Document E644MLoXvrvKDwkM04BXmB2b

Two Week Oral Rangefinding Toxicity Study of T-2509CoC in Rats Experiment No.: Dosing Started: Recovery Intervals Completed: Study Director: Testing Facility: Sponsor: 179RRO23 October 3, 1979 November 15, 1979 G. R. Steffen, BS Safety Evaluation Laboratory Riker Laboratories, Inc., .St. Paul, Minnesota W. C. McCormick Toxicology Services 3M Company St. Paul, Minnesota G. R. St,4iffen,/YS Senior Toxicofo-gist Study Director Date x;@ol@rlec) M. T. Case, DVM, PhD Date Manager, Patholocjy-Toxicology Safety Evaluation Laboratory TABLE OF CONTENTS Page 1 Sunanary . . . . . . . . . . . . . . . . . . . . . . . . . . . Introduction .. . . . .. . . .. . . . .. . . .. . . . . 2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . 22 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . 23 Summary The oral rangefinder toxicity study of T-2509CoC in rats was completed in two parts. In Part I the acute oral ALD50 was defined in male CD rats. Six groups having six rats/group, 51-59 days of age when dosed, were administered single doses ranging from 250-10,000 mg/kg. In Part II, T-2509CoC was administered by daily gastric intubation to male and female CD rats. Twelve rats/sex/group, 51 days of age on dose day 1, were dosed at 200 or 75 mgAg/day. Dosing continued for five days at the high level (200 mg/kg/day) and for nine days at the low level (75 mgAg/day). All surviving high dose animals and half of the low dose group had a 14-15 day recovery interval following dosing. Selected low dose animals were euthanatized on day 10. All surviving animals in both groups were euthanatized at the end of the recovery interval. Brain, stomach, liver, adrenal and kidney tissues were evaluated microscopically. The acute oral ALD50 of T-2509CoC in male rats was 878 mgag with 95% confidence limits of 558-1399 mgag. Gastrointestinal irritation was the most common effect caused by high acute doses. Handling-precipitated convulsions occurred in some dose groups 2-7 days following dosing. The lowest dose group (250 mgag) was free of treatment-related effects. Daily gastric intubation of rats with T-2509CoC resulted in treatment-, dosageand sex-related effects. The primary effects were gastric irritation, handlingprecipitated convulsions and microscopic liver changes. These effects contributed to dosage-related deaths. Secondary effects at both levels were a tucked-up appearance, an unkempt appearance, soft feces, body weight loss during the dosing period, head tremors and hyperactivity. The high dose group, but not the low dose group, had histologic evidence of gastric irritation. There were no microscopic changes in the brain sections from either level to correlate with the CNS effects. The treatrnent-related liver effects consisted of focal necrosis and fatty changes. The males generally had earlier occurring deaths, more convulsive episodes and more animals with liver changes. Reversal of the toxic effects occurred after dosing stopped. Severe delayed CNS effects did not occur during recovery. The gastric lesions and other secondary effects subsided during recovery. All recovery animals had weight gain. The recovery animals did have mild fatty liver changes but not liver necrosis. 2 Introduction The oral rangefinder toxicity study of T-2509CoC in rats was completed in two parts. Part I evaluated the acute oral ALD50 in male rats. Part II evaluated the toxic effects and recovery following daily oral dosing in male and female rats using dose levels selected from the results in Part I. Toxicology Services, 3M Company, St. Paul, Minnesota was the study sponsor. The study was conducted by the Safety Evaluation Laboratory, Riker Laboratories, Inc., St. Paul, Minnesota. Acute dosing started on October 3, 1979 and the repeat dose portion was completed on November 15, 1979. The protocol with amendments and a list of principal participants and supervisory personnel are found in Appendices I and II respectively. Since the study was a rangefinder experiment, it was excluded from Good Laboratory Practice (GLP) regulations, however, the study was conducted in accordance with our Standard Operating Procedures which incorporate GLP requirements. The storage location for specimens, raw data and final report is maintained in the record archives for the Safety Evaluation Laboratory. Methods Part I: The approximate oral ALD50 was determined in male CD rats obtained from Charles River Laboratories, Inc. The rats were conditioned in-house for approximately one week prior to dosing. They were housed six/wire-top cage with food and water available ad libitum. Animal identification was bv individual numbers and a color coding system. The animal housing room was temperature and humidity controlled and had a 12 hour light/dark cycle. Six rats/dose group were selected from the conditioned animals. The animals were 51-59 days old and weighed 179-262 grams when dosed. A constant dose volume of 10 ml/kg was used at each level. Distilled water was used to dilute the test article to the appropriate concentrations. The animals were observed 3-6 hours following dosing and daily thereafter for 14 to 20 days. Body weights were obtained just prior to dosing and on the day of necropsy. Gross necropsy abnormalities were recorded for all animals that died or were killed at the end of the observation period but tissues were not saved. Part II: Male and female CD rats were obtained from Charles River Laboratories, Inc. and conditioned in-house for two weeks prior to dosing. The animals were individually housed in hanging cages having wire mesh fronts and bottoms. Food and water were available ad libitum. Animal identification was by individual ear tags and cage cards. The room was temperature and humidity controlled and. had a 12 hour light/dark cycle. 