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F-xperiment No.: Conducted At: Dates Conducted: Conducted By: Reviewed By: Acute Oral Toxicity Screen with T-3421 in Albino Rats 0883A.RO287 Safety Evaluation Laboratory Riker Laboratories, Inc. St. Paul, Minnesota August 2, 1983 to September 7, 1963 'I) ,U4-, . D. M. Markoe, Jr., BS" Toxicologist Study Director @O/Zi E(ate K. D. O'Malley, BS Senior Toxicologist Acute Toxicology Date i@.@L.-Eb-bent'-, ES Date Supervisor, Toxicology Testing dc: M. T. Case f it:li---Z W. C. M,--Cormick Summary The acute oral toxicity screen with T-3421 was conducted from Aug-ust 2, 1983 to September 7, 1983 at Riker Laboratories, Inc., St. Paul, Minnesota using male and female albino rats ranging in body weight from 191-258 grams. The test article was administered by gastric intubation at dosage levels of 5,000, 2,000, 500 and 200 mg/kg body weight with mortalities of 10/10, 10/10, 10/10 and 2/10 noted respectively from one hour to six days post dose administration. The untoward behavioral reactions which occurred during the 14 day observation period generally consisted of hypoactivity, lethargy, prostration, diarrhea and unkempt appearance with the onset occurring from 1-30 minutes to day four post dose adndnistration. Clonic convulsions were noted in two animals prior to death, trancient alopecia was noted in two animals while dyspnea and salivation were noted in one animal during the study. All reactions subsided by day eleven or death precluded recovery. Body weight gains were noted in animals which survived the study period. Necropsies performed at termination of the study revealed no visible lesions while hyperemic or hemorrhagic lungs and/or hemorrhagic intestinal tract generally were noted in the animals which died during the conduct of the study. The acute oral LD50 of T-3421 is greater,than 200 mg/kg and less than 500 mg/kg in male and female albino rats. Introduction The objective of this study was to determine the acute oral LD50 of T-3421 in albino rats. This study was conducted in accordance with the Food and Drug Administration's Good Laboratory Practice Regulation of 1978. The raw data generated by the Study Director and the final report are stored in the conducting laboratory's archives. 2. Method and Results ta Young albino ra s- were used in this test. All animals were held under quarantine for several days prior to testing with only animals which appeared to be in good health and suitable as test animals at the initiation of the study used. The rats were housed in stock cages in temperature and humidity b controlled rooms and permitted a standard laboratory diet@ plus water ad libitum except during the 16 hour period immediately prior to gastric intubation when food was withheld. Five male and five female rats were adrlinistered the test material at preselected dosage levels. The doses were administered at a constant volume of 10 ml/kg directly into the stomachs of the rats using a hypodermic syringe eauipped with an intubation needle. After gastric administration of the test material, the rats were returned to their cages and observed for the following 14 days. Initial, seven day and final body weights, mortalities (Table 1) and adverse reactions (Table 2) were recorded. A necropsy was conducted on all animals that died during the study as well as those euthanatized at the end of the 14 day observation period (Table 1). The protocol, principal personnel involved in the study, composition characteristics and Quality Assurance statement are contained in Appendices I - IV. a King Labs, Oregon, WI Ralston Purina Laboratory Chow, Ralston Purina, St. Louis, MO TA.BLF- 1 ACUTE ORAL TOXICITY S7JDY - ALBINO RAFS with