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7 Oral Teratology Study of FM-3422 in Rats Experiment No-: Conducted At: Inclusive Dosing Period: Study Director: 0680TR0010 Safety Evaluation Laboratory Riker Laboratories, Inc. St. Paul, Minnesota August 19 to September 4, 1980 E. G. Gortner l-za-r) E. G. Gortner Senior Research Technologist Animal Reproduction-Teratology Study Director Date phiAX 1 - 2 . 2 - 8 ) E. G. Lamprecht, DVM, PhD Date Research Veterinary Pathologist f n ^ r & jl 'fa/fz M. T. Case, DVM, PhD Date Manager, Pathology-Toxicology Safety Evaluation Laboratory 004233 Summary 1. Oral administration of FM-3422 at 75, 37.5 and 25 mg/kg/day to pregnant Sprague-Dawley rats during days 6 through 15 of gestation (period of organogenesis) was teratogenic to rat fetuses. Teratogenic changes included a developmental eye abnormality, cleft palate, blood in the kidney parenchyma and sternebrae malformations. 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 of the secondary lens fibers of the fetal nucleus. The proportions 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. The sternebrae changes, although normally considered skeleton aberrations, were viewed as compound-related 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 or reproductive tract contents of the dams. FM-3422 was maternally toxic to the 75 and 37.5 mg/kg/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. 004234 2. Introduction This teratology study -- in rats was conducted to evaluate the embryotoxic and teratocrenic effects of orally administered FM-3422. The study was sponsored by 3M Commercial Chemical Division, St. Paul, Minnesota and was conducted by the Safety Evaluation Laboratory, Riker Laboratories, Inc., St. Paul, Minnesota. 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 flgors and fronts in a temperature and humidity controlled room. Food- 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 mg/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. Riker Experiment No. 0680TR0010 -- Purina Laboratory Chow, Ralston Purina Company, St. Louis, MO 004235 3. Results and Discussion FM-3422 was maternally toxic to the high and mid dose groups (75 and 37.5 mg/kg/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 weighings 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 significantly 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 followings 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 arid 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, although 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 following: sternebrae asymmetrical, sternebrae bipartite, sternebrae scrambled, sternebrae enlarged, sternebrae 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 004236 4 significantly higher proportion of fetuses with bipartite sternebrae than the control qroup. 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 teratogenic 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). 004237 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 processes, but also by extrinsic influences exerted by neighboring tissues-- . 