Document 3eDZVkKO4O00r9n1KxNJpBdjD

oral Terstoloa Study of T-2999C*C in RALbbits Zxperiment No.: Gonducted At: Dosing Period: Study Director: 06SITBO212 Safety Zvaluation Laboratory Fikex 'fa ratorl", Inc* ft. Paul, minnesota May 10, 1961 through June 26, 1981 So Go GortnSr E. G. Gortner Senior Research Technologist Animal Teratology Reproduction Date 3. G. Lamprecht, VM, PhD Date Fase&rZb VeterinarY Pathologist ])VM, Fhv..@ M. T. Canei Safety Evaluation LaboratorZ Date summary The oral administration of T-2999CoC at 0, 15, 5 and 1.5 mg/kg/day to pregnant New Zealand White/Minikin rabbits during gestational days 6 through 18 (period of organogenesis) was not teratogenic. Compound-related teratogenic effects were not seen on gross, internal or skeletal examinations of rabbit fetuses. T-2999CoC was embryotoxic at the 15 mg/kg/day level. This embryotoxicity was seen as a high incidence of abortions or total resorptions, increased numbers of resorption sites and low numbers of viable fetuses. The 15 mg/kg/day fetuses experienced poor neonatal survival during a 24 hour incubation.' The 5 and 1.5 mg/kg/day dose levels were free from the embryotoxicity and poor neonatal survival seen in the higher dose level. T-2999CoC caused maternal toxicity of failure to gain not mean body weight between the initiation of dosing and the termination of the study only in the 15 mg/kg/day level. No compound-related clinical signs were unique to T-2999CoC treatment of pregnant rabbits. Fetal rabbit lenses from all dose groups were evaluated histologically. T-2999CoC did not cause lens abnormalities in fetal rabbits exposed to the compound in utero. 2. Introduction This teratology study-a in rabbits was conducted to evaluate the embryotoxic and teratogenic effects of orally administered T-2999Cocb-. 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, minesota. Two sets of compound administration groups were dosed between May 10 and June 26, 1981. 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 Cperating 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 Sexually mature New Zealand White/minikin female rabbits were obtained from Dutchland Laboratoiies, Inc., Denver, PA, and assigned cages according to a computer-generated random numbers table. The rabbits, ranging in weight from 1601 to 2917 grams, were than divided into four groups of 18 animals each. The rabbits were housed individually in stainless steel cages in a temperature and humidity controlled room. FoodE and water were available ad libitum. The lights were on a 12 hour light/dark cycle. Each female rabbit was injected with 3 mg of pituitary luteinizing hormone Via the ear vein before breeding. The does were then artificially inseminated with 0.5 ml of pooled diluted semen. The day of insemination was designated day 0 of pregnancy. The animals were observed daily from days 3 through 29 of gestation for abnormal clinical signs. Body weights were recorded on gestational days 3, 6, 9, 12, 15, IS and 29. The four groups were dosed with T-2999CoC suspended daily in corn oil at 0, 15, 5 and 1.5 mg/kg/day. The suspensions were administered daily using a constant dose volume of I ml/kg by oral intubation with a syringe and rubber catheter on gestational days 6 through 18. T-2999CoC characterization was provided by 3M Commercial Chemical Division, St. Paul, Minnesota and corn oil clearance was provided by Analytical Chemistry, Riker Laboratories, Inc., St. Paul, Minnesota (Appendix IV)* All surviving animals were euthanatized on gestational day 29. 