3 Twelve rats/sex/level were selected from the conditioned animals and assigned to the 200 or 75 mg/kg/day group. On day 1 the animals were 51 days old and the males weighed 154-211 grams and the females weighed 144-185 grains. The test article solutions were prepared daily using distilled water as the diluent. A constant dose volume of 10 mlag was used for each dose level. Dosing continued for five days at the high dose level (200 mg/kg/day) and for nine days at the low dose level (75 mg/kg/day). The low dose group had six animals/sex retained for recovery after dose day 9 and the remaining animals were euthanatized on day 10. All surviving animals in the high dose group were retained for recovery after dosing stopped. The recovery interval was 14 days for the low dose group and 15 days for the high dose group. The animals were observed daily for signs of toxicity. Body weights were obtained predose, biweekly during dosing and once or twice a week during recovery. All animals that died or were euthanatized were subjected to a gross necropsy and tissues were saved for histopathological evaluation. The dose levels for Part II were selected at the completion of Part I. The 75 mgAg/day level for the repeat dose portion of the study was selected to deliver a cummulative 14 day dose approximately equal to the ALD50 value. The 200 mgAg/day level was selected to be close to the acute no-effect level and to deliver a cummulative 14 day dose at least two times greater than the ALD50 value. Results Part 1: The acute oral ALD50 of T-2509CoC in male rats was 878 mgag 95% confidence limits of 558 to 1399 mg/kg. having Dosage-related gastrointestinal toxicity was the primary T-2509CoC effect. Clinical signs included a tucked-up appearance, soft feces/diarrhea and hyporeactivity/hypoactivity (Table 1). Most deaths at the higher levels occurred overnight on the day of dosing (found dead on study day 2) while those at the lower levels were delayed through study day 8 (Table 2). Most animals found dead had gross evidence of reddened or hemorrhagic gastrointestinal mucosa (Table 3). Delayed CNS toxicity was evident at some dosage levels. One animal at each of the 5000, 2000 and 1000 mgag levels had clonic/tonic convulsions on days 6, 2 or 7 respectively. Each episode was precipitated by handling and the animals were found dead within 24 hours. Hypereactivity occurred three to nine days following dosing at the 2000, 1000 and 500 mgag levels but these animals recovered. The observation period was extended to 20 days at the 1000 and 500 mg/kg levels because of the delayed deaths but no additional effects occurred. The lowest dose group (250 mgag) was free of treatment-related effects and these animals showed normal weight gain. All animals at each dose level that survived for the duration of the study had acceptable weight gain and were free of obvious gross lesions at necropsy. observation Appeared normal Salivation Lacrimation Hyporeactive Ptosis Diarrhea Chromodacryorrhea Hypoactive Tucked-up abdomen Deaths NE = Not evident Table 1 Acute oral Toxicity Study of T-2509CoC in Male Rats 10,000 mg/kg Group Number of Animals Affected Hours 1 1-2 2-4 4-6 2 3 2 NE 3 2 NE NE NE 2 2 1 NE 2 4 NE NE NE 5 4 NE NE 5 4 NE NE 1 NE NE NE 3 4 NE NE NE 1 0 0 1 0 observation Period Days 3 4 5 6 7 8 9 10 - - - - - - - - 5 (overnight) Table I (continued) Acute Oral Toxicity Study of T-2509CoC in Male Rats 5000 mg/kg Number of Animals Affected observation Hours 0-1 1-2 2-4 4-6 Appeared normal 4 4 1 1 Salivation 3 1 NE 1 Bloody nares & mouth 2 NE NE 1 Hyporeactive 2 1 5 5 Dyspnea Soft feces 1 NE NE NE NE 1 5 5 Ptosis Tucked-up abdomen NE 1 1 '1 NE NE 2 3 Clonic/tonic convulsion NE NE NE NE Deaths 0 0 0 0 observation Period Days 2 3 4 5 6 7 8 9 10 2 2 2 2 1 NE - - - NE NE NE NE NE NE - - - NE NE NE NE NE NE - - - NE NE NE NE NE NE - - - NE NE NE NE NE NE - - - NE NE NE NE NE NE - - - NE NE NE NE NE NE - - - NE NE NE NE NE NE - - - NE NE NE NE l@!