Document X11mX9gM63dMZoxXN2knvbXg

\iro~o2% RECc. Oral Teratology Study of T-2999CoC in Rabbits yJS . %OJ;".. T*^WV/M W Experiment No.: O661TB0212 Conducted At: Safety Evaluation Laboratory Riker Laboratories, Inc. St. Raul, Minnesota - Dosing Period: May 10, 1981 through June 26, 1981 Study Director: S. G. Gortner A C - S*z E. G. Gortner Date Senior Research Technologist Animal Teratology Reproduction TULo '/7/sa. M. T. Case/ DVM, PhD'' Date Safety Evaluation laboratory 1/ 7/92 E. G. Laaprecht, D W , PhD ' Date Research Veterinary Pathologist BEST COPY AVAILABLE 004192 1. 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 net 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. 004193 2. Introduction This teratology study, in rabbits was conducted to evaluate the embryotoxic and teratogenic effects of orally administered T-2999CociU. The studty 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 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 Sexually mature New Zealand White/Minikin female rabbits were obtained from Dutchland Laboratories, 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 of18animals each. The rabbits were housed individually in stainless steel cages in a temperature and humidity controlled room. Food and water were availableadlibitum. 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, 18 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 1 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 37 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 g- Riker Experiment Number 0681TB0212 -- FM-3924 (technical grade FM-3422) -- Purina Rabbit Chow, Ralston Purina Co., St. Louis, MO 004194 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 (0 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 mid (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 V I ) . 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 ltea 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. Two fetuses from separate control dose group does had clubbed forepaws (Table 4, Appendix VII) . Small and runted fetuses were seen in the control, mid and low dose groups, but not in the high dose group. No internal abnormalities 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 004195 4. with 13 ribs spurred in the low dose group than in the control group (Table 5, Appendix VIII). Hie 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 eyeB. All nine abnormal lenses were from fetuses of control doe NIB1196. 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. 004196 .Table 1 Oral Teratology Study of T-2999CoC in Rabbits Mean Body Weight Gain or Loss (g)- of Pregnant -Rabbits Between Weighings with Standard Deviations DRV 6 9 12 15 18 29 0 mgAg/day MERN 83 -36 -9 8 36 234 ST AN. DEV 47. 8 66. 4 75. 0 68. 4 92. 3110. 6 15 mgAg/day ME R N 56 -1205- -56 1 10 159 STRN. DEV 31. 5 67. 7 63. 4 73. 5 72. 4137. 8 5 mgAg/day MERN 47 -48 -24 -23 5 244 STRN. DEV 49. 3 48. 2 75. 1 68. 2 66. 0113. 3 1.5 mgAg/day MEAN 63 -28 1 21 56 178 STRN. DEV 25. 9 77. 0 55. 7 66. 1 62. 1 59. 