T-3421 Mortality, Necropsy and Body weight Data E)ose a (,-.g/lkgS)ex 5:)Oo m Animal Nt=ber 3P,3553 3R3554 3Tz3555 3R3556 3R3557 5000 3R3573 3R35'14 3P-3575 3R3576 3R3577 zooo m 3R3556 3R3559 3R3560 3R3561 3R3562 2C)OC 3P,3578 3R3579 3R3560 3R3581 3R3562 Soo m 3R4070 3R4071 3R4072 3R4073 3R4074 500 F 3R4106 3R4107 3R4106 3R4109 3R4110 individual Body Weights (g) Test Day Number: Nu=,ber Dead 0 7 14 Number Tested 208 (Day 6) - 5/5 210 (Day 2) - 197 (Day 6) - 219 (1 Hour) - 214 (Day 6) - 191 (1 Hour) - 5/5 207 (1 Hour) - 207 (1 Hour) - 192 (1 Hour) - 209 (Day 1) - 195 207 209 217 207 199 216 201 205 203 210 212 213 217 216 204 200 205 221 198 (Day 6) (Day 6) (Day 6) (Day 6) (Day 2) - (Day 6) (1 Hour) (1 Hour) (Day 1) (illiour)- (Day 3) (Day 4) (Day 6) (Day 5) (Day 4) - (Day 6) (Day 4) (Day 5) (Day 3) (Day 4) - 5/5 5/5 5/5 5/5 3. Percent Dead 100 100 100 100 100 100 TA.BL7- 1 (concluded) ACIJTE C)PAL TOXICIN STUDY - ALBII:D PATS with T-3421 Mortality, Necropsy and Body Weight Data Dose (,mg/kg) Sex 200 m Animal *4LLr@be r 3R4075 3R4076 3R4C)77 3R4078 3R4079 200 F 3R4111 3R4112 3R4113 3R4114 3R4115 I.ndividual Body Weights (9) Test Day Nu=@>er: N L=.ber Ce ad 0 7 14 '4u--t@>re Te s te d 251 (Day 6) - 1/5 236 274 297 253 303 336 256 302 345 248 249 297 220 230 243 1/5 212 242 242 217 236 236 238 253 257 225 (Day 5) - 4 Percent I>ead 20 20 '@ote Figures in parenthesis indicate time of death a Test article administered as a suspension in water. The approximate oral IZ50 is greater than 200 mg/kg and less than 500 mg/kg in fasted male and female albino rats. Necropsy Necropsy of the animals which survived the observation period revealed no visible lesions 'while necropsy of those animals which died during the conduct of the study generally had hyperemic or hemorrhagic lungs. Hemorrhagic small intestine was noted at the 500 mg/kg dose level and one animal from the 200 mg/')..dgose group. One incidence of mottled liver was noted at the 5,000 mg/kg level. TABLE 2 ACUTE ORAL TOXICITY SCREEN - ALBINO RATS with T-3421 Summary of Reactions Reactions Dose n-q/kq 5000 Sex m Hypoactivity Lethargy Salivation Dyspnea Diarrhea Unkempt. Appearance 5000 2000 F Hypoactivity Lethargy Prostration m flypoactivity Lethargy Prostration Diarrhea Unkempt Appearance 2000 F ilypoactivity Lethargy Prostration Diarrhea Unkempt Appearance Minutes 1-30 60 120 2/5 0/4 2/5 3/5 4/4 1/5 0/5 1/5 0/4 1/5 0/3 4/5 1/3 0/1 2/3 1/1 2/5 2/5 3/5 3/5 1/5 2/5 2/5 1/2 0/2 3/5 1/2 0/2 2/2 Observation Periods Number Affected/Nvmbcr D - sed Days 1 2 3 4 5 6 7 9 3/3 3/3 - - 4/4 0/3 4/4 3/3 3/3 3/3 3/3 - 5/5 4/4 4/4 0/5 0/5 5/5 4/4 4/4 - 4/4 4Z4 - 1/1 1/1 1/1 - 0/1 1/1 1/1 1/1 1/1 1/1 TA13LE 2 (conclude(l) ACUTI'@ ORAT, TOXICITY SCRF,:PN - ALBINO RAT',; with T-3421 Summary of Reactions Reactions Dose Sex rq/kq Minutes - 1-30 60 120 500 500 200 200 m Hypoactivity Cdnvulsions (clonic) Hypoactivity Ataxia m Convulsions (clonic) F Alopecia Observation Periods -- Number Af fegtgd/Nmmbvx 2gs-Z - Days 1 2 3 4 5 6 7 a 9 2/3 1/1 * 1/3 0/1 3/3 2/2 * 1/4 0/3 1/5 0/4 2/4 2/4 Key: Blank indicates no significant reactions observations Total Death inadvertenly missed over weekend APPENMIX 1 PROTOCOL Riker Experiment No.: TEST: @'C SPONSOR: 3M CONDUCTED BY: SafetyEvaluation Laboratory,Riker Laboratories,Inc.,St.Paul,Minnesota TEST ARTICLE: CONTROL ARTICLE-. PROPOSED STARTI NG,,ICOM PLET ION DATE OF TEST: TEST SYSTEM: SOURCE: Sex: Number: Weight Range: L 7. Division OBJECTIVE: METHOD: The objectiveof thistestwillbe to characteriztehe acute toxicitoyf the test articlein albino '-,::F .