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g6H- oe - 373 / H U 3 6 -ogs$ 'b'T--^ Cotitohit Mo CBI Two Week Oral Rangefinding Toxicity Study of T-2509COC in Rats o o T c: cm rs> '"O .n <n Experiment No.: Dosing Started: Recovery Intervals Completed: Study Director: Testing Facility: Sponsor: EPA-OTS 000811796W GDllTTblil 179RR023 October 3, 1979 November 15, 1979 G. R. Steffen, BS reecrs=raj>> CO m O cm "D m I ~o O --H m '12nS * ro cn Safety Evaluation Laboratory Riker Laboratories, Inc., St. Paul, Minnesota W. C. McCormick Toxicology Services 3M Company St. Paul, Minnesota Study Director Safety Evaluation Laboratory 003695 TABLE OF CONTENTS Page Summary ..................................................... 1 Introduction .............................................. 2 Methods ..................................................... 2 Results ..................................................... 3 D i s c u s s i o n ................................................... 22 A p p e n d i c e s ................................................... 23 003696 1 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 ad ministered single doses ranging from 250-10,000 m gAg. 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 mg/kg/day. Dosing continued for five days at the high level (200 mg/kg/day) and for nine days at the low level (75 mg/kg/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 m g A g with 95% con fidence limits of 558-1399 m g A g - 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 con tributed 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 treatment-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. 003697 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 by indivi dual 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. 003698 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 grams. The test article solutions were prepared daily using distilled water as the diluent. A constant dose volume of 10 ml/kg 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 mg/kg/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 mg/kg/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 I : The acute oral ALD50 of T-2509CoC in male rats was 878 mg/kg having 95% confidence limits of 558 to 1399 mg/kg. 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 mg/kg 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 mg/kg levels but these animals re covered. 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 mg/kg) 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. 003699 Observation Appeared normal Salivation Lacrimation Hyporeactive Ptosis Diarrhea Chromodacryorrhea Hypoactive Tucked-up abdomen Deaths Table 1 Acute Oral Toxicity Study of T-2509CoC in Male Rats 10,000 mg/kg Group Number of Animals Affected Observation Period Hours Days 0-1 .1-2 2-4 4-6 2 3 4 5 6 7 8 9 10 11 12 13 14 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 5 (overnight) NE = Not evident oo CO * v! O O Table 1 (continued) Acute Oral Toxicity Study of T-2509CoC in Male Rats 5000 mg/kg Number of Animals Affected Observation Observation Period ________Hours________ ______________________________Days c>-l 1-2 2-4 4-6 2 3 4 5 6 7 8 9 10 11 12 13 14 Appeared normal 4 4 1 1 2 2 2 2 1 NE - - - - -- - -- Salivation 3 1 NE 1 NE NE NE NE NE NE - - - - - - - Bloody nares & mouth 2 NE NE 1 NE NE NE NE NE NE - - - - - - - Hyporeactive 2 1 5 5 NE NE NE NE NE NE - - - - - - - Dyspnea 1 NE NE NE NE NE NE NE NE NE - - - - - - - Soft feces NE 1 5 5 NE NE NE NE NE NE - - - - - - - Ptosis NE 1 1 1 NE NE NE NE NE NE - - - - - - - Tucked-up abdomen NE NE 2 3 NE NE NE NE NE NE - - - - - - - Clonic/tonic convulsion NE NE NE NE NE NE NE NE 1^ NE - - - - - - - Deaths 0 0 0 0 3100 11 NE = Not evident -- Precipitated by handling Table 1 (continued) Acute Oral Toxicity Study of T-2509COC in Male Rats 2000 mg/kg Number of Animals Affected Observation Observation Period ________Hours________ ______________________________Days 0-1 1-2 2-4 4-6 2 3 4 5 6 7 8 9 10 11 12 13 14 Appeared normal 666 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 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 - - - - - - 