1 -- Significantly lower than the control (Dunnett's t test p < 0.05) 004197 Dose Group 0 mgAg/day 15 mgA9/day 5 mgAg/day 1.5 mgAg/day Number of Animals 18 18 18 18 Table 2 Oral Teratology Study of T-2999CoC in Babbits Reproductive Performance and Fetal Survival Conception Rate No. % 15 83 16 89 16 89 14 78 Incidence of Total Abortions or Resorptions 2/15 7/16 1/16 2/14 24 Hour Incubation .Mortality Rate % 13 16/81 20 44 14/28- 50 6 14/79 18 14 14/58 24 6Tfr00 -- Significantly higher than the control (Chi-square with Yates correction p <0.05) <n Table 3 Oral Teratoloby Study of T-2999CoC in Rabbits Mean Litter Data and Fetal Weights with Standard Deviations GRP NO. OF VIABLE FETUSES ANIMALS M F TOTAL 0 mg/kg/day 12 STAN DEV 4. 8 2. ? 1. 8 1. 7 6. 7 2. 3 15 mg/kg/day 8 STAN DEV 2. 0* 1. 5 1. 2 1. 3 3.^ 1. 8 5 mqAg/day 14 STAN DEV 2. 9 2. 8 1. 3 1. 3 5. 6 1. 8 1.5 mqAg/day 18 STAN DEV 3. 2 2. 6 1. 4 1. 3 5. 8 2. 3 DEAD RESORPTION FETUSES SITES 0. 1 0. 3 0. 2 0. 9 0. 4 0. 7 1.3* 1. 5 0. O 0. 0 0. 6 0. 8 0. 0 0. 0 0. 5 1. 0 IMPLANTATION CORPORA MEAN WT. SITES LUTEA FETUS<G) 7. 1 2. 1 7. 4 38. 8 1. 7 5. 5 5. 4 2. 1 6. 5 34. 8 1. 6 7. 1 6. 2 6. 5 36. 0 2. 2 1. 5 5. 0 6. 3 2. 1 6. 7 1. 3 36. 9 5. 2 004199 -- Significantly different than the control (Dunnett's t test p <0.05) si Table 4 Oral Teratology Study of T-2999CoC in Rabbits a Number of Fetuses with Gross Findings-- 8. Findings 0 mg/kg/ 15 mg/kg/ day day 5 mg/kg/ day 1.5 mg/kg/ day Total Fetuses examined Small Runted Clubbed right forepaw Total normal fetuses Total abnormal fetuses 81 4 (5) 1 (1) 2 (3) 74 (91) 7 (9) 28 28 (100) 0 (0) 79 2 (3) 2 (3) 75 (95) 4 (5) 58 3 (5) 2 (4) 53 (91) 5 (9) a -- Treatment groups were not significantly different from the control group (Chi-square with Yates correction p < 0.05) ( ) percent of total examined 04200 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/ . 1.5 mg/kg/ day_____ day______ Fontanel not closed Frontal not ossified Parietal not ossified Interparietal not ossified Holes in both parietals Holes in one parietal 14 (17) 10 (12) 10 (12) 2 (3) 3 (4) 3 (4) 4 (14) 7 (25) 7 (25) 2 (7) 2 (7) Sternebrae not ossified Sternebrae asymmetrical Sternebrae bipartite Sternebrae fused Sternebrae scrambled Sternebrae misshapen One sternebrae missing Extra sternebrae Extra sternebrae not ossified 23 (28) 7 (9) 3 (4) 4 (5) 2 (3) 4 (5) 1 (1) 2 (3) 6 (21) 2 (7) 1 (4) 1 (4) 13 ribs 13 ribs spurred 13 ribs floating Ribs forked Ribs fused 10 ribs Extra ribs Ribs misshapen 9 (11) 6 (7) 5 (6) 1 (1) 1 (1) 1 (1) 4 (14) 5 (18) 1 (4) Vertebrae scrambled Vertebrae fused Vertebrae misshapen Caudal vertebrae scrambled 1 (1) 1 (1) 1 (1) 1 (1) Total Normal Fetuses 25 (31) 8 (29) Total Abnormal Fetuses Total Number Fetuses 56 (69) 20 (71) 61 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) 13 (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 004201 10. TITLE: Appendix I a Protocol for Oral Teratology Study of T-2999CoC--in Rabbits (Riker Experiment Number 0681TB0212). 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 recomendation6 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. STUD* 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 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: C o m 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. -- FM-3924 (technical grade FM-3422) 004202 11. 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 study. No dietary contaminants are known to interfere with the test article. On the day of breeding, each female will be given am 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 six rabbits at each dose level. The samples will be labeled, frozen and tremsferred 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, 18 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 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. 