-P - were selectedas a testsystem for reproducibiliotfy response, historicaulse, ease inhandlingand generalavailability. The animals willbe housed instainlesssteelsuspended wire mesh cages intemperatureand humidity controlledrc>omsduringboth the quarantineand testperiods,withfoodq and water offeredad libitumt. Each animal willbe identifiebdy colorcoding, according to the laboratory'sstandard operatingpro- cedure,which willcorrespond tothe animal numbers on a card affixedtothe outsideofthecage. A single dosage of ; mglkg willbe administeredeach animal,however, itthisdosage leveldoes not adequately characterizethe toxicitoyf the testarticlea,dditionalanimalswillbe administeredthe test articleat supplemental dosage levels.Any additionadlosage levelswillbe documented and filedwith thisprotocolT.he testarticlweillbe administeredtothe animals inthe form receivedfrom the sponsor. Afteradministratioonf the testarticlet,he animalswillbe returnedtotheircages and observed forany untoward behavioralreactionsforthe following14 days. Initialnd finalbody weightswillbe recorded.A gross necropsy which willinclude,but not be limitedto heart,lungs,liverk,idneysand generalgastro- intestinatlractwillbe conducted on allanimalswhich die duringthe conduct ofthe testas wellas the animals survivingthetestperiod.Any gross abnormalitieswhich areobserved duringthe conduct ofthe necropsy willbe recorded with specificmention to the organ and/orsiteobserved.The acute medial lethaldose (LD.-;o)fthe testarticlweillbe ca)culatedi,fpossible,usinga probitanalysismethod atthe end of the observationperiod.Allraw data generated by the study directoarnd the finalreportwillbe storedinthe RikerLaboratoriesA'rchive,St.Paul,Minnesota. PurinaLaboratoryChow, RalstonPurina,St,Louis,Missouri LA.'-T-H L.L@-F"-r:-- F--jru7@1-l7I!1.V9V3E EDD JUN 1 1.OJ33 C. -)rl-.ci'@Ll Sponsor Date Study Director Date FDm, 1917i-i5-ok-Pwo Riker Experiment No. : 0883A-TZ0267 APPENDIX Ceviations and/or I (concluded) A=!endments to protocol 1. Weekena observation for Aucrust 6 ana 7 were ina,-3vertentlv missed ana on SeDteml>er 3rd. D. M. Markc>e, ljlr. Study Director 10/5/83 Date 2. Due to a aelav in stuav conduct the propose8 completion aate shoula be &-nended to 1/64. D. M. Markoe, Jr. Study Director 12/29/83_ :,at e 3. Study Direczor C-a ::e 4. Study Director [)at e S. Study Director Uto 9. APPEN'DRX rr Principal Part.Lcipating per3ornal Tnvolved in =t,.eScud_y Na= e 0. M. Markoe, jr., 8S K. L. Ebbens, RS -K. 0. O'Ma.Iley, B.S @7,.C. Pecore Eun c ti on Toxicologist Sttjdy D-Lrector Suporvisor TO xi c o 1 c@gy Te s tx ng Senior Tbxicoloq3.St Acute Toxicology Supervisor AnL=al Laboratory APPENDIX III lo. Test and/or Control Article Characterization fo 1< 1. The identity strength, uniformity, composition, purity or other pertinent characterizations of the test and/or control substances ?@ave been determined and documented as of 2. The method of synthesis or origin of the test and control substances, including their amount and the method of bioassay (if applicat)le) is documented. yes no 3. The stability of mined or will be The above cords. information the test and/or control substances have been deter- determined as of ;?--5Tt.,r3 r,@@7@: and doclnentation are located in the sponsor's re- "' Sponsor 93 E)ate Z- r V4 9S33 r:jujUi,-Ji-1 C-V Li APPENDIX IV QUA@-ITY ASSURANCE STATEMENT Acute Toxicology Study No.: Laboratory Studies This short term study was audited by Compliance kudit, and the The final re7)ort examined against the raw data on -7 results of the audit were reoorted to the study director and .0 managenent on In addition'to t-he data audit, different significant phases for studies un,:Ierway in the Acute Toxicologv Laboratcr-v are inspected weeklv on a recurring cycle, and t-he facilities are examined by Comoliance Audit on a three month schedule. 17 i Complia-n ce Audit Date