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 during the early phases of induction. While the nature of the inductive influence remains unknown, 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^the 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 invaginates 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. Following 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 toward the ectoderm, which do not elongate further, form the lens epithelium-- The cuboidal lens epithelial cells which face the cornea continue to grow 004238 6. after the lens vesicle forms. As the cells rotate through the equator region, they take their places on the surface of the growing 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 controlled by factors which control the number, size and shape of the lens cells-- . The teratogenic lens effect of FM-3422 probably occurred during the portion of organogenesis between differentiation of lens tissue from ectoderm and the formation of secondary lens fibers surrounding 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 2ccur spontaneously in rat fetuses but with a very low incidence of 1.2% . The abnormality resembles the Fraser developmental lens abnormality gf a mutant mouse strain which results from degenerative primary lens cells-. 004239 7. References 1. Couloiribre AJ, Couloiribre 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. Couloiribre 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 Fuer Toxikologie 32 : pp 199-20 7, 1974. 5. Hamai Y, Kuwabara T: Early cytologic changes of Fraser cataract. An electron microscopic study. Investigative Ophthalmology 1 (7) : pp 517-527, 1975. 004240 8 Table 1 Oral Teratology Study of FM-3422 in Rats Mean Body Weight Gains of Pregnant Rats Between Weighings with Standard Deviations Dose Group 0 mg/kg/day 75 mg/kg/day 37.5 mgAg/day 25 mg/kg/day Gestation Day 6 9 12 15 20 MEAN 17 26 29 71 STAN. DEV 5. 5 7. 5 5. 8 4. 9 12. 1 MEAN 3:0 05 65 25 69 SIAM. DEV 14. 2 14. 6 19. 8 17. 8 15. 1 MEAN 65. 17 145 69 STAN. DEV 5. 4 10. 9 9. 8 10. 4 1.5. 8 MEAN 11 20 22 73 STAN. DEV 11. 9 15. 3: 8. 9 5. 4 11. 6 -- Significantly lower than the control (Dunnett's t test p 0.05) 004241 Table 2 Oral Teratology Study of FM-3422 in Rats Mean Litter Data with Fetus Weights and Standard Deviations Dose Group No. of VIABLE FETUSES DEAD RESORPTION IMPLANTATION CORPORA MEAN NT. Animals M F TOTAL FETUSES SITES SITES LUTEA FETUS <0;- 0 mg/kg/day 18 3. S 5. 4 8. 9 1 .6 1 .8 2. 0. 0 0. 0 0. 7 1 .0 75 mg/kg/day 17 5. 1 4. 7 9 8 0. 1 0. 5 2. 1 2 .3 2. 1 0. 2 0. 6 37.5 mg/kg/day 20 4. 4 5. 4 9. 7 0. 0 0. 7 2. 1 2. 1 1. 9 0. 0 0. 9 25 mg/kg/day 21 4. 3 5. 8 10. 1 0. 0 0. 5 1.6 1 .9 1. 9 0. 0 0. 5 9. 6 2. 5 10. 4 1. 9 10. 4 1 .6 10. 7 2. 0 9. 9 2. 1 10. 5 2. 2 10. 5 1. 7 11. 3 1. 9 4. 4 0. 5 3. 7 0. 5 4 Qa 0. 3 4. 01 0. 3 -- Significantly lower than the control (Dunnett's t test p <0.05) Zi'ZI'OO VO Table 3 Oral Teratology Study of FM-3422 in Rats Number of Fetuses with Gross Findings-- 10. Finding Total Fetuses Examined Runted Umbilical hernia Total Normal Fetuses Total Abnormal Fetuses 0 mg/kg/day 161 -- 1 160 1 75 mg/kg/day 167 2 -- 165 2 37.5 mg/kg/day 195 -- -- 195 0 25 mg/kg/day 213 2 2 209 4 -- Treatment groups were not significantly different from control (Chi-square p <. 0.05) 004243 11. 