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, fetal weights and sex, and gross fetal abnormalities. The fetuses were than placed in an incubator at 370 C for a 24-hour incubation period. Following the incubation period, all fetuses were killed and examined for internal abnormalities. The fetuses were then preserved in alcohol for clearing and staining of the skeleton with alizarin red to detect skeletal abnormalities. A total of 458 fetal rabbit eyes were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 5-6 microns, stained with hematoxylin and eosin and evaluated histologically. This was done because a a Riker Experiment Number 0681TBO212 FM-3924 (technical grade FM-3422) Purina Rabbit Chow, Ralston Purina Co., St. Louis, MO 3. lens artifact in rat teratology studies was being evaluated concurrently with this study. The rabbit was used as a second species in case the lens finding was a true abnormality in the rat. Results and Discussion The oral administration of T-2999CoC to pregnant rabbits during the period of organogenesis caused maternal toxicity of low mean body weight gain. The high (15 mg/kg/day) dose does lost significantly more weight than the controls (o mg/kg/day) between days 6 and 9 of the study (Table 1, Appendix V). They also failed to gain net body weight between the first day of dosing and the end of the study. The ulid (5 mg/kg/day) and low (1.5 mg/kg/day) dose group mean maternal body weight gains and losses were never significantly different from the controls group. No compound-related clinical signs were unique to T-2999CoC treatment of rabbits. Maternal deaths occurred during the study. No deaths could be attributed directly to compound-related toxicity, even though the compound caused maternal toxicity of low body weight gain. A compound-related high incidence and percentage of abortions or total resorptions occurred only in the high dose group rabbits; however, the increased incidence was not statistically significant (Table 2). The incidence and percentage of abortions or total resorptions of the mid and low dose groups were not different from the control group. T-2999CoC was embryotoxic at the high dose level. The T-2999CoC treated high dose does had a significantly lower number of viable fetuses and significantly higher number of resorption sites than the control group (Table 3, Appendix VI). The numbers of viable fetuses and resorption sites of the mid and low dose groups were not significantly different from the controls. The following litter data measurements were not affected by compound administration to the does: number of dead fetuses, number of implantation sites, number of corpora lutea and mean fetus weight. The fetuses of T-2999CoC treated high dose does experienced poor neonatal survival. The high dose fetuses had a significantly higher mortality rate than control fetuses (Table 2) during a 24 hour Incubation. Survival of mid and low dose fetuses was comparable to controls. No major gross malformations were observed to be compound-related. from separate control dose group does had clubbed forepaws (Table VII). Small and runted fetuses were seen in the control, mid and groups, but not in the high dose group. No internal abnormalities Two fetuses 4, Appendix low dose were found. The incidences of skeletal findings among fetuses of rabbits receiving T-2999CoC were generally not different from the findings for the control fetuses. The only exception was a significantly higher incidence of fetuses 4. with 13 ribs spurred in the low dose group than in the control group (Table 5, Appendix VIII). The oral administration of T-2999CoC to pregnant rabbits was not teratogenic at the dose levels tested. Nine abnormal lenses were found on histological examination of hematoxylin and eosin stained fetal rabbit eyes. All nine abnormal lenses were from fetuses of control doe NlBll96. The remainder of the fetal lenses either had no significant changes, were not readable, or had autolysis. T-2999CoC did not cause lens abnormalities in fetal rabbits exposed to the compound in utero. 5. -Table 1 4Dral Teratolc>gY Study of T-2999COC in Rabbits Mean Body Weight Gain or Loss (g).of Pregnant Jtabbits Between weighings with Standard Deviations DAY 6 -Q 12 :L5 IS 29 ---------------------------------------------- 0 MgAg/day MEAN STAN. DEV 15 vq/kg/day MERN STAN. DEV 83 -36 -9 47.8 60.4 75.0 56 -1202@ -50 31. 5 67.7 63.4 8 30 234 68.4 92.3ilO.6 1 :LO 1.59 73.5 72. 4:L37. 8 5 mcl/kg/day MEAN STAN. DEV 47 49.3 -48 -24 -23 5 244 48.2 75. 1 68.2 66. 0:1:133. 1.5 mci/kg/day MEAN STAN. DEV 63 -28 25.9 77.0 1 21 55.7 60.1 50 :178 62. 