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 - - - - - - Deaths 0 00 30 10 10 1 NE = Not evident -- Precipitated by handling o CJ o (J\ Table 1 (continued) Acute Oral Toxicity Study of T-2509COC in Male Rats 1000 mg/kg Number of Animals Affected Observation Observation Period ________Hours________ ______________________________Days 0-1 1-2 2-4 4-6 2 3 4 5 6 7 8 9 10 11 12 13 14-20 Appeared normal 6 6 6 6 6 6 6 5 4 3 NE NE 3 3 3 3 3 Clonic/tonic convulsion NE NE NE NE NE NE NE NE NE & NE NE NE NE NE NE NE Thin NE NE NE NE NE NE NE NE NE 1 NE NE NE NE NE NE NE Hypereactive NE NE NE NE NE NE NE NE NE NE 3 3 NE NE NE NE NE Tremors NE NE NE NE NE NE NE NE NE NE 1 1 NE NE NE NE NE Deaths 0 0 0 00011010 0 0 0 0 0 NE = Not evident a -- Precipitated by handling Observation Appeared normal Hypereactive Deaths NE = Not evident Observation Appeared normal Deaths Table 1 (concluded) Acute Oral Toxicity Study of T-2509CoC in Male Rats 500 mg/kg Number of Animals Affected Observation Period ________Hours________ ______________________________Days 0-1 1-2 2-4 4-6 2 3 4 5 6 7 8 9 10 11 12 13 14-20 6 6 6 6 6 6 6 6 5 5 NE NE 5 5 5 5 5 NE NE NE NE NE NE NE NE NE NE 5 5 NE NE NE NE NE 0 0 0 0 00001000 0 0 0 0 0 250 mg/kg Number of Animals Affected Observation Period ________Hours _____ ______________________________Days 0-1 1-2 2-4 4-6 2 3 4 5 6 7 8 9 10 11 12 13 14-20 666 66666666 6 6 6 6 6 000 00000000 0 0 0 0 0 Table 2 Acute Oral Toxicity Study of T-2509COC in Male Rats Death Pattern 9 Dose Level Deaths/Day(s) 1 2 3 4 5 6 7 8 9-Term 10000 mg/kg~ 15 5000 mg/kg-- 0 3100 1 2000 mg/kg 0 30101 iOOO mg/kg 000011 500 mg/kg 000001 250 mg/kg 000000 ALD50 = 878 mg/kg 95% Confidence Limits = 558 to 1399 mg/kg-- 1-- 01 01 00 00 -- -- 0 0 0 Total Deaths N o . Dosed 6/6 6/6 6/6 3/6 1/6 0/6 -- Data from this level not used for ALD50 calculations -- Computations by Duluth Probit Analysis, Environmental Research Laboratory Program 03705 Table 3 Acute Oral Toxicity Study of T-2509CoC in Male Rats Necropsy Results Number of Animals Affected Gross Observation Dose Group (mg/kg) 10,000 5000 2000 1000 500 250 No. Dead/No. Dosed 6/6 6/6 6/6 3/6 1/6 0/6 No Visible Lesions Euthanatized Found Dead 0 0 0 3 56 0 2 1 1 00 Reddened Stomach Mucosa 4 2 2 0 0 0 Hemorrhagic Stomach Mucosa 2 2 1- 1 10 Hemorrhagic Small Intestinal 0 0 2 0 00 Mucosa Cannabilized 0 0 0 1 00 -- With possible ulcer 11 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 con vulsions 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 than for the high dose females. Seven of 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 003707 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 per forated esophagus. 003708 13 Tabi* 4 Gross and Microscopic Tissue Observations MOTE; Following complete gross necropsy, histologic examination was done on brain, liver, stomach, adrenals and kidneys. If a tissue is not listed, no significant changes were found, ie only abnormalities are listed. Dose Group and Rat No. Organ Gross Observations Microscopic Observations 200 mg/kg Males 9R17285 (died day 6) Stomach Small hemorrhagic areas (ulcers?) At the outer regions of the gastric mucosa there were scattered focal areas of congestion and hemorrhage. These hemorrhagic foci were not associated with any inflammation nor with any particular necrosis of the gastric 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 some times, but not Always, had a central lobular dis tribution . 9R17286 (died day 8) Stomach Small black areas (hemorrhages ?) Scattered foci of congestion and hemorrhage were present in the outer regions of the gastric mucosa Adrenals Dark (hemorrhagic?) Rather marked congestion was present, particularly in the medulla but in the cortex as well. 9R17287 (died day 6) Stomach Large hemorrhagic areas (ulcers?) Scattered foci of congestion and hemorrhage were present in the gastric mucosa. 9R17288 (died day 7) Stomach Large black spots (hemorrhages?) Scattered foci of congestion and hemorrhage were present in the gastric mucosa. At one of these foci there was some necrosis of the gastric epitheliun (development of a shallow hemorrhaaic ulcer). 