AD IITRI Project No. L6121--Study No. 6 DETERMINATION OF THE CHRONIC MAMMALIAN TOXICOLOGICAL EFFECTS OF RDX: (Twenty-four Month Chronic Toxicity/Carcinogenicity Study of Hexahydro-1,3,5-trinitro-1,3,5-triazilne (ROX) In the Fischer 344 Rat) FINAL REPORT--PHASE Volume I N• Barry S. E. Marianna VIadIslava Donovan E. Paul M. (t - V Levine Furedl S. Rac Gordon Lish November 1983 Supported by U.S. Army Medical Research and Development Command 21701-5012 Fort Detrick, Frederick, Maryland Contract No. DAMD17-79-C-9161 DT [c - ELECTE OCT 2 W5 I IT Research Institute 10 West 35th Street Chicago, Illinois 60616 B Project Officer: Jesse J. Barkley, Jr. U.S. Army Medical Bloengineering and Research Development Laboratory 21701-5010 Fort Detrick, Frederick, Maryland Approved for publ ic release; distribution unlimited The findings In this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. 85 10 01 * *:-.* -- 052 *. -_ ° Unclassified SECURITY CLASSIFICATION OF THIS PAGE (W"en Date Entered) I. REPORT NUMBER _ __ __ _ _ 2. GOVT ACCESSION NO. _::__ A READ INSTRUCTIONS BEFORE COMPLETING FORM REPORT DOCUMENTATION PAGE _ _ 3. RECIPIENT'S CATALOG NUMBER TITLE (ald Subtitle) DETERMINATION OF THE CHRONIC 5. TYPE OF REPORT & PERIOD COVERED MAMMALIAN TOXICOLOGICAL EFFECTS OF RDX TwentyPHASE V FINAL REPORT four Month Chronic Toxicity/Carcinogenicity Study October 1980 to November 1983 of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in 6. PERFORMINGORG. REPORT NUMB".. the Fischer 344 Rat L6121 Study No. 6 4. 7. AUTHOR(a) 6. Barry S. Levine, E. Marianna Furedi, Donovan E. Gordon, Vladislava S. Rac, Paul M. Lish 9. PERFORMING ORGANIZATION CONTRACT OR GRANT NUMBER(*) DAMD17-79-C-9161 NAME AND ADDRESS 10. PROGRAM ELEMENT. PROJECT, TASK AREA & WORK UNIT NUMBERS IT Research Institute 10 West 35th Street Chicago. IL 62720A.3El62720A835.00.101 60616 ,_. II. CONTROLLING OFFICE NAME AND ADDRESS 12. U.S. Army Medical Research and Development Command Fort Detrick Frederick, 14. Maryland REPORT DATE November 1983 13. NUMBEROF PAGES 21701-5012 _ MONITORING AGENCY NAME & ADDRESSQI different from Controlting Office) IS. SECURITY CLASS. (of this report) Uncl assi fied 15.. 16. DECL ASSI FI CATION/DOWNGRADING SCHEDULE DISTRIBUTION STATEMENT (of this Report) Approved for public release; distribution unlimited. I7. DISTRIBUTION STATEMENT (of the abetract entered In Block 20, It different from Report) IS. SUPPLEMENTARY NOTES b 19. KEY WORDS (Continue on reveres side it neceseary end Identify by block number) RDX Hexahydro-l,3,5-trinitro-1,3,5-triazine Fischer 344 rats CAS Reg. No. 121-82-4 Chronic toxicity i ".20. ABTrRACTr (rcafu@ m overs firrPtnageey a iderttifyby block number) -- This study was cunducted to evaluate the toxicity of the munition compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX" CAS Reg. No. 121-82-4) in Fischer 344 rats when administered in their diet for up to 24 months. Groups of 75 rats per sex received RDX at doses of 0, 0.3, 1, 5, 8, or 40 mg/kg/day. Ten rats/ sex/dose were killed following 6 and 12 months on test with surviving animals killed after 24 months of treatment. Toxicologic endpoints included clinical signs, body weights, food consumption, hematology, clinical chemistry, ophthalmology, )rqan weights, and gross and tissue morphology-___ (continued) 1403 DO IOR" DO " n EM-no. o,, Io NOV isISOBSOLETE Unclassi fied SECURITY CLASSIFICATION OF THIS PAGE (When Date Entered) *. . . . . . . . . . . "-" . . .. ." ,a'-.. i. . . . ii"''" .. ...... .. . " *"" •; ;.'"" S \:"~UnclIass if ied SECURITY CLASSIFICATION OF THIS PAGE(Wlihe Data Entered) "ýthe major toxic effects observed during the administration of RDX to F344 rats for up to 24 months included anemia with secondary splenic lesions, hepatoxicity, possible CNS involvement, cataracts and urogenital lesions. Based on the observance of increased levels of a hemosiderin-like pigment deposited in the spleen (a secondary response to a hemolytic-type anemia) and