@ NE - - - 3 1 0 0 1 1 NE = Not evident 2@Precipitated by handling Table I (continued) Acute Oral Toxicity Study of T-2509CoC in Male Rats 2000 mgag Number of Animals Affected Observation Hours 0-1 1-2 2-4 4-6 Appeared normal 6 6 6 Hyporeactive NE NE 1 Dyspnea NE NE 1 Bloody nares NE NE NE Tucked-up abdomen NE NE NE Clonic/tonic convulsion NE NE NE Soft feces NE NE NE Ptosis NE NE NE Hypereactive NE NE NE Thin NE NE NE Hypoactive NE NE NE Deaths 0 0 0 Observation Period Days 2 3 4 5 6 7 8 9 10 NE 2 2 NE NE NE NE - - 4 NE NE NE NE NE NE - - NE NE NE NE NE NE NE - - 2 NE NE NE NE NE NE - - 3 NE NE 1 1 1 NE - - a 1 NE NE NE NE NE NE - - 1 NE NE NE NE NE NE - - NE NE NE 1 NE NE NE - - NE 1 NE NE NE NE NE - - NE NE NE 2 1 1 NE - - NE NE NE 1 NE NE NE - - 3 0 1 0 1 0 1 NE = Not evident 2@Precipitated by handling Table 1 (continued) Acute Oral Toxicity Study of T-2509CoC in Male Rats 1000 mg/kg Number of Animals Affected observation 0-1 Appeared normal 6 Clonic/tonic convulsion NE Thin NE Hypereactive NE Tremors NE :Deaths 0 Hours 1-2 2-4 4-6 6 6 6 NF, NE NE NE NE NE NE NE NE NE NE NE 0 0 Observation Period Days 2 3 4 5 6 7 8 9 10 11 6 6 6 5 4 3 NE NE 3 3 NE NE NE NE -NE 1a NE NE NE N NE NE NE NE NE 1 NE NE NE N NE NE NE NE NE NE 3 3 NE N NE NE NE NE NE NE 1 1 NE 0 0 0 1 1 0 1 0 0 0 NE = Not evident a Precipitated by handling Observation Appeared normal Hypereactive Deaths NE = Not evident Observation Appeared normal Deaths Hours 0-1 1-2 2-4 6 6 6 NE NE NE 0 0 0 Table I (concluded) Acute Oral Toxicity Study of T-2509CoC in Male Rats 500 MgAg Number of Animals Affected observation Period -Days --6 2 3 4 5 6 7 8 9 10 6 6 6 6 6 5 5 NE NE 5 NE NE NE NE NE NE NE 5 5 NE 0 0 0 0 0 1 0 0 0 0 250 mgag Number of Animals Affected Hours 0-1 1-2 2-4 4-6 Observation Period Days 3 4 5 6 7 8 9 10 6 6 6 6 6 6 6 6 6 6 6 6 0 0 0 0 0 0 0 0 0 0 0 0 9 Table 2 Acute Oral Toxicity Study of T-2509CoC in Male Rats Death Pattern Dose Level Deaths/Day(s) 1 2 3 4 5 6 7 8 9-Term 10000 mg/kg' 5000 mg/kg' 2000 mg/kg 1000 mg/kg 500 mg/kg 250 mg/kg 1 5 --- --- --- --- --- --- 0 3 1 0 0 1 1 --- 0 3 0 1 0 1 0 1 0 0 0 0 1 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 ALD50 = 878 mg/kg b 95% Confidence Limits = 558 to 1399 mg/kg- ------- 0 0 0 Total Deaths No. Dosed 6/6 6/6 6/6 3/6 1/6 0/6 a Data from this level not used for ALD50 calculations computations by Duluth Probit Analysis, Enviroranental Research Program Laboratory Gross Observation No. Dead/No. Dosed No Visible Lesions Euthanatized Found Dead Reddened Stomach Mucosa Hemorrhagic Stomach Mucosa Hemorrhagic Small Intestinal Mucosa Cannabilized Table 3 Acute Oral Toxicity Study of T-2509CoC in Male Rats Necropsy Results Number of Animals Affected 10,000 6/6 Dose Group (mgag) 5000 2000 6/6 6/6 0 0 0 0 2 1 4 2 2 2 2 12@ 0 0 2 0 0 0 1000 3/6 3 1 0 1 0 1 2@With possible ulcer Part II: Treatment- and dosage-related gastric irritation was the most prominant effect caused by daily oral administration of T-2509CoC. The high dose group, but not the low dose group, had histologic evidence of gastric irritation. This irritation was present in 11 of 12 males and 8 of 12 females (Table 4). In some instances early hemorrhagic gastric ulcers were found. Clinical signs associated with gastric irritation were present in both dose groups and included a dosage-related tucked-up appearance, dosage-related soft feces for the male only and an unkempt appearance more common in the low dose group than the high dose group (Tables 5 and 6). Better survival at the low dose level no doubt allowed greater expression of a secondary effect, such as an unkempt appearance. Abdominal pain (tucked-up appearance) continued for the first few days of recovery. The surviving high dose animals and some low dose animals appeared unkempt throughout the recovery interval. Dosage-related CNS effects occurred at both treatment levels. These effects were expressed as convulsions and isolated instances of head tremors and hyperactivity. Histopathological brain lesions were not found in animals at either level. The brain sections were obtained at the fore-, mid and hind brain levels. Tonic or clonic/tonic convulsions occurred in twice the number of males compared to females: six high dose males, three high dose females, two low dose males and one low dose female. The less severe CNS effects, head tremors or hyperactivity, occurred in only one animal in each dose group except that no high dose males had these signs. One low dose female continued to be hyperactive during the recovery interval. The convulsive episodes were generally precipitated by handling and did not increase in frequency after dosing was stopped. Handling-precipitated convulsions started on dose day 5 at the high dose level and each animal that convulsed was dead within 24 hours. The low dose female convulsed on study day 9 and survived. The low dose males convulsed during handling just prior to planned necropsies on days 8 and 10. One high dose male convulsed the third day (day 8) after dosing was stopped but the frequency of convulsive episodes were not increasing compared to the rats dosed acutely in Part I. All animals were handled (picked up) as much during recovery as they were while being dosed. Treatment- and dosage-related deaths resulted from the treatment-related toxicities. Deaths occurred earlier in the study for the high dose males thanforthehighdosefemales.Sevenof 12 males and two of 12 females were dead by day 6 and by day 8 all the males and