Document r6B67r0KnKmqN9v02LokLVDMe

Oral Teratology Study of FC-95 in Rats Experiment No.: Conducted At: Dosing Period: Study Director: 0680TROOOB Safety Evaluation Laboratory Riker Laboratories, Inc. St. Paul, Minnesota July 14, 1980 through July 24, 1980 E. G. Gortner E. G. E;rtner Date Senior Research Technologist Animal Reproduction-Teratology Study Director E. G. Lamprecht Research Veterinary Pathologist Date M. T. Case, DVM, PhD Date Manager, Pathology-Toxicology Safety Evaluation Laboratory Summary Oral administration of FC-95-at doses of 10, 5 and 1 mg/kg/day to pregnant Sprague-Dawley rats during days 6 through 15 of gestation (period of organogenesis) resulted in fetuses with teratogenic changes in the lens of the eye. 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. The lens abnormality occurred in all rc-95 dose groups, but the proportion of fetuses with the lens changes was significantly higher than the control group only in the 10 mg/kg/day group. FC-95 administration was maternally toxic only to the 10 mg/kg/day group. At gestation days 12 through 20 their mean maternal body weights were significantly lower than the controls. FC-95 was not maternally toxic to the 5 and 1 mg/kg/day groups. FC-95 was not embryotoxic and did not affect the ovaries or reproductive tract contents of the dams. The compound did not produce an increase in the number or proportion of abnormal fetal skeleton aberrations. 2 Introduction a This teratology study-@-o-f FC-95 in rats was conducted to evaluate the embryotoxic and teratogenic effects of orally administered FC-95. The study was sponsored by 3M Commerical Chemical Division, St. Paul, Minnesota and was conducted by the Safety.Evaluation Laboratory, Riker Laboratories, Inc., St. Paul, Minnesota. The compound administration period was from July 14 through July 24, 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 Gc>odLaboratory Practice (GLP) regulations and the Safety Evaluation Laboratory Standard Operating Procedures (see Appendix III for Quality Assurance Unit statement). 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 Spraque-Dawley derived rats were obtained from Charles River Breeding Laboratory and assigned cages according to a conputer-generated random numbers table. The rats were then divided into four groups of 22 animals weighing 175 to 261 grams. The rats were housed individually in hanging stainless steel cages withbwire mesh floors 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 FC-95 (Lot 640) suspended daily in corn oil at Or 10, 5 or 1 mg/kg/day. rc-95 was administered daily by oral intubation with a syringe ec,-Uippedwith a ball-tipped intubation needle to the rats on days 6 through 15 of gestation (day 0 indicated by ppe=-positive vaginal smear). FC-95 analytical characterization (see Appendix IV) was provided by 3M Commercial Chemical Division, St. Paul, Minnesota. All animals were sacrificed on day 20 by cervical dislocation and the ovaries and uterus, including its contents, were e.-.ariined to determine the following: number of corpora lutea, number of viable fetuses, number of resorp- tion 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. 