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 Stemebrae nonossified 80 Stemebrae asymmetrical 10 Sternebrae bipartite 2 Stemebrae scrambled Stemebrae enlarged Stemebrae misshapen One sternebrae missing 23 Two stemebrae 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 (24) (1) (1) (19) (19) (12) (71) (9) (2) (20) (2) (1) (3) (4) (7) (26) (15) Total Normal Fetuses 9 (8) Total Abnormal Fetuses 104 (92) Total Fetuses Examined 113 75 mg/kg/day 37.5 mg/kg/day 26 (22) 1 (1) 25 (18) 62 (53)5. 62 (53)-- 54 (47)5. 1 (1) 100 (86)-- 42 (36)4 37 (32)2. 1 (1) 1 (1) 32 (28) 16 (14)2. 1 (1) 1 (1) 3 (3) 32 (28)2: 8 (7) 12 (10) 15 (13)5. 70 (51)2. 70 (51)4 46 (33)4 102 (74) 34 (25)2. 6 (4) 1 (1) 1 (1) 31 (22) 9 (7) 3 (2) 28 (20)5. 4 (3) 5 (4) 21 (15)5. 4 (3)-- 5 (4)5. 1 (1) 25 mg/kg/day 28 (19) 75 (50)2. 74 (50)4 59 (40)-- 111 (75) 36 (24)5. 5 (3) 33 (22) 16 (ll) 5 (3) 9 (6) 2 (1) 7 (5) 30 (20) 3 (2)5. 2 (1) 1 (1) 1 (1) 2 (2) 114 (98) 116 6 (4) 132 (96) 138 7 (5) 142 (95) 149 Significantly higher than the control (Chi-square p <. 0.05) -- Significantly lower than the control (Chi-square p <. 0.05) ( ) = percent of total examined 004244 12. Table 5 Oral Teratology Study of FM-3422 in Rats Number and Percent of Fetuses with Internal Findings Internal Finding 0 75 mg/kg/day____ tng/kg/day 37.5 mg/kg/day 25 xngAg/day Fetuses with eye abnormalities 0 Discoloration running through the lens of one eye Discoloration running through the lens of both eyes Discoloration running 1/2 to 3/4 through the lens of one eye Discoloration running 1/2 to 3/4 through 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 atriums Enlarged renal pelvis area in 5 (10) the kidney Blood in the kidney parenchyma Abdominal cavity full of blood 1 (2) 35 (69)7 (1-3) 29 (51)2 (4) 16 (3D -- 13 (23)- 5 (10) 1 (2) 1 (2) 5 (10) 7 (12)- 1 (2) 1 (2) 1 (2) 5 (9) 1 (2) a 7 (14)- 3 (5) 1 (2) a 11 (22)-- 3 (6) 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 -- Significantly different from the control (Chi-square p < 0.05) 27 (42)1 (2) 1 (2) in (16)- 5 (8) 2 (3) 4 (6) 1 (2) 3 (5) 1 (2) 2 (3) 3 (5) 1 (2) 32 (50) 32 (50) 64 004245 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 Identification 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 conputer-generated random numbers table. 004246 Appendix I (Concluded) 14. Control Article C o m 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 c o m 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 Dose Level Group Size High Mid Low Control 75 mg/kg/day 37.5 mg/kg/day 25 mg/kg/day 0 mg/kg/day 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 mlAg 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). 004247 Appendix II Oral Teratology Study of FM-3422 in Rats List of Principal Participating Personnel NAME Edwin G. Gortner Eiden G. Lamprecht Cathy E. Ludemann Gary C. Pecore Loren 0. Wiseth FUNCTION Study Director Veterinary Pathologist Coordinator-Histology Supervisor-Animal Care Technician 004248 Appendix III STATEMENT OF QUALITY ASSURANCE 16. STUDY NUMBER: TITLE; 0680TR0010_______________ Oral Teratology Study of FM-3422 in Rats Audits and/or inspections were performed by the Riker Quality Assurance Unit for the above titled study, and reported to the study director and 