seven of 12 females were dead. The high dose females continued to die through day 12 (recovery day 7) with two surviving throughout the recovery interval. The only treatmentrelated death at the low dose level was a female on day 9. Treatment-related microscopic liver toxicity showed evidence of reversal following the recovery interval. The liver effects consisted of fatty changes and focal areas of necrosis. Minimal to moderately severe fatty 12 changes were present in five of 12 males and three of 12 females at the high dose level and three of five males at the low dose level. Focal areas of necrosis occurred in two of 12 males and one of 12 females at the high dose level and three of five males and two of five females at the low dose level. These low dose effects reflect only those rats killed for the interim necropsy. The last three high dose females that died during the recovery interval (died on days 9, 11 or 12) had no histopathological changes. The two females in the high dose group that survived had one animal with only fatty changes at necropsy. The low dose recovery animals did not have focal liver necrosis but they still had minimal to mild fatty changes in five of six males and one of six females. The greater number of low dose rats compared to high dose rats with liver changes reflected the better survival rate at the low dose level and the subchronic nature of the liver response. Treatment-related body weight loss occurred during dosing and body weight gain occurred during the recovery interval. The weight loss was similar at both dose levels but the females lost more weight than the males (Table 7). Animals in both dose groups also appeared thin. The surviving high dose females and many of the low dose recovery animals continued to appear thin during the recovery interval even though they were gaining weight. Food consumption for all animals appeared normal throughout the study. The only other treatment-related histopathological change was congested and hemorrhagic adrenals in one of 12 males and two of 12 females at the high dose level. The adrenal effect was probably due to stress caused by one or more of the other treatment-related toxicities. An infrequent treatmentrelated clinical effect was urinary incontinence in four high dose males. The incontinence occurred for several days prior to death but was not associated with microscopic kidney changes. One low dose male was euthanatized on day 8 having a soft subcutaneous lump caused by an intubation-induced perforated esophagus. 13 Table 4 N=t Following kidneys. Dose Group and Rat No. Gross and KLcroscopi c Tissue Observations complete gross necropsy, histologic examination was done on brain, liver, stomach, adrenals and If a tissue in not listed, no significant changes were found, is only abnormalities are listed. Organ Gross Observations Kicroscopic observations 200 mg/kg Rales 9Rl7285 (diedday 6) Stomach Small hemorrhagic areas (ulcers?) At the outar region& of the qastric mucosa there were scattered focal areas of congestionand hemorrhage. These hemorrhagicfoci were not associatedwith any inflammationnor with anv particular necrosis of the 9"tric mucosa. Liver no significant changes Moderately severe fatty changes were present, as approximately one-third of the hepatocvtes had cytoplasmic fat vacuoles. The involved cells sometimes, but not always, had a central lobular dis- tribution. 9Rl7286 (diedday 8) Stomach Adrenals Small black areas (hemorrhages?) Dark (hemorrhagic?) Scattered foci of congestion and hemorrhacfe were present in the outer regions of the gastric mucona. Rather marked congestion was present, Darticularly in the medulla but in the cortex as well. 9Rl72a7 (died day 6) gRl7288 (died day 7) Stomach Stomach Large hemorrhagic areas (ulcers?) Large black spots (hemorrhages?) Scattered foci of congestion " present in the gastric mucosa. hemorrhaqe were Scattered foci of congestion and hemorrhage were present in the gastric mucosa. At one of these foci there wam some necrosis of the gastric epithelium (development of a shallow hemorrhacic ulcer). 9R17289 (diedday 8) Adrenals Small Intestine Liver Dark (hemrrhagic?) Reddened areas No significant changes No significant changes Tissue not processed for examination Kild fatty changes were indicated as anaroximatelv 10-15% of the hopatocytes had cytoplasmic fat vacuoles. 9R17290 (diedday 6) Stomach Dark lines (hemorrhages?) Focal areas of congestion and hemorrhage were present in the gastric mucosa. In one instance this was associatedwith some necrosis of the gastric epithelium (developmentof an early hemorrhagic ulcer). Liver no significant changes Kiniml fatty changes were present as approximately 5% of the hopatocytes had cytoplasmic fat vacuoles. 