0680TROO08 b Purina Laboratory Chow, Ralston Purina Company, St. Louis, MO 3 Results FC-95 administered during the period of organogenesis was toxic to the high dose group (10 mg/kg/day) maternal rats. The mean body weights of all dose groups were similar at gestation days three through nine (Table 1, Appendix V). At gestation days 12 through 20 the high dose group rats weighed significantly less than controls (0 mg/kg/day). The mean maternal body weights of mid (5 mg/kg/day) and low (1 mg/kg/day) dose groups were not different from the controls throughout the study. Even though FC-95 was maternally toxic at the high dose level, no compound-related clinical signs were observed in any of the dose groups. FC-95 was not embryotoxic and did not affect the ovaries or reproductive tract contents of the dars. The mean number of male, female, total and dead fetuses; the mean number of resorption sites, inplantation sites, corpora lutea and mean fetus weights of the three FC-95 dose groups were not significantly different from the controls (Table 2, Appendix VI). The high dose group did have a lower mean number of viable male, female and total fetuses than the other three groups which resulted from a lower number of embryos at the start of the study. Contributing pieces of evidence to the lower number of high dose embryos are the low mean number of implantation sites, corpora lutea, resorption sites and the absence of dead fetuses. FC-95 did not cause compound-related abnormal gross fetal findings (Table 3), nor did FC-95 treatment produce an increase in the number or proportion of abnormal fetal skeletal aberrations. Fetal skeleton results of the three compound treated groups were not significantly different from the control group (Table 4). The incidence and proportions of sternebrae nonossified and associated changes of sternebrae assymetrical, sternebrae bipartite and one sternebrae missing were unusually high in all dose groups of this study including the control group. FC-95 was teratogenic in 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 of the lens near the anterior margin to a dark colored oval area, often containing a cleft, extending from beneath the lens epithelium to half-way through the lens posteriorly. Histologically the discolorations were due to presence of lens vesicle remnants surrounding the abnormal embryonal.lens nucleus. One of the most severly affected eyes had most of the embryonal lens nucleus replaced by sinus spaces containing red blood cells. Also contributing to the discolorations were primary lens fibers which appeared to have not elongated. These lens fibers were tortuous and lacked nuclei in a normal lens bow of nuclei. Secondary lens fiber development progressed normally except immediately surrounding the abnormal embryonal nucleus. Secondary aberrations of secondary lens fibers included the bending of the fibers around the abnor-mal oval area, the subsequent formation of prominant anterior and posterior Y sutures of the converging fibers and lens vesicle remnants surrounding the embryonal nucleus. 4 The lens abnormality occurred in all dose groups except the control group. The proportion of fetuses with the lens abnormality in one or both lenses was significantly higher in the high dose group than the control (Table 5). The lens abnormality recorded for one control fetus under the dissecting microscope was an artifact when evaluated by transmission light microscopy. A no-effect dose level for the teratogenic lens abnormality was not established in this study. Discussion optimal visual functional requirements are met during embryonic development by the differentiation of highly specialized populations of cells from undifferentiated precoursers and by the coordinated morphogenesis of the resulting tissues. Both processes are controlled to a remarkable extent by interactions which occur a ng emerging tissues. Each tissue of the eye is brought to its final state of differentiation, its cell population size and its definitive ge6netry, not only by intrinsic processes, but also by extrinsic influences exerted by neighboring tissuesi. 