9R17289 (died day 8) Adrenals Small Intestine Dark (hemorrhagic?) Reddened areas No significant changes Tissue not processed for examination Liver No significant changes Mild fatty changes were indicated as aooroximatelv 10-15 of the hepatocytes had cytoplasmic fat vacuoles. 9R17290 (died day 6) Stomach Dark lines (hemorrhages?) Focal areas of congestion and hemorrhage were present in the gastric mucosa. In one instance this was associated with some necrosis of the gastric epithelium (development of an early hemorrhagic ulcer). Liver No significant changes Minimal fatty changes were present as approximately 5% of the hepatocytes had cytoplasmic fat vacuoles. 9R17291 (died day 6) Stomach Small black specks (hemorrhages?) No significant changes 9R17292 (died day 7) Stomach Red lines Focal areas of congestion and hemorrhage were present in the gastric mucosa. Liver No significant changes Mild fatty changes were present as approximately 10-15* of the hepatocytes had fine cytoplasmic fat vacuoles. 9R17293 (died day 6) Stomach Long black lines (hemorrhagic?) Focal areas of congestion and hemorrhage were present in the gastric mucosa. 003709 Dose Group and Rat Mo. Organ 200 m q A g Males (con't) 9R17294 (died day 7) Stomach Liver 9R17295 (died day 5) Stomach Liver 9R17296 (died day 6) Spleen Stomach 200 m g A g Females 9R17343 (died day 8) Stomach Adrenals 9R17344 (died day 7) Stomach Liver 9R17345 (died day 7) Stomach liver 9R17346 (died day 11) 9R17347 (died day 7) 9R17348 (died day 6) 9R17349 (died day 7) 9R17350 (died day 6) Stoautch Stomach Stomach Stomach Table 4 (continued) Gross and Microscopic Tissue Observations 14 Gross Observations Microscopic Observations Large black spots (hemorrhages 7) No significant changes No significant changes White band on left lateral lobe Appears pale Large black spots (hemorrhages 7) There were focal areas of congestion and hemorrhaq in the gastric mucosa. In one ares 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 5t of the hepatocytes had fine cytoplasmic fat vacuoles. Tissue not processed for examination. Focal areas of congestion and hemorrhage were present in the gastric mucosa and at several areas were associated with necrosis of the gastric epithelium (a developing hemorrhagic gastric ulcer Large dark area (hemor rhages? ) Dark (hemorrhagic?) Small black spots (hemorrhages 7) No significant changes Dark red lines No significant changes (none) Dark red streaks No significant changes Large black spots (hemorrhages?) Thin dark lines (hemorrhages 7) 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 some of these necrotic foci. No significant changes Mild fatty changes were present as approximately 10 of the hepatocytes 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 congestion and hemorrhage were present in the gastric mucosa. No significant changes 003710 Dose Group and Rat Ho. Organ 200 mg/kg Peaales (con't) 9R17351 Uver (on compound 5 days, terminated day 20) 9R17352 (died day 9) Stomach Adrenals Liver 9R17353 (on compound 5 terminated day 20) 9R173S4 (died day 12) 75 sxf/kq Males 9R17297 Liver (on compound 9 days, terminated day 23) 9R17298 * Liver (on compound 9 days, terminated day 23) 9R17299 Liver (on compound 9 days, terminated day 10) - 9R17300 Liver (on conpound 9 days, terminated day 10) * Adrenals 9R17301 (on compound 9 days, terminated day 10) Adrenals Liver 9R17302 Liver (on compound 9 days, terminated day 10) Tabla 4 (continued) Gross and Microscopic Tissue Observations 15 Gross Observations Microscopic Observations Mo significant changes Patty changes were present as approximately 30-40' of the hepatocytes had cytoplasmic fat vacuoles. Black lines (hemorrhages?) Enlarged and dark (hemorrhagic) No significant changes (none) .No significant changes Areas of congestion and hemorrhage were present, Mild fatty changes were present as about 10% of the hepatocytes had cytoplasmic fat vacuoles. (none) No significant changes No significant changes Mild fatty changes were present as approximately 10-20% of the hepatocytes had cytoplasmic fat vacuoles. Mild fatty changes were present as approximately 10% of the hepatocytes had cytoplasmic fat vacuoles No significant changes Pale