9Rl7291 (diedday 6) 9Rl7292 (died day 7) Stomach Stomach Liver 9Rl7293 (died day 6) Stomach Small black @per-ke (hemorrhages?) Red lines No significant changes Long black lines (hemorrhagic?) no significant changes Focal areas of conqestion and hemorrhaqe were present in the gastric sucosa. Mild fatty changes were present as approximately 10-154 of the hopatocytes had fine cytoplasmic fat vacuoles. Focal areas of congestion and hemorrhage were present in the gastric mucosa. Does Group and Rat No. Organ 200 mg/kq males (con't) 9Rl7294 (died day 7) Stomach Liver 9R17295 (died day 5) Stomach Liver 9Rl7296 (died day 6) Spleen Stomach 200 wq/kq Females 9Rl7343 (died day 8) Stomach Adrenals 9Rl7344 (died day 7) Stomach Liver 9Rl7345 (died day 7) Stomach Liver 9RI7346 (died day 11) 9R17347 (died day 7) 9R17348 (died day 6) 9R17349 (died day 7) 9R17350 (died day 6) Stm@ch Stomach Stomach Stomach 14 Table 4 (continued) Gross and Microscopic Tissue observations Gross observations Microscopic observations Large black spots (hemorrhages?) So significant changes no significant changes White band on left lateral lobe Appears pale large black spots (hemorrhages?) There were focal areas of congestion and hemorrhage in the gastric mucosa. In one area there were several small foci of coagulation necrosis. There were focal areas of congestion and hemorrhage in the gastric mucosa. The area observed -rossly appeared to be a longitudinal area of coagulation necrosis. in addition, there were minimal fatty changes as approximately 5% of the hepatocytes had fine cytoplasmic fat vacuoles. Tissue not processed for examination. Focal areas of congestion and hemorrhage were present in the q"tric mucosa and at several areas were associated with necrosis of the gastric epithelium (a developing hemorrhagic gastric ulcer). Large dark area (hemorrhages?) Dark (hmorrhagic?) Small black spots (hemorrhages?) No significant changes Dark red lines No significant changes (none) Dark red streaks No significant changes Largo black spots (hemrrhages?) Thin dark lines (hewrrhages?) A hemorrhagic ulcer was present with localized necrosis of the gastric epithelium. Rather extensive congestion and some areas of hemorrhage were present. Foci of congestion and hemorrhage were present in the upper portion of the gastric mucosa. Scattered foci of coagulation necrosis were present. Infiltration of mononuclear cells were present within acme of these necrotic foci. so significant changes Mild fatty changes were present as approximately In% of the hopatocvtes had cytoplasmic fat vacuoles Foci of congestion and hemorrhage were present in the upper regions of the gastric mucosa. A small, shallow hemorrhagic ulcer was present. Small focal areas of oonqestion and h were present in the gastric mucosa. no significant changes rrhage Dme Group and Rat No. Organ 200 ag/kg Females (con't) 9R17351 (on compound 5 days, terminated day 20) Liver 9Rl7352 (died day 9) Stomach Adronals Liver 9Rl7353 (an compound 5 terminated day 20) 9R17354 (died day 12) 75 mg/kg Malos 9RI7297 (on ccmpound 9 days, terminated day ;L3) Liver 9RI7298 (on ccopound 9 days, terminated day 23) Liver 9RI7299 (on compound 9 days, terminated day 10) Liver 9RI7300 (on ccupound 9 days, terminated day 10) Liver Adronals 9Rl7301 (an compound 9 days, tera@ inatod day 10) Adronals Liver 9Rl7302 (on compound 9 days, terminated day 10) Liver 15 Table 4 (continued) Gross and Kicroscopic Tissue observations Gross Observations nicroscopic Observations No significant changes Slar-k lines (hemrrhages?) Enlarged and dark (hemorrhagic) no significant changes (none) (none) IPatty changes were present as approximately 30-40% of the hopatocytes had cytoplasmic fat vacuoles. No significant changes Areas of congestion and ha-rrhage were present. Kild fatty chanqes were present as about 10% of the hopatocytes had cytoplasmic fat vacuoles. No significant changes No significant changes No significant changes Pale streaks and foci Dark, enlarged? Enlarged? Palo streaks No significant changes Kild fatty changes were present as approximately 10-20% of the hapatocytes had cytoplasmic fat vacuoles. kild fatty changes were present as approximately 10% of the hopatocytes had cytoplasmic fat vacuoles. Yoderately severe fatty changes were present as approximately 50% of the hepatocytes had cytoplasmic fat vacuoles. In addition, in one section there were several small, focal areas of older coagulation necrosis with infiltration of numerous sononuclear cells cleaning up the cellular debris. The lesions observed grossly were focal areas of coagulation necrosis. A limited amount of acute to subacute inflammatory response was present adjacent to and within some of these foci. Areas of congestion and hemorrhage were r)resent in the cortex. No significant changes The areas observed grossly were areas of coacjulation necrosis, frequently accompanied by a limited amount of acute to subacute inflammatory response. Approximately fatty changes vacuoles. 