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. Alternative or sequential 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 probabililty 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 retina2. During the early stages of the inductive process, the ectodermal cells immediately overlying the tip of the optic vesicle elongate prependicularly 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 temporaty 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 epithelium2. OL 5 The cuboidal lens epithelial cells which face the cornea continue to grow 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 lens cells2. The teratogenic effect of FC-95 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 abnormality3. A similar-appearing structural lens abnormality has been reported to occur spontaneously in rat fetuses, but with a very low incidence of 1.2%4. The abnormality resembles the Fraser developmental lens abnormality of a mutant mouse strain which results from degenerative primary lens cells5. References 1. Coulombre AJ, Coulombre 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. Coulombre AJ: The Eye, in DeHaan RL, Ursprung H (eds): Organogenesis. New York, Holt Rinehart and Winston, 1965, pp 227-232. 3. Mann 1: 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 199-207, 1974. 5. Hamai Y, Kuwabara T: Early cytologic changes of Fraser cataract. An electron microscopic study. Investigative ophthalmology L4 (7): pp 517-527, 1975. 6 Table I oral Teratology Study of FC-95 in Rats Mean Maternal Body Weights with Standard Deviations Dose Group 0 mg/kg/day 10 mg/kg/day 5 mg/kg/day 1 ing/kg/day Gestation Day 3 6 9 12 is 20 MEFIN 20C4 22--< 247 272 30!1, 38C, STFit-4. E'-EV 16. 7 :17. 6 2 0. '-:@2 Li.5 2 -4.4 8 tiEFit-4 19'-; !FITHt-4.C)EV 11. 8 22 24--,* 25i-,A 27'C"A 8 18.2 16. 2 1 S. 6 --4<. MEFit-4 21-71!t7'---"28 24,-:4 2 6 Sl Fit4. C-EV 2C'. 0 1 E,.4 :12. E. I --e.2 2'4-;4 ----7 82 8 tiERN 2t-i5 2 2 25 2'f@ 7 :1*-;.5 "='@4.6 Significantly lower than the controls (Dunnett's t test p 0.05) Dose Group 0 mg/kg/day 10 mg/kg/day 5 mg/kg/day 1 mg/kg/day Table 2 Oral Teratology Study of FC-95 in Rats Mean Litter Data and Pup Weights with Standard Deviationsa No. of Animals 20 17 17 19 IHE'LE FE t [11 F -["Cf-HIL- 2 4. '-4 1 Cl. I. 2. 1 2. 8 -z. 9 2. 8 2. 6 4. 5. C, 1.9 5. 5 2.0 10. 5 2. 2 4. 7 -1. 7 5. 4 2. J- :LO. :L 2- 8 F'E S Li 0. 0 Cl. 0 0. 0 0.0 0. 1 0. 2 RE!@-CIF.P'TI ON I I EE. ILI. ILI9. 0. et 0. 6 0. 7 I.o 0. t 0. 8 I14PLHN I'Ht10 I'T"ES 2. 5 S. 1 4. -z@ 11. 2 2.2 10. 6 2. 7 Treatment groups were not significantly different from controls (Dunnett's t test p 8 Table 3 Oral Teratology Study of FC-95 in Rats Number of Fetuses with Gross Findingsa Finding No. of fetuses examined Umbilical hernia Runted Total Normal Fetuses Total Abnormal Fetuses 0 mg/kg/day 201 1 --200 1 10 mg/kg/day 131 --- 1 130 1 5 mg/kg/day 178 1 --177 1 1 mg/kg/day 192 --- 1 191 1 Treatment groups were not significantly different from the control (Chi-square p -,0.05) C) 9 Table 4 Oral Teratology Study of FC-95 in Rats Number and Percent of Fetuses with Skeleton Findings@l Skeleton Finding 0 mgAg/day Fontanelle not closed 10 (7) Frontal nonossified Parietal nonossified 4 (3) 2 (1) Interparietal nonossified 3 (2) Occipital nonossified 1 (1) Sternebrae nonossified 114 (81) Sternebrae asymmetrical 53 (38) Sternebrae bipartite 7 (5) One sternebrae missing 30 (21) Two sternebrae missing 10 (7) 13 ribs 5 (4) 13 ribs spurred 7 (5) Wavy ribs 1 (1) Protrusion on ribs 6 (4) One body of the vertebrae 32 (23) bipartite Two bodies