E. ci 1 ci :1-,: ci C@OF' 15 4 5 QOP 15 4 ---'4 11 ci 1 C-1 ci C,!C,P: :L 5 4, =-, rqOT PFE[3r-lHt-41 QC.IF: :147-171 5 :li Q F. 14772 .5 4 F- F: 1477-@ 2 5 0 QOR 14774 4 7 ci OOR 14775 6 9 0 QOR 14791 5 E. 11 0 C! 1.7F1: 14792 -I:z E: 11 0 OOF' 14 7'-=.4 4 E: 1 QOF' :14794 5 G!OF 14795 I ci 0 C@ t--Fi. 14799 7 4 11 0 I li ci I--:- 0 11 1 10 1 ci 12 0 9 1 12 1 11 1 12, cli 5 :1 r:: :L io 1 12 0 11 1 :17. ci a 1 11 C-i 11 9 12 :Ll 1 ci :14 1 11 1 :1-,: 9 1 C-1 8 12 12 12 10 :li 4. -7. 7 4. 5 4. 0 E. 4. C' 4. Fj 4. 5 4. C, 4. 4 4. 1 4, 4. 0 4. 4. 'Li 4. 1 1. 5 4. 4 4. 0 t4. ri 4. 4. 1 4. C-1 4. 4. 1 1. cj 3. 9 4.1 f@ 4. 5 -q 4. 4. 5 4. 2 3. 8 3.-q 5 4. E. 4. 0 4. -.:@ 4. 5 7 4. 2 4.2 7 4. 4. 4 4. !f*