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 f j ?'E * Qrterstrom Laboratory Quality Assurance Riker Laboratories, Inc. January 22, 1981 Date 004249 Appendix IV 17. Test and/or Control Article Characterisation for uot 794 1. T3ie identity strength, uniformity, composition, purity or other per tinent characterizations of the test and/or control substances have JtMj/ S. / _______ .been determined and documented as of 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 y no _____ 3. The stability of the test and/or control substances have been deter mined or will be determined as of AKf/dtou a~f Thx. The above information and documentation are located in the sponsor's re cords. f r jL s jL ______s / * (jo Sponsor Date 004250 Appendix V Oral Teratology Study of FM-3422 in Rats Individual and Mean Body Weights of Rats With Standard Deviations 18 Dose Group and Rat No. ___________ Study Day_________ 3 6 9 12 15 20 0 MG/KG/DAV NOR 14756 204 280 248 276 304 386 NOR 14757 136 224 242 278 304 377 N0R 14760 212 250 257 286 210 253 NOR 14776 184 203 222 242 278 **.*" NOR 14777 222 262 274 207 241 426 NOR 14778 186 213 222 264 237 3 7 ? NOR 14780 226 255 271 2O0 225 336 NOR 147`36 136 220 222 254 280 34 7 NOR 15285 137 221 251 271 301 3-y4 NOR 15287 188 216 228 264 232 1 O NOR 15288 136 228 254 286 322 406 NOR 15283 132 2cii 263 232 346 NOR 15465 184 283 213 225 260 210 NOR 15406 135 226 240 261 233 273 NOR 15407 228 267 272 287 312 230 NOR 15408 223 258 278 306 331 401 NOR 15403 132 218 240 263 237 273 NOR 15425 154 171 206 222 255 213 MEAN 2O0 223 245 271 300 271 STAN . DEV 21. 8 22. 4 20. O 22. 1 22. 6 20. 7 NON PREGNANT ANIMALS NOR 14758 212 244 253 273 268 232 NOR 14753 21 222 226 242 243 264 NOR 14773 134 222 227 255> 243 '25 NOR 15386 132 225 243 244 252 280 004251 BEST COPY AVAILABLE Appendix V (Continued) Oral Teratology Study of FM-3422 in Rats Individual and Mean Body Weights of Rats With Standard Deviations 19 Dose Group and Rat No. _____________ Study Day 36 9 12 15 20 75 MG/KG/I::-Av O0R 14761 215 247 '-* A 255 '7* 307 O0R 14762 224 252 218 217 243 --2 S O0R 14763 188 211 208 230 246 320 00R 14764 193 220 220 245 250 309 O0R 14765 230 26 267 292 3G3 384 O0R 14782 202 223 09 204 21G 267 O0R 14783 207 245 237 264 262 317 O0R 14785 28 246 249 281 202 370 OOR 14797 188 214 210 237 225 291 OOR 15390 176 209 222 226 186 231 OOR 15391 e.!0*4 S' 228 191 168 OOR 15392 212 225 233 .-5Cm 225 c.0trr|* O0R 15393 234 252 251 263 265 311 O0R 15394 194 ">">o 227 23 7 240 39 O0R 15410 185 211 215 185 182 260 O0R 15411 140 221 231 216 237 313 00R 15414 219 240 261 255 259 351 O0R 15426 195 216 243 243 276 368 MEAN 201 231. cijl'd 2 -:;V,b 240-- 31.3-- STAN. DEV 22. 1 16. 6 17. 6 23. y j5. 7 4. 2 NON PREGNANT ANIMALS BEST COPY AVAILABLE O0R 14781 200 243 208 165 0 0-- 00R 14784 195 221 194 1 1'r 204 239 0GR 15412 224 245 22,;y 179 149 0- 00R 15413 241 248 240 242 258 ~ Rat died -- Significantly lower than the control (Dunnett's t test p <0.05) 004252 Appendix V (Continued) Oral Teratology Study of FM-3422 in Rats Individual and Mean Body Weights of Rats With Standard Deviations 20 Dose Group and Rat No. _____________ Study Day 3 6 9 12 15 20 37.5 MG/KG/D P0R 14766 183 214 '18 237 254 301 P6R 14767 208 250 240 