of the vertebrae 18 (13) bipartite Three bodies of the vertebrae 4 (3) bipartite Four bodies of the vertebrae --- bipartite Total No. Normal Fetuses 7 (5) Total No. Abnormal Fetuses 1.33 (95) Total No. of Fetuses Examined 140 10 mgAg/day 10 (11) 1 (1) 1 (1) --1 (1) 77 (85) 23 (25) 4 (4) 13 (14) 2 (2) 2 (2) 7 (8) 2 (2) 9 (10) 21 (23) 7 (8) 1 (1) --- 3 (3) 88 (97) 91 5 !ng/kg/day 7 (6) --1 (1) ---100 (81) 36 (29) 5 (4) 26 (21) 4 (3) 2 (2) 8 (7) 3 (2) 25 (20) 11 (9) 1 (1) --- 10 (8) 113 (92) 123 1 mg/kg/day 5 (4) 1 (1) 1 (1) 1 (1) --107 (81) 39 (29) 6 (5) 26 (20) 6 (5) 6 (5) 4 (3) 1 (1) 8 (6) 32 (24) 9 (7) 3 (4) 1 (1) 10 (8) 123 (92) 133 -2@Treatment groups were not significantly p < 0.05) percent of total examined different from the control (Chi-square 10 Table 5 Oral Teratology Study of FC-95 in Rats Number and Percent of Fetuses with Internal Findings Internal Finding 0 mg/kg/day Eye abnormality Thoracic cavity full of blood Enlarged atria Enlarged renal pelvis in the kidney Abdominal cavity full of blood area l@1(2) --- 1 (2) 3 (5) 4 (7) Total No. Normal Fetuses 52 (85) Total No. Abnormal Fetuses 9 (15) Total No. of Fetuses Examined 61 10 Fig/kg/day 14t(35)S 1 (3) ----- 2 (5) 5 mg/kg/day 4t(7) --5 (9) 23 (57) 17 (43) 40 47 (85) 8 (15) 55 1 mg/kg/day 2b(3) --3 (5) 2 (3) 53 (90) 6 (10) 59 Eye abnormality was an artifact and was not considered for statistical evaluations Eye abnormalities were developmental lens abnormalities with secondary lens aberrations significantly higher than the control (Chi-square p 0.05) percent of total examined Appendix I Oral Teratology Study of FC-95 in Rats Protocol objective A teratology study will be used to evaluate the embryotoxic and teratogenic effects of orally administered FC-95 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 July, 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 available. Purina Laboratory Chow and water will be available ad libitum. 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. 12 Appendix I (Continued) Randomization The animals will be assigned cages according to a computer-generated random numbers table. Control Article Corn oil. Test Article FC-95. 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 Dose Level Group Size High Mid LOW Control 10 mg/kg/day 5 mg/kg/day 1 mg/kg/day 0 mg/kg/day 22 @ 22 * 22 2 22 @ The oral route of administration will be used because of metabolism studies showed radiolabeled FC-95 was well absorbed. 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 of 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 13 Appendix I (Concluded) 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 fourth quarter, 1980). 14 Appendix II oral Teratology Study of FC-95 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 Ap pendix III STATEMENT OF QUALITY ASSURANCE STUDY NUMBER: TITLE: 0680TROO08 Oral Teratolocrv Studv of FC-95 in Rats Audits and/or inspections were performed by the Riker Quality Assurance Unit for the above titled studyand reportedto the study director and to management as follows: Date Perfomed 18 July :L980 28 July 1980 15 December *1980 17 December 1980 Date Reported 21 July 1980 28 July 1980 17 December '1980 17 December 1980 E. Orterstrom Laboratory Quality Assurance Riker Laboratories,Inc. December 17, 1980 Date APPMMIX Iv 16 Test and/or Control Article Characterization for Loi 6,4o 1. The identity strength, uniformity, composition, purity or other per- tinent characterizations of the test and/or control substances have been determined and documented as of k-,(-gdO 2. The method of synthesis or origin of the test and control substances, including their amount ynd the method of bioassay (if applicable) is documented. yes no 3. The stability of the test and/or control substances have been deter- mined or will be determined as of CAJ OX 7-OA The above information and documentation are located in the sponsor's records. Sponsor Date Form 19793 PWO 17 Appendix v Oral Teratology Study of FC-95 in Rats Individual Body Weights (g) Dose Group and Rat No. 3 Cl tIG/KG,-'[.