streaks and foci Dark, enlarged? Enlarged? Pale streaks Mo significant changes Moderately severe fatty changes were present as approximately 50% of the hepatocytes had cyto plasmic fat vacuoles. In addition, in one section there were several small, focal areas of older coagulation necrosis with infiltration of numerous mononuclear 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 seme of these foci. Areas of congestion and hemorrhage were present in the cortex. No significant changes The areas observed grossly were areas of coagula tion necrosis, frequently accompanied by a limited amount of acute to subacute inflammatory response. Approximately 10-20% of the hepatocytes had fatty changes as indicated by cytoplasmic fat vacuoles. 003711 Table 4 (continued) Groes and Microscopic Tissue Observations 16 Dose Group and Rat Mo. Organ 75 mg/kq Males (con't) 9R17303 ' Uver (on compound 9 days, terminated day 23) 9R17304 , Liver (on compound 9 days, terminated day 23) 9R17305 , Uver (on compound 9 days, terminated day 23) 9RX7306 (euthanatised day 6) Esophagus > Adrenals 9R17307 (on compound 9 days, terminated day 23) 9R17308 Uver (on compound 9 days, terminated day 10) 75 m g A g Females 9R17355 (on compound 9 days, terminated day 10) , 9R17356 Adrenals (died day 10) 9R17357 Liver (on compound 9 days, terminated day 10) * 9R17358 Uver (on compound 9 days, terminated day 10) 9R17359 (on compound 9 " days, terminated day 23) Gross Observations Microscopic Observations No significant changes Minimal fatty changes were present as approximated 1-2% of the hepatocytes had cytoplasmic fat vacuoli 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 hepatocytes had cytoplasi fat vacuoles indicating mild fatty changes. Puncture hole just anterior to thoracic in let; 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 approximately 10% of the hepatocytes had cytoplasmic fat vacuoles (none) Enlarged? Possible pale area Pale streaks (none) Congestion and focal hemorrhages were present in the cortex and medulla. No significant changes The lesions observed grossly were focal areas of coagulation necrosis accompanied by infiltration of an acute to subacute inflammatory exudate. 003712 Table 4 (concluded) Groaa end Microscopic Tissue Observations 17 Dose Group end Rat No. ,, 75 ma/kq Females (con't) 9R17360 Uver 1 (on compound 9 days, terminated day 10) Adrenals 9R17361 , Uver (on compound 9 days, terminated day 23) 9R17362 (on compound 9 days, terminated day 23) 9R17363 (on compound 9 days, terminated day 10) , 9R17364 (on compound 9 ' terminated day 23) 9R17365 (on compound 9 terminated day 23) 9R17366 t (on compound 9 terminated day 23) Gross Observations Microscopic Observations Pale streak Appear enlarged Mo significant changes (none) There were focal areas of coagulation necrosis which were usually accompanied by a subacute inflammatory axudate. No significant changes There were minimal fatty changes as approximately 1-2% of the hepatocytes had cytoplasmic fat vacuoles, (none) (none) (none) (none) 003713 Table 5 Two Week Oral Rangefinding Study of T-2509CoC in Rats Repeat Dose Effects Animals Affected/Day Effect 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 200 mg/kg/day Males Appeared normal Soft feces Tucked-up appearance Thin Urinary incontinence Tonic convulsion-- Unkempt appearance Deaths 200 mg/kg/day Females 12 12 3 4 NE NE NE NE NE NE 7 NE NE 4 1 1 NE NE 4 8 io- 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 00001632 J Appeared normal Tucked-up appearance Thin Urinary incontinence Tonic convulsion^. Unkempt appearance Bloody nose and mouth Hyperactive Deaths 12 12 11 6 NE NE NE NE NE NE NE 1 6 ; 12 11 6 4 1 NE NE NE NE NE 11 6 5 4 NE NE NE NE NE 2 1 1 NE NE NE NE NE 1 2 NE NE NE NE NE NE NE NE 1 NE NE 1 NE NE NE NE NE NE 1 NE NE NE NE NE NE NE NE NE NE NE 0 0 0 00 2 4 1 1 NE "HE "HE 1 :.i :.i "HE "HE '"NE NE "HE 1 NE NE NE NE NE NE NE NE NE NE 43211122222 NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE NE 1 NE NE 1 1 1 1 1 1 1 1 NE NE NE NE NE NE NE NE NE NE NE 1 1 NE NE NE NE NE NE NE NE NE 01100000000 75 mg/kg/day Males e Appeared normal C*J Soft feces *\1 