004203 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 and pup weights, number of fetuses, number of resorption sites, number of implantation sites and number of corpora lutea; Chi square for percent abnormalities. TOie proposed date for the final report is 2-3 months after detailed pup examinations have been completed (approximately fourth quarter, 1981). 004204 13. Appendix II List of Principal Participating Personnel NAME Edwin G. Gortner Elden G. Lamprecht Gary C. Pecore Bud G. Schmidt Loren 0. Wiseth FUNCTION Study Director Veterinary Pathologist Supervisor - Animal Care Biker - Analytical Technician 004205 APPENDIX III STATEMENT OF QUALITY ASSURANCE STUDY NUMBER: 0681TB0212__________________ 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 004206 rCtepilance Audit Riker Laboratories, Inc. DT-- f --L -f-------------------- APPENDIX IV 15 Test and/or Control Article Characterization for T-2999COC 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 j .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. Xyes no 3. The stability of the test and/or control substances have been deter mined or will be determined as of The above information and documentation are located in the sponsor's re cords . 'Sponsor Date * CCD Analytical Reg. No. 16474 October 28, 1980 I fo rm 1*793 fW O 004207 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 004208 Appendix V Oral Teratology Study of T-2999CoC in Rabbits Individual Body Weights (g) with Mean Body Weights and Standard Deviations for Pregnant Animals DAV 3 6 9 12 15 18 29 0 MG/KG/DAV NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB 1178 2237 2367 2313 2369 2458 2399 2661 1179 2279 2447 2359 2392 2292 2474 2758 1188 2483 2484 2421 2439 2306 2420 2695 1181 2015 2140 2025 2014 2016 2075 2374 1194 1781 1828 1813 1842 1922 1978 2149 1195 1718 1768 1578 1517 1522 1543 1706 1196 1842 1899 1928 1966 1942 1928 2198 1197 2917 3033 2973 2731 2705 2560 2939 1374 2248 2334 2316 2272 2338 2407 2671 1375 2364 2443 2410 2411 2345 5- 1390 2461 2687 2625 2594 2654 2689 2882 1391 2136 2156 2155 2233 2325 2209 2468 1392 1888 1977 1979 1994 2820 2692 2888 1393 1935 2011 2049 2886 2861 2871 2256 I486 2086 2130 2112 2121 2117 2267 2612 MEAN 2158 2241 2204 2193 2201 2221 2455 STAN. DEV328. 8337. 9342. 8314. 8384. 1301. 3355. 9 NON PREGNANT ANIMALS NIB 1218 2038 2015 1971 2084 2117 2139 2277 NIB 1376 1763 1908 1881 1717 1702 1738 1791 NIB 1377 2028 2116 2165 2198 2174 2201 2481 Animal died - intubation error 4209 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 DAV 3 6 9 12 15 18 29 15 MG/KG/DAV 01B OIB 01B OIB OIB OIB OIB OIB OIB OIB OlB OIB OIB OIB OIB OIB 1183 1765 1834 1769 1763 1716 1827 2036 1184 2209 2282 2048 2131 2219 2212 2419 1185 2127 2177 1991 1884 1850 1798 1594 1198 1890 1999 1899 1901 1829 1789 1914 1199 1826 1890 1737 1686 1762 1823 2002 1200 2055 2102 1967 2008 2084 2152 2152 1201 1896 1928 1671 1561 1613 1580 1753 1211 1903 2018 1943 1940 1988 1992 2205 1378 2316 2338 2158 2030 1953 1851 2175 1379 1931 2006 1864 1824 1811 1863 1970 1380 1805 1898 1797 1686 1712 1840 2087 1381 1958 2001 1945 1870 1706 02 0 1394 20O8 2028 1921 1789 1796 1676 1995 1395 2001 2805 1926 1835 1947 1949 2216 1396 1953 1993 1935 1868 1826 1896 2109 1407 1724 1770 1758 1745 1732 1742 1754 MEAN I960 2017 1896 1845 184? 