1 59. 1 significantly lower than the control (Dunnett'st test p < 0.05) Dose Group 0 mg/kg/day 15 mg/kg/day 5 mg/kg/day 1.5 mg/kg/day Number of Animals is is 18 18 Table 2 Oral Teratology Study of T-2999CoC in Rabbits Reproductive Performance and Fetal Survival Conception No. Rate % is 83 16 89 16 89 14 78 incidence of Total Abortions or . Resorptions 2/15 7/16 1/16 2/14 24 H mo 13 44 6 14 Significant]Ly higher than the control (Chi-square with Yates correction p < 0.05) Table 3 Oral Teratoloby Study of T-2999CoC in Rabbits Mean Litter Data and Feta:l Weights with Standard Deviations GRP NO. OF VIABLE FETUSES ANIMALS m F TOTAL DEAD FETUSES 0 mgAg/day 12 STAN DEV 4.0 2.7 6.7 :L.8 :L.7 2.3 0.1 0.3 15 mgAg/day 8 STAN DEV 2. e :L.2 :1.5 :L.3 3. !!!, 1. 8 0.4 0.7 5 mg/kg/day 14 STAN DEV 2.9 1. 3 2.8 :1.3 5.6 1. 8 0.0 0.0 1.5 mg/k@;/day :LO STAN DEV 3.2 :1.4 2.6 1.3 5.8 2.3 0.0 0.0 RESORPTION SITES 0.2 0.9 1. 5s 1. 5 0.6 0.8 0.5 JL.0 ITIPLANTATION SITES 7.1 2.:1 5.4 2.1 6.2 2.2 6.3 2. 1 CORPORF LUTER 7.4 1. 7 6.5 1:.6 6.5 1. 5 6.7 1. 3 Significantly different than the control (Dunnett's t test p < 0.05) Table 4 Oral Teratology Study of T-2999COC in Rabbits a Wumber of Fetuses with Gross Findings- Findings 0 mg/kg/ day 15 mg/kg/ day 5 mg/kg/ day .1.5 mg/kg/ day Total Fetuses examined Small Runted Clubbed right foripaw Total normal fetuses Total abnormal fetuses al 28 4 (5) 1 (1) 2 (3) 74 (91) 28 (100) 7 (9) 0 (0) 79 75 (95) 4 (5) 58 53 (91) Treatment groups were not significantly different (Chi-square with Yates correction p< 0.05) percent of total examined from the control group 9. Table 5 Oral Teratology Study of T-2999CoC in Rabbits Fetal Skeletal Findings Findings 0 mg/kg/ day 15 mg/kg/ day 5 mg/kg/ day 1.5 mg/kg/ day Pontanel not closed Frontal not ossified Parietal not ossified Interparietal not ossified Holes in both parietals Holes in one parietal Sternebrae not ossified Sternebrae asymmetrical Sternebrae bipartite Sternebrae fused sternebrae scrambled Sternebrae misshapen one sternebrae missing Extra sternebrae Extra sternebrae not ossified 13 ribs 13 ribs spurred 13 ribs floating Ribs forked Ribs fused 10 ribs Extra ribs Ribs misshapen Vertebrae scrambled Vertebrae fused Vertebrae misshapen Caudalvertebrasecrambled Total Normal Fetuses Total Abnormal Fetuses Total Number Fetuses 14 (17) 10 (12) 10 (12) 2 (3) 3 (4) 3 (4) 23 (28) 7 (9) 3 (4) 4 (5) 2 (3) 4 (5) 1 (1) 2 (3) .9 (11) 6 (7) 5 (6) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 1 (1) 25 (31) 56 (69) 81 4 (14) 7 (25) 7 (25) 2 (7) 2 (7) 6 (21) 2 (7) 1 (4) 1 (4) 4 (14) 5 (18) 1 (4) 8 (29) 20 (71) 28 16 (20) 5 (6) 5 (6) 1 (1) 4 (5) 15 (19) 5 (6) 4 (5) 2 (3) 1 (1) 1 (1) 5 (6) 3 (4) 18 (23) 7 (9) 1 (1) 1 (1) 7 (12) 8 (14) 8 (14) 2 (4) 16 (28) 1 (2) 1 (2) 4 (7) 7 (12) 1A (22) 29 (37) 50 (63) 79 24 (41) 34 (59) 58 Significantly higher than the control (Chi-square with Yates correction p <0.05) percent of total examined 10. Appendix I TITLE: Protocol for Oral Teratology Study of T-2999COClin Rabbits (Riker Experiment Number 0681TBO212). OBJECTIVE: A teratology study will be used to evaluate the embryotoxic and teratogenic effects of orally administered T-2999CoC to pregnant rats during the period of organogenesis. The procedure complies with the general recomendations of the FDA issued in January, 1966 ("Guidelines for Reproduction Studies for Safety Evaluation of Drugs for Human Usem). 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: May, 1981. TEST SYSTEM: Sev6nty-two sexually mature New Zealand White/Minikin rabbits from Dutchland Laboratories, Inc., will be housed in stainless steel cages with wire mesh floors in a temperature and humidity controlled room. This strain of rabbit will be used because historical control data is available. Purina Rabbit 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. RANDOMIZATION: The animals will be assigned cages according to a computer-generated random numbers table. CONTROL ARTICLE: Corn oil. TEST ARTICLE: T-2999CoC. 