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 1111111111111 1 NE NE NE NE NE NE NE NE NE NE NE NE 3 1 1 NE NE NE NE NE NE NE NE NE NE NE = Not evident -- Convulsive episodes generally occurred during handling (dosing, weighing, etc.) 003714 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 Tonic convulsions-- Unkempt Appearance Bloody nose and mouth Head tremors Lump on left side Deaths 75 mg/kg/day Females Appeared normal Tucked-up appearance Thin Respiratory congestion Clonic/tonic convulsion-- Unkempt appearance Hyperactive Hair loss on abdomen Deaths Study Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 NE NE 3 3 NE 2 3 3 3 NE NE NE NE NE NE 2 5 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 0 0 0 0 0 0 0 0 3 NE NE 1 1 2 2 2 2 2 2 2 2 5 2 2 3 3 3 3 3 3 3 NE NE NE 1 NE NE NE NE NE NE NE NE NE NE NE NE 7445555555555 1 NE NE NE NE NE NE 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 NE NE NE NE NE NE NE 5- 0 0 0 0 0 0 0 0 0 0 0 0 12 12 12 12 12 9 7 4 4 NE NE NE NE NE 3 4 5 6 NE NE NE NE NE NE 4 7 8 NE NE NE NE NE NE NE NE 1 NE NE NE NE NE NE NE NE 1 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 000000000 4222222222222 5 1 1 NE NE NE NE NE NE NE NE NE NE 7442222222222 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 4224444444444 NE NE NE 1 1 1 1 1 1 1 NE NE NE NE 1 1 1 1 1 1 1 1 1 1 1 1 6~ 0 0 0 0 0 0 0 0 0 0 0 0 003715 NE = Not evident a k Convulsive episodes generally occurred during handling --c Euthanatized -- Five euthanatized, one found dead (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 cf 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 d 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 003716 Table 7 Two Week Oral Rangefinding Study of T-2509CoC in Rats Group Mean Body Weights Dose Group -6 200 mg/kg/day d 138.5 9.21 200 mg/kg/day ? 133.0 5.43 75 mg/kg/day d 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 6 134.219.03 114.110.25 170.9 20.23 143.0 8.32 Study Day 9 13 -- -- . mm 124.8 10.05 165.519.39 129.515.16 154.0 14.4 175.3 16.33 148.3 16.58 20 ___ 186.5 0.71 224.7 17.19 182.4 9.03 % Change from Day +1 23 End of Dosing End of Recovery . -25% ______ -31% +13% 245.7 - 5% 18.58 190.7 -21% 13.80 +41% +16% -- Dosing discontinued following day 5 -- Dosing discontinued following day 9 003717 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 recovery interval. The later-occurring deaths in the high dose female group were probably due to a generalized debilitated condition because none of these animals had microscopic 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. 003718 Appendix I Two Week Oral Rangefinding Toxicity Study of T-2509CoC in Rats Protocol 23 TITLE: Protocol for a Two Week Oral Rangefinding Toxicity Study in Rats (Study Number 179RR023) 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, 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 constant dose volume of 10 ml/kg will be used for each dosage level. Distilled water will be used for all necessary dilutions of the test article. 003719 Appendix I (continued) 24 Dose Group High Low Dosage Levels (2) (to be selected after the acute portion has been completed) Group Size 12 e, 12 $ 12 <S, 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 tempera ture 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). 003720 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 and examined histologically will be selected by the pathologist at the conclusion of the gross necropsy. The tissue selections will be reviewed with the sponsor prior to processing (5) . 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). 003721 Appendix I (concluded) 26 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 ob served for approximately two more weeks. 2. The selected high dose level was 200 mg/kg/day and the selected low dose level was 75 mg/kg/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. . 003722 Appendix II Two Week Oral Rangefinding Toxicity Study of T-2509COC in Rats List of Principal Participating Personnel 27 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 003723 DISTRIBUTION LIST M. T. Case K. L. Ebbens F. D. Griffith W. C- McCormick R. A. Nelson G. R. Steffen (2) 003724