*,HY Study Day 6 9 12 15 20 NOF.' :LE.'6. L,12 2 t- 254 2 NCif@: :129 22e4 2t '1 2 .'9 2'0t- 3*45 N t-I NOR :1 :i;6'l 2 -,'8. 2 4 @-I -:15 21 2-'.'2 271 2,--,t Cl2 224 24ti 5 t4LIF, :L --,'10 t'. t-4ClF.'Li18 tior 12019 t-4@-4I F-,-<Ci;L:k--i N CiF. I.-:@Cl *--E'.-'-: NOR :1.-C@l4 Li IE'2 LiLl',-'2,--:4 2 tst-3 1 i"t, L14E. 19--,: 219 2 2 1'--44 221 2 27'1@ 22c: 222 2,i:4 I L t@."1:: 2 2 1-i'-. 2i,4 Z-77 09 319 --@22 29-, t-4t-iF;: I Li4 1 2 1'-z@ @-2 Li 2 0 2!-. 2 t-a': 28? L@ 4 N t-I@' 1-::,Ci,14 2@".* 22c: i:4 2 290-'-. t4@:'F. I C-IE -L, 2 2 4 'i.' 2 1 t-@ --@4: t4C!F: ILI I l-@4 212 2 1 2 5'-; 297 NOR 13062 210 229 222 272 302 NOR 1.-; 6 1-4C!F.'.I -7*'z.Cic:C' IE:t., 20c-: 1 211. 17'-:4 l-@44 24-e 2T,7 2t-t-I 25E. 2 289 2 8'-@: ;L5l E. 3 4 Z4 9 4i -@4.2 4 @-0I 4C'S -81 ----.'5 42E. to 4 4. --6" 362 --,,42 --6-.-:'i' --:.-:21. rIEFit-4 2 CiC' 2'-'---' 24@r, 2 -f f@l!: 8 C' T At-4. E)E@@' 1E.. 17. P-'.2 @'j.-:@ 2 L@. 2 4. 4 NOt-4 PREi3t-4HI.4'iFlNll*lHL';-z@ N-0k: :1-@.,017 18E. 21c'*" 2--'.'42 t, NOR 13042 188 209 2E.0 L@'4-(2'55 272 18 Appendix V (Continued) Oral Teratology Study of FC-95 in Rats Individual Body Weights (g) Dose Group and Rat No. 3 Study Day 6 9 12 is 20 :10 MG,,-KGe'C-fqY OOR 13CiCil :1 OOR 1,002 1,-4 0 @-F@.':1 0 Cl I 9':-P CIOF, I C.t,-I4 1 @o *---' OOR :1:-'.CI05 2C'l f@, 22'17' OOR 12022 212 OOR 1 0 2 180 C.IOF' O.:-@'t, 2 0 LIOF: I _-0;, 1 -i-, CiO@-., 1.7@,C- ,4 OCIP, 1 0 4 OOF: I.L@065 204 0 t'-Fi..' 2 @--i OOR l,'067 210 OOR 13069 203 CIOR 13081 194 22Z, 217 222 1" 2 22!:-. Z-t 244 20E. 2-@9 230 224 21E. 26 24 24,., 258 21.4 2 9 2 1 t---, 22:i. 2f-@5 22E.' 27e;. 22E. 27ti 2,:4 222 228 262 209 238 25-1: 281 2 5 28-. 2i-'.to 2 @.,L-1 2 3 -". 255 26:1 28ti 2.'@'8 2 9 -7. 2,.-.:8 '-@'ll 22-t' 24ti 8 2:@.4 25L, 274 25,-:4 2 24e. 2 2 2E.--*. 2 268 278 262 2 8 237 251 347 356 8:1 2,6 @i --6< C, 402 285 82 357 -fE.1 --@I:I 04 322 38 281 MEFIN 1 *-9@4 223 2 4"--.*: 2ti7 2-r'7 4 -;I*Ht-4.C)E'-ej'.1. E. 1 8 18. 2 ItE.2, 1S. 4. E.* t-40t-4PF!El3t-iHN'T At-4IriFiLS OOF.' 0 @--Fi: 001@.' OCIF' OOF* :1 2 4 1304t--. I-RO47 13C-149 1 -7:CiE.,E: :19t, I e. 1:?,4 21 2:1 21*1-" 2 2C'9 24, 2 C-1i 244 23-@' Z,4 2 --'2 2 3 2 22E. 221 2tk'-@;l 2-7e9 242 2 2-:-@ 251 250 2L 2 t*, 2 19 Appendix V (Continued) Oral Teratology Study of FC-95 in Rats Individual Body Weights (g) Dose Group and Rat No. 3 Study Day 6 9 12 is 20 L-1L--i 27 F,i-iP I CiI C, i-Ik-I i. ci i@0-4 212 p C, t.@-Li 17E. F'C-IF' I C, "-'1$ 2;i P l@: 1. Clt, 2 *@-t',: laOb4 19*7 :L L, 2 r,-'4 P C,I-.'.I I 2LO F*C'F* I -e'2Ie. 21;L 22L@i 2 21@-1 t-4I 24_--;24c: 224 2E@E. 2 Z' --'-.*,. 211 L*74 c-;.t'42t,l 2 241 ;m.i,-.i.-@i 2 21--.6 242 2 %L-,' Ll4 5 2 rzLti 2 .4 .04 2E2 2!f,4 2 E. 2 8 2t-'-.9 2 &.:--: 4 4 2*., 2E@l: L-17 t'--i 294 4'--, E. ;L,4E. 2 2'@-I; 2 t,'-0@ 2 25 4 L, :@2t- 4LE. :i"*'9 14'-@-0 2 l,'O4 29.2 4 1 @-I 178 -@6 I-lEAt.4 .*:@,-LI., ,:--.THI.i.L.,E%.@L'L1,. Li :1P--,4. 24:@ Zo P-'.: 294 7. :1 2 i: 2 t-4L't-4F'F*Ei:iNHr4l f-it-r4i1FIL--. F'@-11-;':I --@':Ll:.'t:*:'. POi-, l3Ob2 F'1-11-.::1 0 a, F't-l@' :1 r@l 4 *:-@'*-*(i' 218 2c"t. L, 19@-, 2 2-7:7 1 2-7,:4 214 29,1 252 250 244 24C, 2t-2 248 250 257 225 2t-,2 25,@ 24,1 272 @L- 2 26,@l25@o 240 20 Appendix V (Concluded) Oral Teratology Study of FC-95 in Rats Individual Body Weights (g) Dose Group and Rat No. 3 Study Day 6 9 12 is 20 II I t-1-1-4 C@Cil@' C,I t, Li 1'*-*:. 