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. The sponsor will be responsible for retention of test article sample as required in GLP regulations. PM-3924 (technical grade FM-3422) 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 rabbits on days 6 through 18 of gestation according to the following: Dose Level 15 mg/kg/day 5 mg/kg/day 1.5 mg/kg/day 0 mg/kg/day Group Size is 18 18 18 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. on the day of breeding, each female will be given an intravenous injection of 1 mg of pituitary luteinizing hormone to induce ovulation. The does will then be artificially inseminated with 0.5 ml of pooled diluted semen collected from New Zealand White/Minikin male rabbits. A blood sample will be taken pre-dose, day IS and day 29 of gestation from the same six rabbits at each dose level. The samples will be labeled, frozen and transferred to the sponsor for blood levels analyses. The sponsor will be responsible for the analyses of the samples. The animals will be observed daily from day 3 through day 29 of gestation for abnormal clinical signs. Body weights will be recorded on days 3, 6, 9, 12, 15, IS and .29 of pregnancy and the rabbits dosed accordingly using a constant dose volume of I ml/kg of body weight. The females will be killed on day 29 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. The pups will be placed in an incubator at 370 C for a 24 hour survival check. The following day the pups will be terminated and their viscera examined for any internal abnormalities. The eyes will be removed, fixed in buffered formalin for possible processing and histologic examination. The pups will then be fixed in ethyl alcohol for subsequent skeletal examination after clearing and staining with alizarin red. 12. DATA ANALYSIS AND FINAL REPORT: The proposed statistical methods to be used for analysis of the data are: Dunnett's t test for dam andpup 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, 1981). 13. Appendix 11 List of Principal Participating Personnel NAME Edwin G. Gortner Elden G. Lamprecht Gaxy C* Pecore Bud G. Schmidt Loren 0. Wiseth FUNCTION Study Director Veterinary Pathologist Supervisor - Animal Care Riker - Analytical Technician APPENDIX 111 14. STATEMENT OF QUALITY ASSURANCE STUDY NUMBER: 0681TBO212 TITLE: Oral Teratology Study of T 2999CoC in Rabbits 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 14 and 19 May 1981 2 June 1981 12 June 1981 18 June 1981 7 July 1981 7-8 July 1981 28 December 1981 30 December 1981 21 May 1981 3 June 1981 12 June 1981 25 June 1981 9 July 1981 16 July 1981 4 January 1982 4 January 1982 et%m-p'l-i-ancAeu-dit Riker Laboratories, Inc. I)ate 15. p@ibpENDIX rV Test-"/or Control Article for T-2999COC Characterization uniformity, composition, purity or other per- 1. The identity strength# tinent characterizations of the of been dete=Lned and documented Ab5i nces have synthasis or origin of the test and control subst&nc&&*is 2. The Method Of thod of bioassay (if aPPlicILbl*) including their amount and the me documontud- yes -lz, no 3. The stability Of the tast and/or control -m-t"ex. 9 4ILve b4jen dO ter- subst&ncg mined or will be determined as of above The is. information and documentation are located in the sponsor-s re- --@ponsor Data CCD Analytical Reg- NO- 16474 october 28# 1980 Form 1*793PWO ..... 16. Appendix IV (Continued) Oral Teratology Study of T-2999COC in Rabbits Prestudy and Poststudy Analysis of the Corn oil Control Article Analyses Peroxide Value Refractive Index Prestudy 1.2 meq/kilo 1.4734 Poststudy 1.8 meq/kilo 1.4740 17. Appendix V Oral Teratology Study of T-2999CoC in Rabbits Individual Body Weights (g) with Mean Body Weights and Standard Deviations for Pregnant Animals DAY 3 6 9 12 :L5 IS 29 -------------------------------------------------- 0 MG/KG/DAY NIB :L178 2237 2367 23:L3 2369 2450 2399 2661 NIB :L:L79 2279 2447 2359 2392 2292 2474 2758 NIB JL:LBO 2483 22418440 22402215 22483194 223001.66 221402705 22639754 NIB NIB :iial2015 1194 1781 1820 1813 1842 1922 1970 2149 NIB 1195 1710 1768 11597288 11591676 11592422 11594230 12710968 NNIIBB 11119967 12894127 13809393 2973 2731 2705 2560 2939 NIB 1374 2248 2334 2316 2272 2338 2407 2671 NIB 1375 2364 2443 22461205 22451914 22635445 2689o@@ 2882o NIB NIB 1390 2461 2607 1391 2136 2156 2155 2233 2325 2209 2460 NIB 1392 1880 1977 1979 1994 2020 2092 2008 NIB 1393 1935 2011 2049 2006 2061 2071 2256 NIB 1406 20B6 2130 2112 212l 2117 2297 2612 MEAN 2158 2241 2204 2193 2201 2221 2455 STAN. DEV320. 