268 261 335 POP 14768 208 234 238 264 287 368 P0R 14768 218 245 48 73 84 -i'82 POP 14770 212 242 251 286 288 377 POP 14787 187 215 223 250 267 338 POP 1*41*'yy 176 204 208 226 245 305 P0P 14788 187 222 212 23-4 246 303 POP 14780 182 221 25 251 78 336 POP 14788 186 228 210 236 238 303 POP 15385 182 284 227 240 262 --**v-- d POP 15386 181 ci'12 233 235 243 316 POR 15387 217 245 266 3 Oel 367 382 P0R 15 231 248 256 268 278 360 P0R 15388 188 217 225 237 245 303 POR 15415 205 238 246 268 282 374 POR 15416 210 243 254 270 285 371 POR 15417 222 44 245 257 262 340 POR 15418 186 231 .252 267 287 355 POR 15418 240 263 57 246 237 340 POR 15427 182 216 231 238 245 268 MEAN 203 230 237 254L 26t. 337L STAN. DE1'/ 16. 8 16. 7 16. 8 17. 3 22. 7 31. 3 NON PREGNANT ANIMALS BEST COPY AVAILABLE POR 14786 188 06 213 214 6 Significantly lower than the control (Dunnett's t test p <0.05) 004253 Appendix V (Concluded) Oral Teratology Study of FM-3422 in Rats Individual and Mean Body Weights of Rats With Standard Deviations-- 21. Dose Group and Rat No. _____________ Study Day -3 6 9 12 15 20 25 14G/KG/DAV 00R 14771 232 261 265 282 295 376 Q0R 14772 212 24G 247 263 273 347 Q0R 14773 192 223 228 251 278 322 Q0R 14774 182 210 215 236 256 326 Q0R 14775 282 238 241 269 289 344 Q0R 14791 217 251 263 291 315 389 Q0R 14792 201 229 242 278 293 373 Q0R 14793 221 254 251 281 308 375 Q0R 14794 216 248 264 291 311 376 Q0R 14795 193 223- CLS.S 250 276 345 Q0R 14799 187 212 287 236 255 340 Q0R 15400 153 131 201 214 242 317 Q0R 15481 191 217 233 245 269 346 Q0R 15402 206 255 269 297 394 Q0R 15483 179 212 226 228 247 311 Q0R 15404 192 229 254 274 388 393 Q0R 15428 214 241 258 2*5 291 367 Q0R 15421 183 207 219 234 255 304 Q0R 15422 185 216 . 231 260 280 361 Q0R 15423 228 253 262 257 282 365 Q0R 15424 227 257 259 280 382 376 MEAN 281 228 240 259 281 355 STAN. DEV 19. 8 28. 8 19. 9 21. 3 21. 6 27. 3 HON PREGNANT ANIMALS Q8R 15428 196 225 23:1 234 236 271 -- Means not significantly different from control (Dunnett's t test p <0.05) 04254 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 VIABLE FETUSES DEAD RESOR IURLAN UURPRA MEAN FETUIS NT M fr TOTAL FETUSES PTION TATION LUTEA RVG M F SITES SITES NOR 15385 NOR 15388 NOR 1538? NOR 15388 NOR 15389 NOR 15405 NOR 15406 NOR 1540? NOR 15408 N0R 15409 NOR 15425 NOR 14756 NOR 1475? NOR 14758 NOR 14759 NOR 14760 NOR 14776 NOR 1477? NOR 14778 NOR 14779 NOR 14780 NOR 14796 4 7 11 NOT PREGNANT 4 8 12 "7; jl.; 11 13 4 -- jj 6 36 9 3S 9 45 9 10 34 4 7 11 2S C' NOT PREGNANT NOT PREGNANT 12 3 3 7 10 7 6 13 7 4 11 NOT PREGNANT 44 8 54 9 Ei Ei Ci O Ei 0 0 0 O O Ei Ei 0 0 0 0 0 O Ci 11 p. 3 7 3. 4 0 12 11 *. f 3. 8 3. 6 1 12 10 4. 5 4. 8 4. 4 04 6 4. 4 4. 5 4. 4 4 10 o 4. 1 4. 5 .t"! 11 10 4. 5 4. 4 4. 5 0 9 11 4. 1 4. 3 4. 1 0 9 12 4. 8 4. 7 4. 9 Ei 10 10 4. 2 4. 3 4. 2 07 t' 4. 9 5. Ei 4. 9 0 11 11 4. 2 4. 5 4. O 19 9 4. 4 4. 4 4. 4 14 8 4. 7 4. 5 4. 7' 0 10 1^.' 4. 2 4. 3 4. 1 1 14 14 4. 0 4. 0 Q 0 11 11 5. 1 5. 2 4. 8 1 9 11 5. 5 5. 7 5. 3 1 10 11 3. 9 3. 8 2*. 