1866 2025 STAN. DEV158. 8150. 6125. 3143. 515?. 2163. 3211. 1 NON PREGNANT ANIMALS 01B 1182 1987 2105 1849 1928 1932 1908 2106 01B 1397 1690 1788 1638 1559 1519 1531 1752 - Animal died ocmio 18. 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 DRV 3 6 9 12 15 18 29 5 MG/KG/DAV P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B 1186 2147 2266 2216 2087 2122 2163 2494 1187 2607 2673 2526 2596 2459 2447 2841 1188 2367 2288 2297 2344 2327 2278 2603 1189 1879 1950 1884 1948 1944 1996 2196 1203 2251 2394 2379 2266 226G 2359 2603 1205 1783 1824 1795 1795 1885 1881 2056 1212 2119 2206 2183 2223 2273 2301 2528 1382 2107 2165 2051 2011 1969 1917 2157 1383 2158 2200 2106 2001 1871 1732 1690 1384 2042 2065 2029 2018 2006 2091 2329 1285 2147 2178 2165 2130 2106 2118 2351 1298 1703 1782 1770 1 7 3 9 1724 1793 2 41. 1399 2168 2183 2137 2061 1988 1917 1400 1829 1912 1905 1868 1750 1820 2157 1401 2521 2591 2505 2364 2306 2243 2650 1408 1868 1762 1808 1909 1985 2000 2107 MEAN 2112 2159 2110 2085 2061 2066 2320 STfiN. DEV268. 2270. 7240. 3227. 8215. 0217. 5301. 6 NON PREGNANT ANIMALS P1B 120 2 2073 2147 02. 0 0 0 0 P1B 1204 2252 2287 0* 0 0 0 0 19 Ul Animal died - intubation error Animal died 004211 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 12 15 IS 29 1. 5 M/KG/DA Q1B Q1B Q1B Q1B Q1B Q1B GIB GIB Q1B GIB Q1B GIB GIB GIB 1190 2854 2120 2092 2066 2046 2130 2332 1191 1821 1839 1858 1825 1895 1914 2157 1192 1653 1706 1744 1769 1806 1793 2014 1206 1737 1798 1772 1796 1863 0P 0 1207 1986 2106 2106 2182 2226 2250 2501 1288 1870 1918 1917 1918 1961 2832 2103 1213 2111 2162 2084 2079 1952. 2028 2216 1 j86 1871 1943 1746 1613 # 0 0 1387 2109 2218 2210 2186 2132 2080 2193 1388 2176 2218 2151 2151 2186 2232 2422 1389 2428 2581 2333 2434 2519 2548 2742 1482 1822 1897 1963 1967 2006 2088 2273 1403 2673 2732 2699 0~ 0 0 0 1405 2176 2234 2291 2299 2332 2519 2615 MEAN 2835 2098 2869 2822 2077 2147 2324 STAN. DEV276. 7286. 5266. 3233. 8210. 0230. 7224. 6 NON PREGNANT ANIMALS Q1B Q1B Q1B GIB 1193 1961 1990 1932 1994 1988 1970 2867 1209 2342 2437 2410 2371 2295 2225 2598 1404 1857 1892 1856 1873 1883 1912 2093 1489 1601 1702 1703 1788 1697 1669 1768 Ul Animal died - intubation error Animal died 004212 Appendix VI Oral Teratology Study of T-2999CoC in Rabbits Individual Litter Data with Mean Fetus Weights 21. 0 mgAg/day ANIMAL VIABLE FETUSES DEAD M F TOTAL FETUSES RESOR IMPLAN CORPRA PTION T A T ION LUTEA SITES SITES MEAN AVG NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB NIB 1178 1179 1180 1181 1194 1195 1196 1197 1218 1374 1375 1376 1377 1390 1391 1392 1393 1406 LITTER TOTALLY REABSORBED 41 5 0 42 6 0 71 8 1 32 5 0 30 3 0 24 6 0 45 9 0 NOT PREGNANT 6 4 10 0 DEAD NOT PREGNANT NOT PREGNANT 52 7 0 6 4 10 0 ABORTED 13 4 0 35 8 0 0 0 0 0 3 0 0 0 0 0 0 0 5 6 9 5 6 6 9 10 7 10 4 8 5 44. 1 11 40. 0 7 29. 0 9 38. 8 8 36. 0 6 38. 9 8 34. 9 6 35. 3 7 42. 3 6 28. 6 9 42. 4 7 46. 1 004213 Appendix VI (Continued) Oral Teratology Study of T-2999CoC in Rabbits Individual U t t e r Data with Mean Fetus Heights 22 15 mgAg/day ANIMAL VIABLE FETUSES DEAD M F TOTAL FETUSES RESOR IMPLAN CORPRA PTION TATION LUTEA SITES SITES MEAN AVG 01B 1182 NOT PREGNANT 01B 1182 22 4 0 15 6 42. 4 01B 1184 24 6 0 39 6 32. 4 01B 1185 ABORTED 01B 1198 ABORTED 01B 1199 20 2 0 24 6 42. 4 01B 1260 ABORTED 01B 1201 LITTER TOTALLY REABSORBED 01B 1211 02 2 2 04 7 29. 