8337. 9342. 8314. 0304. :L301.3355. 9 NON PREGNANT ANIMALS NIB 1210 2038 2015 1971 2084 2117 2139 2277 NIB :L376 1763 1908 1881 1717 1702 1738 1791 NIB 1377 2028 2116 2165 2198 2174 2201 2401 Animal died - intubationerror Appendix V (Continued) oral Teratology Study of T-2999CoC in Rabbits Individual Body Weights (9) with Mean Body Weights and Standard Deviations for Pregnant Animals DAY 3 6 9 :L2 15 :LC- 2-q ------------------------------------------------- :15 MG/KG/DAY rj:LB ()IB 018 018 018 OIB O:LB OiS. 018 OIB oiB OiB OIB OIB OiS OIB :L:LS3 .:1765 :LI84 2209 li85 2127 1198 lego 1199 1826 1200 2055 ':L20:L :LS96 1211 1903 1378 2316 1379 1931 t39e 1805 1381 1958 1394 2008 1395 2001 1396 1953 1407 1724 :LB34 1769 2282 2177 1999 2048 :L991 legg 1890 1737 2102 1967 :L-928 :1671 2018 1943 2338 2158 2006 1864 1998 1797 2eei 1945 2028 1921 2005 1926 1993 1935 1770 1758 1763 213:1 1884 :L901 1686 2008 1561 1940 2030 1824 1686 1870 1789 1835 1968 1745 1716 zo@ 1850 1829 1762 2084 :L6i3 1989 1953 1811 1712 1706 1796 1947 1826 1732 1827 2036 22:L2 .2419 1798 :L594 :1789 :L914 1823 2002 2152 2152 1580 1753 1992 2205 1851 2175 1963 1970 1840 2087 e- o 1676 1395 1949 2216 1896 2109 1742 1754 MEAN 1960 2817 1896 1845 1847 1866 2025 STAN. DEY158. 8150. 6125. 3143. 5157. 2163. 321:L. I NON PREGNANT RNIMRLS OIB 1182 1987 2105 1849 1928 1932 1909 2106 OiB 1397 1690 1788 1638 1559 1519 1531 1752 Animal died 19. Appendix V (Continued) oral Teratology Study of T-2999CoC in Rabbits Individual Body Weights (g) with Mean Body Weights and Standard Deviations for Pregnant Animals DAY 3 6 9 :12 :15 is 29 -- -------- --------------------------------------- 5 MG/KGIDAY P:LB .PIB PIS PIB PIB PiB PIB PIB PIB PIB PIB PIB PIB PIB PIB PIB 1196 :LI87 1188 :LI89 1203 1205 1212 1382 1383 :L384 1385 1398 1399 1400 1401 1409 2147 2607 2367 1679 2351 1783 2119 2107 2158 2042 2147 1703 2168 1829 2521 1868 2266 2673 2388 1950 2394 1824 2206 2165 2200 2065 2178 1782 2183 1913 2591 1763 2216 2526 2297 1884 2379 1795 2183 2051 2106 2029 2165 1770 2137 1905 2505 1808 2087 2596 2344 1948 2266 1795 2223 2011 2001 201S 2130 1739 2061 1868 2364 1909 2122 2459 2327 :L944 2260 te85 2273 1969 1871 2006 2106 1724 1988 1750 2306 1985 2163 2447 2278 :1996 2359 198:t 2301 1917 1732 2091 2118 1793 1917 1820 2243 2000 2494 2841 2603 2:L96 2603 2056 2528 2157 1690 2329 2351 204ib OF2157 2650 2107 MEAN 2112 2159 2110 2085 2061 2066 2320 STAN. DEV260.3270.7240.3227.8215.0217.5301.6 NON PREGNANT ANIMALS P:LB 1202 2073 2147 ea o 0 0 0 PiB 1204 2252 2287 es 0 0 0 0 a Animal died - intubation error Animal died 20. Appendix V (Concluded) Oral Teratology Study of T-2999COC in Rabbits individual Body Weights (g) with Mean Body Weights and Standard Deviations for Pregnant Animals DAY 3 6 9 :L2 :L5 :LS 29 -------------------------------------------------- :L.5 MG/KG/DFi OLB :LI90 2054 2120 2092 2066 2046 2130 2332 QiB 1191 1821 1839 1858 1825 1895 :L914 2157 :L744 :1769 :ieo6 1793 2014 Q:LB :LI92 :1653 1706 :1772 1.796 iS63 9 o OQ:ILBB ::LL220067 1:L793876 12i17g09i68e 2119I1D76 211.89128 21292661 22205302 22510013 1951 2028 2216 QIB 1208 1870 QIB 1213 2111 2162 2084 2079 ;@- o o QIB 1366 1871 1943 1746 2210 1613 2186 2132 2080 2193 0Q1i8B 11338878 22110796 222225110001 22313531 22145314 22158169 22253428 22472422 2088 2273 QQIIBB 11348092 21482282 12879372 12966939 1967eP-2006o 0 0 QIB QIB 1403 1405 2673 2176 2234 2291 2299 2332 2519 2615 MEAN 2035 2098 2069 2022 2077 2147 2324 STAN. DEV276.7280.5266.3233-8210.0230.7224.6 NON PREGNANT ANIMRLS Qle I:L93 1961 1990 1932 1994 1988 1970 2067 QIB 1209 2342 2437 2410 2371 2295 2225 2590 QIB 1404 1857 1892 1956 1873 1883 1912 2093 QIB 1409 1601 1702 1703 1700 1697 1669 1760 a Animal died - intubationerror