10-20% of the hopatocytes had an indicated by cytoplasmic fat Dome Group and Rat No. Organ 75 mg/kq Males (con't) 9RI7303 4 Liver (on compound 9 days, terminated day 23) 9Rl7304 (an compound 9 days, terminated day 23) Liver 9RI7305 (on compound 9 Liver days, terminated day 23) 9RI7306 (outhanatized day 8) geophaqus Adronals 9Rl7307 (on compound 9 days, terminated day 23) 9RI7308 Liver (on compound 9 days, terminated day 10) 75 mg/kg Females 9RI7355 (on compound 9 days, terminated day 10) 9Rl7356 (diedday 10) 9Rl7357 (on compound 9 days, terminated Adrenals Liver day 10) 9R17358 (an compound 9 days, terminated Liver day 10) 9R17359 (an compound 9 days, terminated day 23) 16 Table 4 (continued) Gross and Microscopic Tissue Observations Gross observations Microscopic ObsOrV&ticns mo significant changes Minimal fatty changes were present as approximately 1-2% of the hapatocytes had cytoplasmic fat vacuolee no significant changes Minimal fatty changes were present as 1-2% of the hepatocytes had cytoplasmic fat vacuoles. No significant changes Approximately 1-2% of the hopatocvtes had cytoplasmi fat vacuoles indicating nlild fatty changes. Puncture hole Just anterior to thoracic inlet; leads to large necrotic subcutaneous area in left axilla - intubation error No tissue saved Dark (hemorrhagic?), enlarged Congestion and hemorrhage were present. (none) -No significant changes Mild fatty changes were indicated as aporoximatelv 10% of the hepatocytes had cytoplasmic fat vacuoles. (none) Enlarged? Possible pale area Palo streaks (none) Congestion and focal homrrhages in the cortex and swulla. No significant changes were present The lesions coagulation of an acute observed necrosis grossly were focal areas of accompanied by infiltration to subacute inflammatory exudate. Doe* Group " Ut NO.-- Organ 7!5mg/kg Females (con't) 9Rl7360 (on compound 9 days, terminated Liver day 10) Adramls 9R17361 (on compound 9 days, terminated day 23) Liver 9Rl7362 (on compound 9 days, terminated day 23) 9R17363 (on compound 9 days, terminated day 10) 9Rl7364 (on compound 9 terilinatod day 23) 9Rl7365 (on compound 9 0 torminatod day 23) 9R17366 (on compound 9 terminated day 23) 17 Table 4 (concluded) Gross and KicroscopiC TISSUO Observations Gross Observations Palo streak AMG&R onlarged So signifleant changes (none) (none) (none) (none) (none) 14icroscopic observations There were focal areas of coagulation necrosis which war* usually accompanied by a subacute inflammatory exudate. No significant changes There wore minimal fatty changes as approximatelv 1-2% of the hapatocytes had cytoplasmic fat vacuoles. Table 5 Two Week Oral Rangefinding Study of T-2509CoC in Rats Repeat Dose Effects Animals Affected/Day Effect Study Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 200 mg/kg/day Males Appeared normal Soft feces Tucked-up appearance Thin Urinary incontinence Tonic convulsion@@ Unkempt appearance Deaths 200 mg/kg/day Females Appeared normal Tucked-up appearance Thin Urinary incontinence Tonic convulsiorlaUnkempt appearance Bloody nose and mouth Hyperactive Deaths 12 12 3 4 NE NE NE NE NE NE 7 NE NE 4 1 1 NE NE 4 8 10' 5 2 1 NE NE NE 1 NE 5 2 1 NE NE NE 1 NE NE NE NE NE NE NE NE 5 NE NE 1 NE NE NE NE NE 2 1 1 000 016 32 12 12 11 6 NE NE NE NE NE 'NE '-NE '-NE 1 ..l --l '-NE NE NE 1 6 11 11 6 4 1 NE NE NE NE NE 11 6 5 4 1 NE NE NE NE NE NE 432 11 12 NE NE NE NE NE 2 1 1 NE NE NE NE NE NE NE NE NE NE NE NE 1 2 NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE 1 NE NE 1 1 NE NE 1 1 1 1 NE NE NE NE NE NE 1 NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE, NE NE NE 1 1 NE NE NE NE NE 00 00 02 4 1 1 0 110 00 0 75 mg/kg/day_Males Appeared normal Soft feces Tucked-up appearance 12 12 8 9 9 7 5 4 1 NE NE 2 NE NE 1 1 NE 1 NE NE NE NE 3 2 4 5 3 1 1 1 1 1 .1 1 1 NE NE NE NE NE NE 3 1 1 NE NE NE NE NE = Not evident 2@Convulsive episodes generally occurred during handling (dosing, weighing, etc.) Table 5 (concluded) Two Week Oral Rangefinding Study of T-2509CoC in Rats Repeat Dose Effects Animals Affected/Day Effect 75 mg/kg/day Males (con't) Respiratory congestion Thin a Tonic convulsions Unkempt Appearance Bloody nose and mouth Head tremors Lump on left side Deaths 75 mgAg/day Females Appeared normal Tucked-up appearance Thin Respiratory congestion a Clonic/tonic convulsionUnkempt appearance Hyperactive Hair loss on abdomen Deaths Study Day 12 3 4 5 6 7 8 9 10 11 12 13 14 15 16 NE ME 3 3 NE 2 3 3 3 NE NE NE NE NE NE 25 5 NE NE NE NE NE NE NE 1 NE NE NE NE NE NE NE NE NE 7 NE NE NE NE NE NE NE NE 1 NE NE NE NE NE NE NE NE NE NE NE NE NE NE 1 1 1 NE lb 0 0 0 0 '0 0 0 0 3 NE NE 1 1 2 2 5 2 2 3 3 3 3 1 NE NE NE NE NE NE 744 55 55 1 NE NE NE NE NE NE I NE NE NE NE NE NE NE NE NE NE NE NE NE 5!1 0 0 0 0 0 0 12 12 12 12 12 9 7 4 4 NE NE NE NE NE 34 56 NE NE NE NE NE NE 4 78 NE NE NE NE NE NE NE NE 1 NE NE NE NE NE NE NE NE I NE NE NE NE NE NE NE NE 5 NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE 00 0 0 0000 0 4 2 2 2 2 2 2 5 1 1 NE NE NE NE 7 4 4 2 2 2 2 NE NE NE NE NE NE NE NE NE NE NE NE NE NE 4 2 2 4 4 4 4 NE NE NE 1 111 NE 1 1 1 1 1 1 62- 0 0 0 0 0 0 NE = Not evident a Convulsive episodes Euthanatized generally occurred Five euthanatized, one found dead during handling (dosing, weighing, etc.) Table 6 Two Week Oral Rangefinding Study of T-2509CoC in Rats Summary of Clinical Observations Number Affected/Group Clinical Sign No signs through study Treatment-related deaths Soft feces Tucked-up appearance Thin Unkempt appearance Tonic or clonic/tonic convulsions