4 - :I'C-l C!Cli-,:L Li5!f, C@Cl@-* I Lit, :I C, C-iF.., I 'Cl5 130'75 F!OR I --0@ 7 7 21'i@ 1 198 2 @-I 1'-;t;2L'4 1 I &.'-I 2'2 1-11 2k-.14 2C';l 185 198 2 1-&1. 2E.1 224 2 5 k--i 24:'. 2C14 221 @,24- 22 ;Llt,-' 2 41 22C-1 2'-@C- 24t-. 244 .':';'2 254 2Cil. 2 2 5 @'2. '@.CI2 2;LlCl 2 *-2z: 22--,: 2t['-:@ 211 2 224 ;:@ 4 206 283 215 262 22E. 24-,. 279 7 e. 261 2i=2 2 E. 272 24 2b'@*, Zo 2 P*-251 1 21 2 2 t. 2.,Li 257 262 2 oE. 2 I --'0 *-@L:iI -@'12Ea-@2 27 -:@20 2 'i:E.* ;271 4 79 *,,P''*41 411 --'22 40-.17 5 41 2 266, '-@::0; 1 291 296 -@:75 -1:62 a6 @74 Z-'C'!--, 22E. 252 2 7.:7_' L, 7 S"iHt-4. E'EV IE:.8 :I:iO1. 1'-4;. 7 2 4. t-40t@4 C!C4F, Fit4Itif-iL'-z. 11-42 2C4t- 24-e@ 226 2:1'-@ 2.-@6 254 218 228 244 2-@*8 25--,' 278 270 255 2E.6 21 Appendix VI Oral Teratology Study.of FC-95 in Rats Individual Litter Data with Pup Weights Dose Group and Rat No. %.@IFIE:LE FEI'U':-:E--.'I)EFiL.- m F TOI'HL FEl RESOW IMPLFit4 CCIF.'FIRH MF-HN FF-T@-[,--. P'llOt-4 TF-ITION LUTEH Flvl@i t-I F SITES SYTES 0 mg/kg/day 14OR 1299E'. te 4 0 NOR 1291-;47 4 9 :1-Z. 0 NOR :129'-z'8 7 4 :i:l ci T-JCIF* :129'-:4:? 7 4 :1:1 0 NOR 13000 7 7 :14 0 NOR 13016 NOR 13017 4 5 9 0 NOT PF.'El:it-JFit-l'l NOR 1-@018 :It-I 0 NOR 13L419 E. :L 7 0 NOR :1---@@--420 4 8 :12 t-@ NOR 13036 5 5 :1Li f-i NOR 12040 6 7 :L ci NOR 13041 6 1: ci NOR I-Z-042 NOT PREGNHNT N t-Fi: :I.-:C,i4 --@: 4 6 10 El NOI@' :1.-@:044 5 2 7 t-i NCIF.'1--<060 8 5 I.-L' 17-1 NOR 13[161 4 E. I ci 0 NOR 13062 --.c 4 7 0 NOR :13 @_-6i _-@- 15 6 A- NOR 13064 5 7 12 0 NOR 1--Z:oso E. 2 8 0 MEAN 5. 2 STFit4. E.*-EV. 1. 7 4. :IC1.0 2. 1 2. 0. ci 0.0 :1 :14 f3 :L:i 2 i.-@ ci 14 :1 :1ci :1 li 7 L4 :12 :1 :1:1 ci :1cl j. 0 7 1 14 2 12 0 7 0 t---. ci 12 1 !@4 0. 7 0. :10.8 2. t, 9 :1E. :L2 J-2.. :17 9 :1:1 6 :L2 8 :12 j-2 :15 11 12 11 :12 9 11. 2 2. 7 4. '3 E. 4. ---. 4. C-i 4. 1 :1 8 4. 4. :1 4. 2 4. 5 4.. 2 -::'.'@-4 4. 0 4. @-i 4. 5 5. :1 4. 4. :1 -:1.4 4. 2 4. b ti.1. 4. 4. 4. t, 4. 4. Z -q. 4. 4.. 2 4. 1 4. 2 9 4. 1 4. 2 4. 1 4. -:'1.2 4. 4 4. l 4. 1 4. 4. 4 4. 4. 4. 4 4. :1 9 4. 1 2. 8 4. 4. 2 4. R 4. Cl. I 22 Appendix VI (Continued) oral Teratology Study of FC-95 in Rats Individual Litter Data with Pup Weights Dose Group and Rat No. VIFIE'LE FEILI':-;E@-C-E-F.i[.'- m F TCITFIL FETUSES F.ESOF. IMPLHN CURPI-.H PTIOt-4 *TFiTI01-4LL)TEFI SITES SlTEI-@@ tqEHN FLVG FE"ILI'@-:. 1-1 F 10 mg/kg/day oc4R OCIP 1300-1: 0 0 F..':1--<004 0 ciF-.,1--<Clcitl OOR 13021 OOF.' 1--<022 CIOFL 0 @_-Ri 13024 OOR 12025 OOR 13037 OOR 13C145 1--@C146 OOR 13047 OOR 13048 OOR 1--<04 _;o OOR 13065 OOR i-_@066 OOR 13067 OOR 13068 OOF, 12..069 CIOR 13081 4 6 :Lo 6 9 4 7 l:i 7 2 !t. 7 :1 1 4 11 2 1-@: 2 ci 2 NOI' PREGNf-it-4'1' 6 !fl 5 12 ii_;i 4 t-,-. :ILi NCil PF.,EGI-4Ht.4'1 NOI PF.EGr4Ht4l 5 8 NCil PFEi3NHt.41 ci I 1 4 :?. 12 11 2 NCIT PF,.Ei:jNF4tq"I 2 5 01 1 MEFit4 8 9 7. STFIN. DE@o'. 2. 8 2. 6 4. 0 ci ci C4 t-i ILI 0 0 0 0 0 0 0 0. @-_i 0. 1--1 :1 :li 2 0 :Lj- ci 9 0 :12 0 4 ci :1 12 cli io 1 11 1 0 0 1 1 0 0. 4 0. E. 9 :1 12 1 S. 1 4. I ci :I;a 9 :L2 7 14 5 12 :12 11 8 E. :li 8 9.2 4. @l A 4. 2 4. ;L' 4. 4. 1 4. 4. 8 4. 4 4. 2 4.. 4. 1 -4. 2 :i: !f, S 1. 1 4. C, 1. 2 1. 4 C" 4. 4 4. 4. 5 4. 4. t -'1 4. 4 4. 2 4. 4. 0 4. 6 4. A-. C-i 4. 2 4. 4. 4. 5 4.. 1 'LiL.I -4. 1 4. 23 Appendix Vi (Continued) oral Teratology Study of FC-95 in Rats Individual Litter Data with Pup Weights Dose %-7roup and Rat No. VLHE:LE FETUSES E)EFIL) ti F 1'01'HL FETUSES RESOR PTION SITES IMPLFIN CORPF'FL IHIION LUTEH 51TEE. MEHr-4 FEI*U:=, Fivl:i t-1 1-@ 5 mg/kg/day PiLiF, t'-ci .1i: cici--4 17,@027 F,CIF.'