1 01B 1378 LITTER TOTALLY REABSORBED 01B 1379 LITTER TOTALLY REABSORBED 01B 1380 LITTER TOTALLY REABSORBED 01B 1381 DEAD 01B 1294 28 2 0 24 5 40. 1 01B 1295 42 6 0 06 7 28. 9 01B 1396 3 14 0 4 8 10 28. 8 01B 1297 NOT PREGNANT 01B 1407 112 1 03 5 24. 2 004214 23. Appendix VI (Continued) Oral Teratology Study of T-2999CoC in Rabbits Individual Litter Data with Mean Fetus Heights 5 mgAg/day RNIMRL VIRBLE FETUSES DEAD M F TOTAL FETUSES RESOR IMPLAN CORPRA PTION TATION LUTEA SITES SITES MEAN AVG P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B P1B 1186 1187 1188 1189 1202 1283 1204 1205 1212 1382 1383 1384 1385 1398 1399 1400 1401 1408 44 8 0 44 8 0 34 7 0 13 4 0 DEAD 11 2 0 DEAD 33 6 0 43 7 0 43 7 0 LITTER TOTALLV REABSORBED 33 6 0 12 3 0 41 5 0 DEAD 32 5 0 15 6 0 41 5 0 0 2 1 0 0 1 2 0 0 0 0 1 0 1 8 10 8 4 2 7 9 7 6 3 5 6 6 6 8 27. 7 9 36. 1 6 38. 1 6 38. 7 6 46. 2 6 35. 8 6 35. 5 8 26. 2 8 35. 4 4 39. 9 5 35. 1 6 34. 3 8 40. 3 5 34. 4 042l5 Appendix VI (Concluded) Oral Teratology Study of T-2999CoC In Babbits Individual Litter Data with Mean Fetus Weights 24. 1.5 mg/kg/day ANIMAL VIABLE FETUSES DEAD M F TOTAL FETUSES RESOR IMPLAN CORPRfi PTION TATION LUTEA SITES SITES MEAN AVG Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B Q1B 1196 1191 1192 1193 1206 1207 1208 1209 1213 1386 1387 1388 1389 1402 1403 1404 1405 1409 21 3 0 21 3 0 32 5 0 NOT PREGNANT DEAD 35 8 0 44 8 0 NOT PREGNANT 42 6 0 DEAD LITTER TOTALLY REABSORBED 63 9 0 44 8 0 12 3 0 DEAD NOT PREGNANT 32 5 0 NOT PREGNANT 0 3 0 0 1 0 0 0 1 0 3 6 5 8 9 6 9 8 4 5 5 40. 0 6 40. 9 7 35. 0 8 34. 1 9 35. 0 7 35. 4 7 28. 9 7 40. 2 5 47. 2 6 32. 6 004216 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 1406 Findings Total Fetuses examined 5 6 8 5 3 6 9 10 7 10 4 8 Small 4 Runted 1 Clubbed right forepaw 1 I Total normal fetuses 5 5 8 4 2 6 9 10 7 6 4 8 Total abnormal fetuses 0 10110000400 004217 wto 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 1407 46222642 46 2 2 26 4 2 00000000 004218 to c* Appendix VII (Continued) Oral Teratology Study of T-2999CoC in Rabbits Number of Fetuses by Dam with Gross Findings 5 mgAg/day Dam No. Findings Total fetuses examined Small Runted Total normal fetuses Total abnormal fetuses 1186 1187 1188 1189 1203 1205 1212 1382 1384 1385 1398 1400 1401 1408 887426776 35565 11 11 8 77426566 35565 0 10000 2 10 00000 004219 to vj Appendix VII (Concluded) Oral Teratology Study of T-2999CoC in Rabbits Nuniber of Fetuses by Dam with Gross Findings 1.5 mg/kg/day Dam No. Findings Total Fetuses examined Small Runted Total normal fetuses Total abnormal fetuses 1190 1191 1192 1207 1208 1213 1388 1389 1402 1405 335886 9 8 35 11 1 11 3348 7 5 88 34 0 0 1 0 1 1 1 0. 0 1 004220 to oo XZZ^o0 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 parietal Total normal fetuses Total abnormal fetuses Total number fetuses 1179 1180 1181 1194 1195 1196 1197 1374 1390 1391 1393 1406 3 443 21 2 2 3 21 2 2 3 11 6 435311 1312 21 1 111 11 211 1 1 311 1 1 12 11 112 131 1 1 1 1 1 1 1 3 3 360 300 311224 208236696824 5 6 8 5 3 6 9 10 7 10 4 8 to vo <N CN 004222 Appendix VII (Continued) Oral Teratology Study of T-2999CoC in Rabbits Number of Fetuses by Dam with Skeletal Findings 15 mgAg/day Dam No. 1183 1184 1199 1211 1394 1395 1396 1407 Fontanel not closed Frontal not ossified Parietal not ossified Stemebrae not ossified Sternebrae asymmetrical Stemebrae bipartite One stemebrae missing 13 