Animal died 21. Appendix VI oral Teratology study of T-2999COC in Rabbits Individual Litter Data with Mean Fetus weights 0 mg/kg/day 'ANIMAL VIABLE FETUSES DEAD M F TOTAL FETUSES RESUR PTION SITES IMPLAN TATION SITES CORPRA LUTES MEAN AVG NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB 1178 t179 liSO 1181 1194 t195 1196 1197 1210 1374 1375 1376 :L377 1390 1391 1392 1393 1406 LITTER TOTALLY 4 :L 5 42 6 7 :L 8 32 5 30 3 24 6 45 9 NOT PREGNANT 6 4 :LO DEAD NOT PREGNANT NOT PREGNANT 52 7 6 4 io ABORTED 13 4 35 8 REABSORBED 0 0 1 la 0 0 0 0 0 0 0 0 0 5 0 6 0 9 0 5 3 6 0 6 0 9 0 10 0 7 0 io 0 4 0 8 5 i.:L 7 9 8 6 8 6 44.:l 40.0 29.0 38.8 36.0 38.9 34.9 35.3 7 42.3 6 28.6 9 42.4 7 46.1 22. Appendix VI (Continued) Oral Toratology Study of T-2999COC in Rabbits Individual Litter Data with mean Fetus Weicjhts 15 nig/kg/day ANIMRL VIABLE FETUSES DEAD M F TOTAL FETUSES RESUR PTION SITES IMPLAN TATION SITES C-ORPRA LUTER MEAN AVG OIS :LI82 NOT PREGNFKNT 018 :Li83 22 4 0 OIB li84 24 6 0 :L 5 3 9 OJB li85 ABORTED OIB 1198 ABORTED OIB 1199 20 2 2 4 OIB 1200 ABORTED OOIIBB :1L221011 LIT0TER 2 TOTALL2Y REAB2SORBED - 0 4 OIB 1378 OIB 1379 LITTER LITTER TOTALLY TOTALLY REABSORBED REABSORBED OIB 1380 LITTER TOTALLY REABSORBED OIB 1381 DEAD OIB 1394 20 2 0 OIB 1395 42 6 0 DIB 1396 31 4 0 2 4 0 6 4 8 OIB 1397 NOT PREGNRNT OIB 1407 11 2 :L 0 3 6 42.4 6 32.4 6 42.4 7 39. :L 5 40.1 7 28.9 :Lo 28.8 5 24.2 23. Appendix VI (Continued) C)ral Teratology Study of T-2999CoC in PAbbits Individual Litter Data with Mean FetUS WeightS 5 mg/kg/day RNI@1AL VIABLE FETUSES m F TOTAL DEAL) FETUSES RESOR PTION SITES IMPLRN TATION SITES CORPFR LUTER PIB :LI86 4 4 8 0 PIS 1187 4 4 8 0 P:LB 1-tes 3 4 7 0 PIB 1189 1 3 4 0 e8 8 2 10 9 :L a 6 0 4 6 PLB 1202 DEAD PIB 1203 1 1 2 0 0 2 6 PIB :L204 DEAD PIB 1205 3 3 6 0 PIB 1212 4 3 7 0 PIB 1382 4 3 7 0 1 7 6 2 9 6 0 7 8 PIB 1383 LITTER TOTALLY REABSORBED PIB 1384 3 3 6 0 0 6 8 PIB 1385 1 2 3 0 0 3 4 PIB 1398 4 1 5 0 0 5 5 PIB 1399. DEAD PIS 1400 3 2 5 0 PIB 1401 1 5 6 0 PIB 1408 4 1 5 0 1 6 6 0 6 8 1 6 5 MER14 AVG 27.7 36.1 38.1 38.7 46.2 35.8 35.5 26.2 35.4 39.9 35.1 34.:c; 40.3 34.4 24. Appendix Vi (Concluded) Oral Teratology Study of T-2999CoC in Rabbits Individual Litter Data with Mean Fetus weights 1-5 mg/kg/day ANIMAL VIABLE FETUSES M F TOTAL DEAD FETUSES RESOR PTION SITES IMPLAN TRTION SITES CORPRA LUTER GIB 1190 2 1 3 0 Ql.B :L191 2 1 .3 0 Q:LB :L192 3 2 5 0 0 3 5 3 6 '6 0 5 7 QIB 1193 NOT PREGNANT GIB 1206 @DERD GIB 1.207 3 5 8 0 GIB :L208 4 4 8 0 0 8 8 1 9 9 QILB :L209 NOT PREGNANT GIB 1213 4 2 6 0 0 6 7 GIB 1386 DEAD GIB 1387 LITTER TOTALLY REABSORBED GIB 1388 6 3 9 0 0 9 7 GIB 1389 4 4 8 0 0 8 7 GIB 1402 :L 2 3 0 1 4 5 GIB 1403 DEAD Q:LB 1404 NOT PREGNANT GIB 1405 3 2 5 0 0 5 6 QI.B 1409 NOT PREGNANT MEAN AVG 40.0 40.9 35.0 34.1 35.0 35.4 28.9 40.2 47.2 32.6 Appendix VII Oral Teratology Study of T-2999CoC in Rabbits Number of Fetuses by Dam with Gross Findings 0 mg/kg/day Dam No. 1179 1180 1181 1194 1195 1196 1197 1374 1390 1391 1393 1 Findings Total Fetuses examined small Runted Clubbed right forepaw Total normal fetuses Total abnormal fetuses 5 6 8 1 5 3 6 9 10 1 1 7 10 4 4 5 5 8 4 2 6 9 10 7 6 4 0 1 0 1 1 0 0 0 0 4 0 Appendix VII (continued) Oral Teratology study of T-2999CoC in Rabbits Number of Fetuses by Dam with Gross Findings 15 mg/kg/day Dam No. Findings Total fetuses examined Total normal fetuses Total abnormal fetuses 1183 1184 1199 1211 1394 1395 1396 1 4 6 2 2 2 6 4 4 6 2 2 2 6 4 0 0 0 0 0 0 0 Appendix VII (Continued) Oral Teratology Study of T-2999CoC in Rabbits Number of Fetuses by Dam with Gross Findings 5 mg/kg/day Dam No. Findings Total fetuses Small Runted examined Total normal fetuses Total abnormal fetuses 1186 1187 1188 1189 1203 1205 1212 1382 1384 1385 1398 14 8 8 7 4 2 6 7 7 6 3 5 1 1 1 1 8 7 7 4 2 6 5 6 6 3 5 0 1 0 0 0 0 2 1 0 0 0 Appendix VII (Concluded) Oral Teratology Study of T-2999CQC in Rabbits Number of Fetuses by Dam with Gross Findings 1.5 mg/kg/day Dam No. Findings Total Fetuses Small Runted examined Total normal fetuses Total abnormal fetuses 1190 1191 1192 1207 1208 1213 1388 1389 1402 14 3 3 5 a 8 6 9 a 3 1 1 1 1 3 3 4 8 7 5 a a 3 0 0 1 0 1 1 1 0. 