.I CiL.8, F,CiF.' I t-I, F'LiF,' I 1-150 . F',.:)II-*. -1 Fl 1--<Li52 POF 1 4 pof-'.:1 Ci7 @-I PCIR 12071 PLIF: :i Ci 2 F,k-IF.: P ClF-.'I Li'l@.4 F, 1-i@., I CiE.2 12 !:1 4 '-4 4 7 li 14Ci-['PREGNHI-41 8 11 4 8 12 4 7 4 1 F:,i 5 1 ci 4 !fl 9 4 7 11 1-4LI1 PFEi:iNHNI' ti 1,4 4 1 Li t-@. 8 :1.-f ti 4 1? t.4Ci']'F,F,Ei3NFit-JT* r.4cll* F,F'Ef3NFiNl' t-401 F'F:Ei3tqFiNI flEAt-4 5. t:,10. STFit-4.[.',EV. 1. -q 2.0 2. 2 0 ci 0 0 0 Ci 0 0 0 @_--i @-i 0 0 0 1-@ 0 ci ci Cl.0 9 :12 :12 0 9 0 :1:1 2 12 0 7 1 14 0 10 :1 10 2 1:; 0 :14 C-i 10 2 15 1 :1ci 0 9 9 :12 8 12 1 ci :L 1 @'4 14 10 @'4 12 14 li 14 9 9 4. 4. e. 4. 4 4. 4. 4 4. 0 4. ci 4. :1 4. 4. 0 4. 4. e. 4. ti 4. 4 1 .0 4. 4. 1 1. 4 5. .2 el. c4. @* 4. 2 4. 4 4. 4. 4. 4. ;2 9 4. 2 4. 2 4. 2 4. 4 4. 4. 4. 2 4. 7 4. -4 4. 1 4. 2 4. 4. Z: 0. 7 1:1.2 :li.1 4. :1.0 2. 2 2. Ci 0. 24 Appendix VI (Concluded) Oral Teratology Study of FC-95 in Rats Individual Litter Data with Pup Weights Dose Group and Rat No. VIHEILE FETU,-z.ES L.,EHLti F IOTFIL FETUSES RESOR PTION SITES I t-IF,LFItC-:4C'F*F'F.".IHIEFtr-F4E I IJ'-:1.,i1 IFIT'ION LLII'Efl Fi@.@G 1.1 F -;IT'ES I mg/kg/day I'@OP I --<@I_i:Il :I ci12 :io 5 E. :1:1 L'iLi 1-.., !E. 7 :12 4 ,-. :Ili (.'!k:iIF:Ci 7 6 :1 :1 :1 2 C"!Lil-.,I Li 4 :1 2 4 E. C.i 5 to :lt.@ C.@'-!I :1 ci@ito 12 7 4 11 C..*It-@ii@':.1-.*.Clt.L,-" fz.''-@!.-!.'1--@057 !f. 6 45 :li 9 f.@--!FI' L,to:i: NCII* PREi3NFINI' 11!ciF., I -_E-l,5 9 G!OR 12075 f-.: C4 E. 4 :lo 6 4 :lo NCII' PREGf-4Fit-4'f' C-1L,F: I --@Cl 5 8 C.,!C' F, 5 5 11-1 CIOR C!CIF' I --@-'Ll 1-4CIT F'F.Ef3NFIP-41' 4 I:L i.Vl MEAN Sl FiN.DEV. 4. 7 I.7 5. 4 1 1 2. :1 2. 8 0 ci ci t_--i C-1 ci 0 0 ci 1 0 0 0 0 0 0 0.1 0. 2 0 10 ci :1:1 0 1 Z-1 t-i :Lo C4 :iz cl 2 0 :1_1" :L:l 0 12 1 12 1 1.-5 1 10 0 :lo 0 10 1 9 0 10 15 Cl.4 Ci.8 1 Cl. 2. 7 8 :12 :1:1 :1.-@ 9 :14 4 :14 8 :1:1 12 12 11 .12 11 i-I 9 1 ci 15 I t_-i9. 2. E@ 4. 4 4. :1 4. 4 4. to 4. 1 1 4. 4 -:1. 4. 4. 1-@ 4. t-i @:1. 0 4. 6 4. 4. 4. 1 9 1. !fl.:1 1.. 4. l --4. 2 4. E: 4. :1 3. 4. '@'t 1.. 4. 2 1. -4. Ci 9 4. :1 4. 4. 2 4. 2 8 4. 1 4. 4 -1. 4 4. E*. 4. 4. 4 4. 1 4. 1 4. 1. t-I 4. 2 0. 4 DISTRIBUTION LIST X. L. Ebbens - J. D. Henderson E. G. Gortner (original +1) F. Keller (QA file) E. G. Lamprecht G. R. Steffen-+ M. T. Case R. A. Nelson-+ E. L. Mutsch R. E. Ober cc: M. T. Case E . G. Gortner (original + 1) F. Keller (QA File) E. G. Lamprecht E. L. Mutsch R. A. Nelson R. E. Ober li. Pearlson G. R. Steffen -o.J. D. Henderson X. L. Ebbens Amendment to the Final Report of the Oral Teratology Study of FC-95 in Rats Experiment No.: 0680TROOOS Issued: 12/18/80 Please add the amended summary, the amended table 5, results and discussion sections to the above report. changed by this amendment to the report. and the amendment to the The study conclusions were E. G. Gortner Senior Research Technologist Animal Teratology Reproduction Date E. G. Lamprecht, DVM, PhD Date Research Veterinary Pathologist bf. T. Case, DVM, PhD Manager, Pathology-Toxicology Safety Evaluation Laboratory Date 26 Amended Summary (P. 1) to the Oral Teratology Study of FC-95 in Rats Experiment No. 0680TROOOS Oral administration of FC-95 at doses of 10, 5 and 1 mg/kg/day to'pregnant Sprague-Dawley' rats during days 6 through 15 of gestation (period of organogenesis) was not teratogenic. FC-95 administration was maternally toxic only to the 10 mg/kg/day group. At gestation days 12 through 20 the maternal body weights of the high dose females were significantly lower than the controls. FC-95 was not maternally toxic to the 5 and I mg/kg/day groups. FC-95 was not embryotoxic and did not affect the ovaries. or reproductive tract contents of the dA-r-. The compound did not produce pLnincrease in the number or proportion of fetal skeleton variations. 