suppurative inflammation of the prostate for rats administered 1.5 mg/kg/day or greater, the no-effect level under the conditions of the present study is 0.3 mg/kg/day. 1 -. Unclassi fied SECURITY CLASSIFICATION OF THIS PAGE(W"en Date Entered) ... Contract No. DAMD17-79-C-9161 IITRI Project No. L6121 Study No. 6 DETERMINATION OF THE CHRONIC MAMMALIAN TOXICOLOGICAL EFFECTS OF RDX TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE (RDX) IN THE FISCHER 344 RAT * STUDY FINAL REPORT * Prepared by Barry S. Levine E. Marianna Furedi Vladislava S. Rac Donovan E. Gordon Paul M. Lish lIT Research Institute 10 West 35th Street Chicago, Illinois 60616 Supported by U.S. Army Medical Research and Development Command Fort Detrick, Frederick, Maryland 21701 * Project Officer: Jesse J. Barkeley, Paul M. Lish Scientific Advisor Life Sciences Research Jr. Barry S. Levine Senior Toxicologist Life Sciences Research Alan M. Sh ;ni Associate Director Life Sciences Research ( 1' "•i ,. "I11 'lll 1• l "I I I! !I lI l Il llI _ 1'~ 1/ l •• •I - l "Q '' Ii "•111 . 11 i-* "I *l "' '' 'l* -"• "~ "" 1' 1 i "" 1'" •* Ul"''' ~ l" ' " ' I * * * This study was conducted to evaluate the toxicity of the munition compound hexahydro-1,3,5-trinitro-1,3,5-trlazine (RDX CAS Reg. No. 121-82-4) In Fischer 344 rats when administered in their diet for up to 24 months. Groups of 75 rats per sex received RDX at doses of 0, 0.3, 1.5, 8 or 40 mg/kg/day. Ten rats/sex/dose were killed following 6 and 12 months on test with surviving animals killed after 24 months of treatment. Toxicologic endpoints Included clinical signs, body weights, food consumption, hematology, clinical chemistry, ophthalmology, organ weights, and gross and tissue morphology. The major toxic effects observed during the administration of RDX to F344 rats for up to 24 months included anemia with secondary splenic lesions, hepatoxicity, possible CNS Involvement, cataracts and urogenital lesions. Based on the observance of Increased levels of a hemosiderin-Ilke pigment deposited In the spleen (a secondary response to a hemolytic-type anemia), suppurative Inflammation of the prostate and decreased survival in male rats administered 1.5 mg/kg/day or greater, the no-effect level under the conditions of the present study is 0.3 mg/kg/day. 0 Accession For ~T--4~ 1 NTT5 j7 S.i• . ' (.1 (@ . . . . . C..... ................................. ............................................................ EFOREWORD 0 * 6 i ( and Research Bioengineering Army Medical The U.S. Fort Detrick, Frederick, Laboratory (USAMBRDL), Development the MD, has been conducting a research program since 1973 for developing the scientific data base necessary for purpose of unique to compounds recommending water quality criteria for the munitions Industry. A water quality criterion (as defined 1977) Is a qual itative or by the amended Clean Water Act, estimate of the concentration of a pollutant in quantitative ensure a water ambient waters that, when not exceeded, will The to protect a specified water use. quality sufficient criterion is a scientific entity based solely on data and It does not reflect considerations of scientific judgement. Currently, a water feasibility. economic or technological consists of two separate numerical limits, quality criterion for the one for the protection of human health and the other when organisms. These numbers, protection of aquatic can be the translated by the appropriate regulatory agency, discharge or effluent limitations In a enforceable basis of under the Clean Water point source discharge permit issued Act. designated Since a water qual ity criterion is to protect water uses, a diverse, multidiscipli nary research program was that Includes "effects" studies on developed by USAMBRDL domestic animals, wildlife species, aquatic laboratory and In Important crops. plants, and economically organisms, fate and chemical and biological addition, extensive the persistence tests are