0 Appendix VIII oral Teratology Study of T-2999CoC in Rabbits Number of Fetuses by Dam with Skeletal Findings 0 mg/kg/day Dam No. Fontanel not closed Frontal not ossified Parietal not ossified Interparietal not ossified Sternebrae not ossified Sternebrae asymmetrical Sternebrae bipartite Sternebrae fused Sternebrae scrambled Sternebrae misshapen one sternebrae missing Extra sternebrae 13 ribs 13 ribs spurred Ribs fused 10 ribs Extra ribs Ribs misshapen Vertebrae scrambled Vertebrae fused Vertebrae misshapen Caudal vertebrae scrambled Holes in both parietals Holes in one p&Lrietal Total normal fetuses Total abnormal fetuses Total number fetuses 1179 1180 1181 1194 1195 11.96 1197 1374 1390 13 3 4 4 2 1 2 2 2 1 2 2 1 1 6 4 3 5 1 3 1 2 2 1 1 1 1 1 1 1 2 1 1 1 3 1 1 1 1 3 1 1 1 I I 1 1 1 1 1 3 3 3 6 0 3 0 0 3 1 1 2 0 a 2 3 6 6 9 6 5 6 8 5 3 6 9 10 7 Appendix VII (Continued) Oral Teratology Study of T-2999CoC in Rabbits Number of Fetuses by Dam with Skeletal Findings 15 mg/kg/day Dam No. Fontanel not closed Frontal not ossified Parietal not ossified sternebrae not ossified sternebrae asymmetrical Sternebrae bipartite one sternebrae missing 13 ribs 13 ribs spurred 13 ribs floating Holes in both pakietals Holes in one parietal Total normal fetuses Total abnormal fetuses Total number fetuses 1183 1184 1199 12-11 1394 1395 1396 1407 3 1 4 2 1 4 2 2 2 2 1 1 1 I 1 1 2 3 1 1 1 1 1 1 1 3 0 2 2 0 1 0 0 1 6 0 0 2 5 4 2 4 6 2 2 2 6 4 2 Appendix VIII (Continued) Oral Teratology Study of T-2999CoC in Rabbits Number of Fetuses by Dam with Skeletal Findings 5 mg/kg/day Dam No. 1186 1187 1188 1189 1203 1205 1212 i382 1384 1385 1398 1 Fontanel not closed 2 2 Frontal not ossified Parietal not ossified Sternebrae not ossified Sternebrae asymmetrical Sternebrae bipartite Sternebrae fused Sternebrae scrambled Sternebrae misshapen 1 2 2 I 1 1 1 1 Extra sternebrae 13 ribs 4 4 13 ribs spurred 13 ribs floating Ribs forked Holes in both parietals Holes in one parietal 2 Extra sternebrae not ossified 1 1 1 3 2 2 1 2 1 1 2 1 1 2 3 1 3 1 1 1 2 1 1 1 2 1 1 1 1 1 Total normal fetuses 2 3 6 2 2 1 2 3 2 0 2 Total abnormal fetuses Total number fetuses 6 5 1 2 0 5 5 4 4 3 3 a 8 7 4 2 6 7 7 6 3 5 Appendix VIII (Concluded) Oral Teratology Study of T-2999CoC in Rabbits Number of Fetuses by Dam with Skeletal Findings 1.5 mg/kg/day Dam No. Fontanel not closed Frontal not ossified Parietal not ossified Sternebrae not ossified Sternebrae asymmetrical Sternebrae fused Extra sternebrae 13 ribs 13 ribs spurred Holes in both parietals Total normal fetuses Total abnormal fetuses Total number fetuses 1190 1191 1192 1207 1208 1213 1388 1389 1402 14 1 2 2 1 1 2. 5 1 2 5 1 1 3 2 2 3 1 1 1 3 1 1 1 1 1 1 2 1 2 1 3 2 2 1 3 4 5 4 2 2 1 1 2 0 1 3. 4 4 7 7 2 3 3 5 8 8 6 9 8 3 [R E C E IIrVVE D cc: TITLE: M. T. Case E. G. Gortner (original F. Keller E. G. Lamprecht +1) W. C.McCormick R. A. Prokop V. Pothapragada/L. L. o. wiseth T Winter Protocol for Oral Teratology Study of T-2999Co(:@a-in Rabbits (Riker Experiment Number 06SITBO212). OBJECTIVE: A teratology study will be used to evaluate the embryotoxic and teratogenic effects of orally administered T-2999CoC to pregnant rats during the period of organogenesis. The procedure ct-mplies with the general recomendations of the FDA issued in January, 1966 ("Guidelines for Reproduction Studies for Safety Evaluation of Drugs for liuman 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, Inc., St. Paul, Minnesota. Riker Laboratories, STUDY DIRECTOR: E. G. Gortner START OF DOSING: May, 1981. TEST SYSTEM: Seventy-two sexually mature New Zealand White/minikin rabbits from Dutchland Laboratories, Inc., will be housed in stainless steel cages with wire mesh floors in a temperature and humidity controlled room. This strain of rabbit will be used because historical control data is available. Purina Rabbit Chow and water will be available ad libitum. The lights will be on a 12 hour/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 computer-generated random numbers table. to a CONTROL ARTICLE: Corn oil. TEST ARTICLE: T-2999CoC. ANALYTICAL SPECIFICATIONS: The test article, composition and purity will be determined by the Sponsor (3M Counercial Chemical group) prior to the start of the study and at the end of dosing. The sponsor will be responsible for retention of test article sample as required in GLP regulations. 