27 Amendment to the Results and Discussion Sections (p..3-5) of the Oral Teratology Study of FC-95 in Rats Experiment No. 0680TROO08 (This amendment addresses the last two paragraphs of the results section and the entire discussion section.) FC-95 was labeled a teratogen of the lens because apparent lens abnormalities were observed at the 10, 5 and I mg/kg/day dose levels. Based on subsequent studies, particularly Riker Experiment No. 0681TR0362, the interpretations of these observations have been extensively modified. The lens findings observed under the dissecting microscope are now known to be either freehand sectioning artifacts or a normal area of lens cell degeneration; The fetal rat lens findings were incorrectly interpreted as a teratogenic change in this study. The gross finding of a lens cleft was an artifact created by freehand sectioning. It represents a separation between the embryonal nucleus lens cells and the lens epithelium. The gross finding of a lens dark streak was a normal observation of the embryonal nucleus. The embryonal nucleus is an area of normal lens cell degeneration in the gestation day 20 fetus. The gross appearance of the rat lens at day 20 of gestation is determined by the region of the lens which is transected by freehand sectioning. In a subsequent study (Riker Experiment No. 0681TR0362) the compound-related occurence of the lens findings could not be repeated when the fetuses were coded before freehand sectioning and gross evaluation.- The range of gross lens observations and the differences among the dose group incidences were due to the manner and frequency im which the lens cleft artifact was created by freehand sectioning and the limitations inherent in visualizing the embryonal nucleus. In summary, FC-95 in utero exposed fetuses did not have compound-related changes in their lenses. 28 Amended Table 5 (pe 10) Number oral Teratology Study of FC-95 in Rats and Percent of Fetuses with Internal Findings Internal Finding Lens findings! Thoracic cavity full of blood Enlarged atria Enlarged renal pelvis Abdominal cavity full of blood No. of Fetuses Examined 0 mg/kg/day 1 (2) --- 1 (2) 3 (5) 4 (7) 61 10 mg/kg/day 14 (35)12 1 (3) ----2 (5) 40 5 mg/kg/day 4 (7) --- --5 (9) 55 1 mg/kg/day 2 (3) 3 (5) 2 (3) 59 The lens findings observed under the dissecting microscope were either freehand sectioning artifacts or a normal area of lens cell degeneration Significantly higher than the control (chi-square with Yates correction p < 0.05) percent of total examined DISTR3:BtITION LIST X. L. Ebbens E. G. Gortner (original + 1) P. D. Griffith -@-F. A. Ubel F. Keller (QA File) E. G. Lamprecht R. E. Ober -@-R. A. Nelson -@.E. L. Mutsch W. H. Pearlson G. R. Stef fen -@.J. D. Henderson M. T. Case W. C. McCormick Amnded Appendix VII STATEMENT OF QUALITY ASSURANCE STUDY NU@IBER: Amendment to 0680TROO08 TITLE: Amendment to the Final Report of the Oral Teratology Study of rt-9'5-inRats Audits and/or inspections were performed by the Riker Compliance Audit unit for the above titled study, and reported to the study director and to management as follows: Date Performed Date Reported July 16 and 19, 1982 July 22, 1982 July 21, 1982 July 23, 1982 Complia@nde'Audit Riker Laboratories, Inc. I)afe -.7: 7 DISTRIBUTION LIST X. L. Ebbens - J. D. Henderson E. G. Gortner (original +1) P. Keller (QA file) E. G. Lamprecht G. R. Steffen-- M. T. Case R. A. Nelson E. L. Nutsch R. E. Ober