ribs 13 ribs spurred 13 ribs floating Holes in both parietals Holes in one parietal 1 2 1 3 1 3 1 42 14 2 11 1 1 12 11 11 11 Total normal fetuses 30 220 10 0 Total abnormal fetuses 1600 254 2 Total number fetuses >4 6 2 2 2 6 4 2 IaJ o 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 1382 1384 1385 1398 1400 1401 1408 Fontanel not closed 22 Frontal not ossified Parietal not ossified Stemebrae not ossified 1 1 Stemebrae asymmetrical 2 2 Stemebrae bipartite 11 Stemebrae fused 11 Stemebrae scrambled 1 Stemebrae misshapen Extra stemebrae 13 ribs 44 11 13 ribs spurred 13 ribs floating Ribs forked Holes in both parietals Holes in one parietal 2 Extra stemebrae not ossified 32 2 131 1 21 1 1 21 1 1231 3111 1 11 21 1 21 1 1 1 113 122 111 1 1 1 3 Total normal fetuses 2362212320 2112 Total abnormal fetuses 6 5120 5544 3345 3 Total number fetuses 88 7426 776 3556 5 004223 U) M 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 1405 1 2211 1 2. 5 125 1 1322 313 1 1 31 1 11112 12132 4 2 134 54 22111 20 13447724 335e8698 35 004224 w fo r e c e iv e d M c c : M. T. Case W. C. McCormick E. G. Gortner (original +1) R. A. Prokop F. Keller V. Pothapragada/L. Winter E. G. Lamprecht L. 0. Wiseth T>\ TITLE: Protocol for Oral Teratology Study of T-2999CoC--'in Rabbits (Riker Experiment Number 0681TB0212). 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 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: 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 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. # 004225 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 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 18 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, 18 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 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. 004226 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). 3. Gortner Date ior Research Technologist nal Teratology-Reproduction iy Director L'. Case, DVM, PhD Dattie iger, Pathology-Toxicology jty Evaluation Laboratory E. G. Lamprecht, DVM, PhD Date Research Veterinary Pathologist Toxicologist Sponsor Representative 004227 SAFETY EVALUATION LABORATORY ANIMAL TOXICITY STUDY PROPOSAL (type of^tudy) study is to be conducted on 7~ & c . The attached protocol details the various aspects of (compound or material) the study and the study will be conducted according to Good Laboratory Practice regulations. The estimated total cost of the study is $ 2/.* o - Q ____ and if dosing starts fKti. /fie/_____ , the estimated cost by calendar year is $ /9*/ _________________. However, it is understood that the sponsoring division will be charged actual time and materials via the 3M recharge system on project number noo 3/ . The estimated time from the initiation to completion and reporting of the study is 7~7 months (NOTE: time estimate includes, as applicable period between ordering and receipt of animals, prestudy conditioning and examination^ of animals, compound dosing interval, preparation of tissue slides, microscopic examina tion 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 appropriate analytical data, has been received, the animals will be ordered. After this point, cancellation of the study wil] 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 Study Director Approval Signatures Date " v/Wr/ 'Date Safety Evaluation Laboratory Date 004228 Date