Hyperactive Head tremors Urinary incontinence Respiratory congestion Bloody nose and mouth Lump on left side Hair loss on abdomen 200 cr 0/12 12/12 8/12 11/12 6/12 4/12 6/12 0/12 0/12 1/12 0/12 0/12 0/12 0/12 Dose Group (mg/kg/day) 200 75 cr 0/12 10/12 0/12 12/12 11/12 3/12 3/12 1/12 0/12 2/12 6/12 8/12 8/12 2/12 1/12 0/12 3/12 0/12 1/12 0/12 0/12 0/12 1/12 0/12 7/12 1/12 1/12 0/12 20 75 1/12 1/12 0/12 6/12 8/12 6/12 1/12 1/12 0/12 0/12 1/12 0/12 0/12 1/12 Table 7 Two Week Oral Rangefinding Study of T-2509CoC in Rats Group Mean Body Weights Dose Group -6 200 mg/kg/day cr 138.5 9.21 200 mg/kg/day 133.0 5.43 75 mg/kg/day cf 141.3 7.52 75 mg/kg/day 133.5 7.69 +1 177.8 13.33 165.0 9.28 174.8 13.91 163.7 10.63 6 134.2a 19.03 114.12@ 10.25 170.9 20.23 '143.0 8.32 Study Day 9 13 --- --- 124.8 10.05 b 165.5 19.39 129.5 b 15.16 154.0 14.4 175.3 16.33 148.3 16.58 20 --- 186.5 0.71 224.7 17.19 182.4 9.03 23 --- % Cha End 0 Dosin -25% --- -31% 245.7 18.58 190.7 13.SO - 5% -21% a Dosing discontinued b Dosing discontinued following day 5 following day 9 22 Discussion Daily gastric intubation of rats with T-2509CoC resulted in treatment-, dosage- and sex-related effects. The primary effects were gastric irritation, handling-induced convulsions and microscopic liver changes. Dosage-related responses to the gastric effects were a tucked-up appearance, an unkempt appearance and soft feces; only the males had soft feces. Treatment-related responses to the gastric effects were thinness and body weight loss during dosing. The females lost more weight than the males. Gross and microscopic stomach irritation which included a few early hemorrhagic gastric ulcers, was present in the high dose group only. Convulsive episodes (CNS stimulation) occurred in more animals in the high dose group than the low dose group and more males than females had convulsions. Less frequent treatment-related CNS effects were head tremors and hyperactivity. Dosage- and sex-related liver toxicity occurred in the form of focal necrosis and fatty changes. The liver changes occurred in more males than females. Dosage-related deaths were caused by T-2509CoC treatment. More high dose males died than high dose females and the males started dying earlier in the study. The recovery interval resulted in reversal or cessation of the toxic effects. Gastric lesions were not present in the two surviving high dose females. Gastric pain (tucked-up appearance) continued for the first few days of recovery and a few animals continued to appear unkempt and thin. All recovery animals, however, had body weight gain throughout the recover-@r interval. Th e later-occurring deaths in the high dose female group were probably due to a generalized debilitated condition because none of these animals had microsconic changes. Severe delayed convulsions did not occur during recovery. Focal liver necrosis was not present in the recovery animals. The low dose recovery group had more males than females with mild fatty changes in the liver. 23 Appendix I Two Week Oral Rangefinding Toxicity Study of T-2509CoC in Rats Protocol TITLE: Protocol for a Two Week Oral Rangefinding Toxicity Study in Rats (Study Number 179RRO23) TEST ARTICLE: A clear, pale yellow solution identified as T-2509CoC. This chemical will be supplied by Toxicology Services, 3m Company, St. Paul, Minnesota. OBJECTIVE: The primary objective will be to identify any major toxic effects in rats during two weeks of daily gastric intubation of T-2509CoC. Before the doses will be selected to complete the above objective however, the acute oral ALD50 of T-2509CoC in rats will need to be determined. This acute portion will use males only from the same supplier, strain and age range of rats as described below for the repeat dose portion. The general procedures for a Riker Safety Evaluation Method 605A will be used for the acute dosing except that six males/level will be dosed. The sponsor and study director will use the acute data to select the dosage levels for the two week repeat dose study. At the conclusion of the two week dosing interval'r the sponsor and the study director have the option of continuing the observation period for about one-half the males and females at each level. The extended observation period will be two weeks in length and dosing will not occur during that interval. CONTROL ARTICLE: None SPONSOR: Toxicology Services, 3M Company, St. Paul, Minnesota TESTING FACILITY: Safety Evaluation Laboratory, Riker Laboratories, Inc., St. Paul, Minnesota. DOSING INTERVAL: September-October, 1979 DOSAGE LEVELS, ROUTE, GROUP, SIZE, ETC.: Oral gastric intubation will be used because it appears to be an appropriate route for evaluating the systmic toxicity in rats. The rats will be dosed daily for two weeks (1). A constantdosevolumeof 10 ml/kgwillbe used for eachdosage level. Distilled water will be used for all necessary dilutions of the test article. 