conducted to provide information on These in the aqueous environment. behavior of a pol lutant making site-specific kinds of data are especial ly useful for translation of criteria into enforceable discharge limits. mammal ian of the a portion This report represents on developed by USAMBRDL data base being toxicology noted It should be hexahydro-1,3,5- trinitro-1,3,5-triazine. and IV were parallel studies with TNT, II, I1l, that Phases The Phase I DAMD17-79-C-9120. conducted under Contract No. report included subchronic studies with both compounds. were experiments Animal E£erimentatni .6niLmal conducted in accordance with the "Guide for the Care and Use No. (NIH) DHEW Publication of Laboratory Animals" (1978), 78-23, prepared by the Committee on Care and Use of Laboratory Laboratory Animal Resources, the Institute of Animals of and standards Council; the regulations National Research and the Public Law prepared by the Department of Agriculture; 91-579, "Laboratory Animal Welfare Act" (1970). 4 S.... . °.. . • . . , . o • . . . . o = In names Citation of commercial organizations and trade Department of the report does not constitute an official this of services or products the of Army endorsement or approval organizations. these ,.. is '~.6 ACKOLE.UfiMi • * * ,-"7 This report was prepared at IIT Research Institute, 10 West 35th Street, Chicago, Illinois, 60616, under U.S. (I ITRI No. DAMD17-79-C-9161 Army Contract of Department Project No. L06121) entitled "Determination of the Chronic Mammal ian Toxicological Effects of RDX". Mr. Jesse J. Division, USAMBROL, Jr., Health Effects Research Barkley, for served as the Contract Officýrls Technical Representative this program. The work reported herein was conducted In the Toxicology and Division, Life Sciences of the Section and Pharmacology named of the above represents a portion of the overall effort research program. Paul R1. Lish, Ph.D., Scientific Advisor, served as Principal Investigator. Barry S. Levine, D.Sc., Senior Toxicologist, served as study director and was responsible for the overall conduct of the study. Eva M. Furedl-Machacek, DVM, served as study toxicologist and was also responsible for the supervision of the technical support personnel. John M. Burns, DVM, Senior Veterinary Pathologist, Bobby R. Collins, DVM, M.S., and Vladislava S. Rac, DVM, M.S., were consecutively responsible for supervision of gross necropsies. Carol A. Thompson, DVM, M.S., tabulated the gross necropsy data. Drs. Burns and Levine served as consecutive heads of the the clinical pathology laboratory, and Don Reitman, Samuel Terese, B.S. (ASCP-MT), and Debbie L. Sava, B.S. (ASCP-MT), were responsible for generation of clinical pathology data. Donovan E. Gordon, DVM, Ph.D., Consultant, Veterinary Pathology, was responsible for tabulation and evaluation of histopathology data. Bobby R. Collins, DVM, M.S., and Joseph B. Harder, DVM, served as clincal veterinarians and supervised animal care personnel. Joann M. Hinz, B.S., and Robert M. Renaud, B.S., were responsible for the collection of test data. Dorothy Davis (ASCP-HT) was responsible for preparation of histology slides. C. Susan West, DVM, performed the ophthalmic examinations. Reed, M.M., M.S., Supervisor, Quality Assurance, Josephine M. was responsible for the quality assurance program. Robert Remaly, B.S., Senior Engineer, was responsible for preparation of the test article premixes. Hugh J. O'Neill, Ph.D., Manager, Analytical Chemistry, Walter C. Eisenberg, Ph.D., Senior Chemist, and Debbie Cunningham, Assistant Chemist, were responsible for chemical analyses of test article, test article premixes and test diets. Jean Graf provided the particle size analyses. Robert Gibbons, Ph.D., provided statistical and computational assistance. phases of critical Laboratory Inspections covering all the study were conducted on twenty-four occasions during the The dates are as fol lows: December 11, course of the study. 