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 rabbits on days 6 through 18 of gestation according to the following: Dose Level 15 mg/kg/day 5 mg/kg/day 1.5 mg/kg/day 0 mg/kg/day Group Size 18 18 18 18 The oral route of administration will be used because toxicity has been defined by this route in a rangefinder st:udy. No dietary contaminants are known to interfere with the test article. on the day of breeding, each female will be given an intravenous injection of 1 mg of pituitary luteinizing hormone to induce ovulation. The does will then be artificially inseminated with 0.5 ml of pooled diluted semen collected from New Zealand White/Minikin male rabbits. A blood sample will be taken pre-dose, day 18 and day 29 of gestation from the same si' x rabbits at each dose level. The samples will be labeled, frozen and transferred to the sponsor for blood levels analyses. The sponsor will be responsible for the analyses of the samples. The animals will be observed daily from day 3 through day 29 of gestation for abnormal clinical signs. Body weights will be recorded on days 3, 6, 9, 12, 15, IS and 29 of pregnancy and the rabbits dosed accordingly using a constant dose volume of 1 ml/kg of body weight. The females will be killed on day 29 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. The pups will be placed in an incubator at 37 C for a 24 hour survival check. The following day the pups will be terminated and their viscera examined for any internal abnormalities. The eyes will be removed, fixed in buffered formlin for possible processing and histologic examination. The pups will then be fixed in ethyl alcohol for subsequent skeletal examination after clearing and staining with alizarin red. 'A 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, 1981). ;@e7 ;3. Gortner Date ior Research Technologist 'nal Teratology-Reproduction .iy Director L'. Case, DVM, PhD Date iger, Pathology-Toxicology !ty Evaluation Laboratory E. G. Lamprecht, DVM, PhD Date Research Veterinary Pathologist W. C. McCormick, f4S Toxicologist Sponsor Representative Da#te SAFETY EVALUATION LABORATORY ANIMAL TOXICITY STUDY PROPOSAL A ttype -7- (compound or material) the study and the study will of4-dtudy) The attached be conducted study is to be conducted, on protocol details the various aspects of according to Good Laboratory Practice regulations. The estimated total cost of the study is $ &to--i and if dosing starts the estimated cost by calendar year is zlo*.,3 ,z /?.P,/ However, it is understood that the sponsoring division will be charged actual time and materials via the 3M recharge system on project number oo31,P1/ The estimated time from the initiation to completion and reporting of the study is 7-? months (NOTE: time estimate includes, as applicable, period between ordering and receipt of animals, prestudy conditioning and examinationg of animals, compound dosing interval, preparation of tissue slides, microscopic examination of tissues, preparation of fetuses, detailed examination of fetuses, compilation and analyses of data, drafting a report, review and final typing of report). Once the study is approved and the compound, with appropi@-ate analytical data, has been received, the animals will be ordered. After this point, cancellation of the study will pose multiple problems because time, space and people have been committed to complete the study. Written confirmation will be necessary for any cancellation and a charge of up to 25% of the remaining estimated cost could be assessed to compensate the testing laboratory. Testing Laboratory Signatures 'Siud@'Director Safety -I-A--7 C4at Evaluation Laboratory Approval Signatures Date Spon so pr, e Date 0 Di;Fision n4 Date