24 Appendix I (continued) Dose Group High Low Dosage Levels (2) (to be selected after the acute portion has been completed) Group Size 12 cr, 12 12 (@, 12 ANALYTICAL CHARACTERIZATIONS: The sponsor has on file one or more of the following or other pertinent characterizations of the test article: identity, synthesis, strength, purity, stability. Preparation and measurement records for the various dilutions of the test article will be considered adequate verification of content and concentration of the dosing solutions for this rangefinding study. TEST SYSTEM: Twenty-four male and 24 female Charles River CD rats, 45-55 days (3) old on dose day 1, will be used for the repeat dose study. The animals will be housed individually in hanging stainless steel cages with wire mesh floors and fronts. The room will be temperature and humidity controlled with the lights on a 12 hour light/dark cycle. JUSTIFICATION FOR SELECTION OF TEST SYSTEM: Charles River CD rats will be used because of historical in-house data on the strain. TEST SYSTEM IDENTIFICATION: Each animal will be assigned a number which will be indicated on the outside of the cage and on an individual ear tag. RANDOMIZATION OF TEST SYSTEM: The animals will be indiscriminately removed from the shipping boxes by Animal Care personnel and placed in the rack of cages from left to right starting at the top and working down. The study director will assign dose groups by vertical rows. DIET SPECIFICATIONS: Purina Laboratory Chow and water will be available ad libitum throughout the study. BIOAVAILABILITY OF TEST ARTICLE: During previous acute toxicity studies in rats, systemic absorption of the test article appears to have occurred in the form of delayed CNS effects. CLINICAL OBSERVATIONS: The animals will be observed daily throughout the dosing interval for evidence of treatment-related toxicity. Body weights will be recorded approximately one week prior to dose day 1, on dose day 1 and biweekly thereafter. Food consumption will be estimated weekly throughout the dosing interval. If animals are retained for two additional weeks following dosing, they will be observed and weighed as indicated above (4). Appendix I (continued) 25 TISSUE PATHOLOGY: Gross necropsies will be conducted on all rats which die during the study. In animals with autolysis, as a minimum, samples of lung, liver, kidney, brain and any gross lesion will be fixed in formalin. Microscopic examination will determine if the tissues are satisfactory for histologic diagnosis. Any rat in a moribund condition should be terminated to insure satisfactory tissue samples. Approximately 24 hours following the last dose, the selected survivors will be killed for gross necropsy examination. The animals selected for additional observation without dosing, if any are selected, will be killed for gross necropsy examination at the end of that interval. Samples to be fixed in 10% buffered formalin are listed below. 1. Mammary Gland (female) 2. Eyes (2) 3. Thymus 4. Thyroid/Parathyroid/Trachea/Esophagus. 5. Lung 6. Heart 7. Liver 8. Adrenals (2) 9. Kidneys (2) 10. Urinary Bladder 11. Testes/Epididymis or ovaries 12. Uterus or Prostate 13. Spleen 14. Pancreas 15. Stomach 16. Small Intestine (at least two areas) 17. Large Intestine 18. Mesenteric Lymph Node 19. Sciatic Nerve 20. Spinal Cord/Bone Marrow (cervical and thoracic) 21. Brain 22. Pituitary 23. Any Gross Lesion The tissues to be processed selected by the pathologist The tissue selections will processing (5). and examined histologically will be at the conclusion of the gross necropsy. be reviewed with the sponsor prior to DATA ANALYSIS AND FINAL REPORT: The appropriate statistical analyses, if any, will be selected at the conclusion of the study. The proposed date for the final report is 1-2 months after slide reading is completed (approximately 2-5 months after necropsy). 26 Appendix I (concluded) AMENDMENTS: 1. The last day of dosing for the high dose group (200 mg/kg/day) was day 5 and the last dose day for the low dose group (75 mg/kg/day) was day 9. All surviving high dose animals were observed for approximately two weeks following the last dose. Following completion of dosing at the low dose level the appropriate number of males and females were killed to reach a total of 6/sex dead. The remaining 6/sex were observed for approximately two more weeks. 2. The selected high dose level was 200 mg/kg/day and the selected low dose level was 75 mgAg/day. 3. Age range for the acute rats extended to 59 days old on the day of dosing. 4. Body weights obtained once or twice a week during recovery rather than biweekly. 5. The tissues processed for histopathological evaluation were liver, adrenals, stomach, kidneys and brain.. 27 Appendix II Two Week Oral Rangefinding Toxicity Study of T-2509CoC in Rats List of Principal Participating Personnel Name Marvin T. Case James D. Henderson, Jr. Cathy E. Ludemann Gary C. Pecore Inara Porietis Jan H. Skroms G. Ray Steffen Function Veterinary Pathologist Staff Veterinarian Coordinator - Clinical Pathology Coordinator - Animal Care Histopathology Technician Toxicology Technician Study Director I DISTRIBUTION LIST M. T. Case K.-L. Ebbens F. D. Griffith W. C. McCormick R. A. Nelson G. R. Steffen (2)