9 004255 BEST COPY AVAILABLE 23 Appendix VI (Continued) Oral Teratology Study of FM-3422 in Rats Individual Litter Data With Mean Fetus Weights Dose Group VIABLE FETUSES DEAD RESOR IMPLAN CORFF:A MEAN FETT. w t -;:g and Rat No. M F TOTAL FETUSES FT ION TAT ION LUTEA AVG M F SITES SITES 75 mq/kg/day GR 15390 0R 15391 O0R 15392 O0R 15393 O0R 15394 DOR 15410 00R 15411 O0R 15412 O0R 15413 O0R 15414 O0R 15426 O0R 14761 O0R 14762 O0R 14763 00R 14764 O0R 14765 O0R 14781 O0R 14782 O0R 14783 O0R 14784 O0R 14785 O0R 14797 4 J. t*' DEAD (5 9 c. 4 45 6 C| 58 4 1*' ii DEAD NOT PREGNANT =1 11 13 5 *17 12 8 d* 10 8 4 12 oo 10 54 9 7 4 ii DEAD 65 ii 15 6 NOT REGNANT 7 4 ii 5 7 12 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 9. C| 2. 8 cl. 3 2. 6 1 10 3 4c r 1:. . . J;.. 1 I*' 3. 6 5 3. 6 1 10 08 3 3. 8 S. 7 3. 4 o --.s s. 5 3. 0 0 ii 12 2. 4 * _J-. Ji- 0 14 14 4. 1 Ji*. C| 4. 1 0 12 12 4. 2 4. 4 4. 1 0 10 12 3. 4 4J-. _i>. 0 le i 11 . -S J-. Ji* J - . _3- 1 ii 11 w . i 8s . 3. 1 03 3 3. 6 j :- . 5 , 3. 8 1 12 13 4. 1 4. 1 4. 1 0 11 11 3. 3 5 3. 1 17 8 4. 7 5. 4. 6 1 12 13 4. 4 4. I. 4. 5 0 12 11 S. 3-i*. 2. 8 004256 BEST copyAVAILABLE 24. Appendix VI (Continued) Oral Teratology Study of FM-3422 in Rats Individual Litter Data With Mean Fetus Weights Dose Group and Rat No. VIABLE FETUSES DEAD RESOR IMPLAN CORPRA MEAN FETUS NT M F TOTAL FETUSES PTION TATIN LUTEA AVG M F SITES SITES 37.5 mg/kg/day POR 15395 POR 15396 45 35 C| 8 O O 0 C| 08 9 9 3. 7 3. S 39 - p 3. 5 3. 5 POR 1539? 5 6 11 0 0 1 1 1 0 4. 3 4. 5 4. 2 POR 1539S POR 15399 8 11 35 8 O O 1 1 2 1 1 4. 1 4. 4 3. 9 2 10 8 4. O 4. 3 "!* O POR 15415 POR 15416 POR 1541? POR 15418 6 s 12 9 3 1 2 8 " 1 1 cL c IO 0 0 O O i 13 13 3*. 9 4. G 3 0 1 2 1 1 3. . o -- cO 1 1 1 1 4. 2 4. 3 4. 1 1 1 1 1 2 4. 7 5. 0 4. 6 POR 15419 6 8 14 0 " O 14 14 3. 9 4. 1 3. POR 14?66 POR 14?67 POR 1476S POR 1*4 53 8 42 6 0 il 54 9 O 0 0 O wS 1 1 13 3. 7 . o 3. 5 28 8 4. 8 4. 1 3 9 0 1 1 1 1 3. -- 9 _ -, 0 C| 9 4. O 4. O 3. 9 POR 14??0 54 9 O 1 1 0 IO 4. 1 4. 3 3. 9 POR 14786 NOT PREGNANT POR 1478? 45 9 0 0 9 1 0 4. 1 4. 2 4. 1 POR 14788 POR 14789 4 7 11 18 Ci O 0 1 1 2 1 2 4. 4 4. 3 4. 4 1 IO 11 3. 6 3 3. 5 POR 14790 17 8 0 1 9 1 1 4. 4 4. 1 4. 5 POR 14798 72 9 0 09 8 4. 3 4. 3 4. 0 0042S 7 BEST COPY AVAILABLE Appendix VI (Concluded) Oral Teratology Study of FM-3422 in Rats Individual Litter Data With Mean Fetus Weights 25 Dose Group and Rat No. VIABLE FETUSES DEAD RESOR IMPLAN CORPRA MEAN FETUS WT<G> M F TOTAL FETUSES PTION TAT ION LUTEA AVG M F SITES SITES 25 mg/kg/day Q0R 15400 QOR 15401 Q0R 15402 QOR 15403 QOR 15404 QOR 15420 QOR 15421 QOR 15422 QOR 15423 QOR 15424 QOR 15428 QOR 14771 QOR 14772 QOR 14773 QOR 14774 QOR 14775 QOR 14791 QOR 14792 QOR 14793 QOR 14794 QOR 14795 QOR 14799 6 4 10 6 1 2 -! 1 1 1* 7 C| 5 7 12 57 "< 6 12 C| 1 0 13 5 1 1 3 ? 10 NOT PREGNANT 5 6 11 .5 4 9 23 rzj 34 7 63 9 5 6 11 3 A 11 4 8 12 ;* 5 8 T' 3 1 0 7 4 11 O O O O O 0 O 0 0 0 0 0 0 O O O 0 0 0 0 0 1 11 Cl "S " 3. 5 3. 2 0 12 12 4. O 3. 7 0 1 1 1 1 4. 3 4. 4 4. 3 1 1 0 1 0 3. 7 4. 1 3. 5 1 13 14 4. 5 4. 6 4. 4 O 1 2 1 2 4. 0 4. 0 4. O 0 C| Cl 3. 6 Pi 3. 5 0 13 13 4. 0 4. 2 4. O 1 1 2 1 1 4. 0 4. 8 3 9 1 1 1 16 4. 3 4. 4 4. 3 1 1 2 13 4. 1 4. 3 4. O 09 9 4. O 4. 1 3'. 9 0 5 1 0 3. 8 3 8 3. 8 18 8 3. 9 4. 2 3. 7 1 1 0 1 2 3. 9 3. 8 4. 2 1 1 2 1 2 4. 1 3- 9 4. 2 0 1 1 1 2 3. 7 3. 5 - y 1 13 1 2 4. 5 4. 8 4. 3 0 8 1 0 3. 9 4. O 3. 9 1 1 1 1 1 4. 6 4. 7 4. 4 0 1 1 1 2 4. 5 4. 5 4, 5 BEST COPY AVAILABLE