7, 22, and 26, April 30, May 20 and 28, June 1980; January 10 and 24, 29, November 18, July 22, August 28, September 1981; and January 18, February 10, April 21, May 27, July 8, 1982. August 24, October 12, and November 10, 16, 23, and 29, audits were conducted beginning on January 15, 1981 with Data 30 April subsequent audits conducted throughout the study on June 18 January 18 to 21, March 11, 29, 1981; and September to 21, July 8, August 2, September 15 and 23 and October 17 to In addition Chemistry operations and data November 2, 1983. diet samples premix or collection were monitored each time final draft report was analysis. The submitted for were Inspections auolted between January 30 and February 10, 1984. and audits were performed ýy Josephine Reed, Julie McPhilips, Susan Nadolny and Kirit Parikh. The study was founl to meet Specimens and IITRI Life Sciences Quality Assurance criteria. retained In the raw data generated during the study will be In standard Archives as specified Life Sciences I ITRI operating procedures. Josephine M. Reed Supervisor, Quality Assurance .. ......... % ~1 A- V ULUIE 1 I. II. III. 16i IV. V. * INTRODUCTION .................................. 15 MATERIALS AND METHODS ......................... 15 A. B. C. D. E. 15 16 17 19 23 Test Article .............................. Test Diets ................................ Test Animals .............................. Experimental Design ............................ Statistical Analysis ...................... RESULTS ....................................... 24 A. B. C. D. E. F. G. H. I. J. 24 24 24 25 25 25 26 26 27 27 Test Diets ................................ Food and Water Contaminants ............... Mortality/Clinical Observations ......... Body Weight .............................. Food Consumption ................ Hematology ................................ Clinical Chemistry ........................ Ophthalmology ................. Organ Weights ............................. Pathology ................................. DISCUSSION ................................ REFERENCES ......... APPENDIX APPENDI APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX APPENDIX I II III IV V Vi VII Vill APPENDIX IX DISTRIBUTION S~9 . *.... ................ ...... TEST ARTICLE ANALYSIS ................. 5002 CERTIFICATION PROFILE/ANALYSIS... TEl ANALYTICAL CHEMISTRY METHODS ...... HEMATOLOGY METHODOLOGY ................ CLINICAL CHEMISTRY METHODOLOGY ........ INDIVIDUAL ANIMAL DATA ................ CHLORTETRACYCLINE CONTENT OF 5002 ..... NITRATE, NITRITE AND MERCURY CONTENT OF 5002 ............................... CHICAGO WATER CHEMICAL ANALYSIS ....... LIST .................................... 28 29 123 159 163 165 169 173 341 343 345 347 .~~~~~~~~1 . . .. . .. TAB LE D.E APPENDIX X . iLQjE~f±1JI OPHTHALMOLOGY ..- &L QDNL REPORT ...................... 348 VQLUAE JJJ1 APPENDIX XI PATHOLOGY REPORT .................................. 594 * Requests for Volumes II and III should be directed to Health Etfects Research Division, U.S. Army Medical Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, Maryland 21701-5012. 1 10 "- 0 * * " I Male 2 3 Female Actual Doses Received ........................... Test Diet Concentrations of RDX ........................ 4 5 Mean Survival Time ..................................... Male Body Weights ......... .............. 6 Cumulative Male Body Wedght Gains ...................... 7 Female Body Actual Doses Received ............................. Weights ................. 32 36 40 ................ ... 47 ....... ....... 52 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 Cumulative Female Body Weightd G.......... Male Food Consumption Measurements.................. . Female Food Consumpt ion Measurements................... Male Hematology Values - Test Week 13.,................ Female Hematology Values - Test Week 13 ........ o....... Male Hematology Values - Test Week 26.................. Female Hematology Values - Test Week 26 ................ Male Hematology Values - Test Week 52 ................. Female Hematology Values - Test Week 52 ............... Male Hematology Values - Test Week 78 .......... Female Hematology Values - Test Week 78 .......... o..... Male Hematology Values - Test Week 104 ................ Female Hematology Values - Test Week 104 ................ Male Clinical Chemistry Values - Test Week 13 .......... Female Clinical Chemistry Values - Test Week 13........ Male CI inical Chemistry Values - Test Week 26 .......... Female Clinical Chemistry Values - Test Week 26 ........ Male Clinical Chemistry Values Test Week 52............ Female Clinical Chemistry Values - Test Week 52........ Male Clinical Chemistry Values - Test Week 78 .......... Female Clinical Chemistry Values - Test Week 78 ........ 29 Male 30 31 32 33 34 35 36 37 38 Female Clinical Chemistry Values - Test Week 104....... ......... ..... Ora..n.Weiht. Rl.e Cataracts .............. Male Mean Relative Organ Weights - Test Week 27....... Female Mean Relative Organ Weights - Test Week 27 ...... Male Mean Relative Organ Weights - Test Week 52-...... Female Mean Relative Organ Weights - Test Week 52....... Male Mean Relative Organ Weights - Test Week 105....... s -TestWeek 105. .... Female Mean Relative Organ eWe Male Mean Organ Weights - Test Week 27 ............... 40 41 42 43 44 45 Male Mean Organ Weights Female Mean Organ Weights Male Mean Organ Weights Female Mean Organ Weights Statistical Evaluation of Statistical Evaluation of 39 Clinical Female Mean Chemistry Organ Values - Weights - Test Week Test Week 41 42 104 ......... 27 ...... .... 57 62 66 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 o 88 .. o.. 89 90 91 92 93 94 95 96 97 98 Test Week 52 .................. 99 - Test Week 52 ............... 100 Test Week 105 ............... 101 - Test Week 105....o.........102 Histopathologic Lesions (M)..103 Histopathologic Lesions (F)..109 11 .t -'-¢ ----- ¢ 3 --. .'•'3 • -:-: -'' '- '-V . " -' . -- .... .. . .. - .". - -i. - . .. • -• -. .... .? ' -'-' - -- ' .. -•. • .• -i. --- LIST Pf I&BLiS i=N.QLLDED. VI.1 VI.2 Survival Rate Data ...... ....................... ...... 174 Individual Body Weights (g) ............................ 181 Individual Food Consumption Measurements (g/day) ....... 238 Individual Hematology Values - Test Week 13 ............ 295 Individual Hematology Values - Test Week 26 ............ 298 Individual Hematology Values - Test Week 52 ............ 301 Individual Hematology Values - Test Week 78 ............ 304 Individual Hematology Values - Test Week 104 ........... 307 Individual Clinical Chemistry Values - Test Week 13 .... 310 Individual Clinical Chemistry Values - Test Week 26....313 IndI v Idua Clinical Chemistry Values - Test Week 52 .... 316 Individual Clinical Chemistry Values - Test Week 78 .... 319 Individual Clinical Chemistry Values - Test Week 104...322 Individual Organ Weights (g) - Test Week 27 ............ 325 Individual Organ Weights (g) - Test Week 52 ............ 328 Individual Organ Weights (g) - Test Week 105 ........... 331 VI.3 VI.4a VI.4b VI.4c * SVi.4d VI.4e VI.5a VI.5b VI .5c Vl.5d VI.5e Vi.6a VI.6b VI.6c * . S12 . * . * * * * * ,. . . * . . . . . . . * .. . * * *. .... - . * *.. *.. .*. .***. .-. . .*.t.. . . * * * . * . . . . . LIST OF FIGURES Pu~gt ED.- f E~ar.er 3 4 5 Survival Curves for Control and High ... Dose Males ........-.................... Survival Curves for t;ontrol and High Dose Females .............................. Male Cumulative Mean Body Weight Gains (g).. Female Cumulative Mean Body Weight Gains (g) Mean RBC Values ............................. 6 Mean Platelet Values ................. 0...... 117 Mean Mean Mean Mean Mean 118 119 120 121 122 1 2 7 8 9 10 11 Glucose Values....**.........0-0...... Cholesterol Values... ............. #... Trlglycerlde Values.................... Female Total ProteinValues............ Female Albumin Values.................. 112 113 114 115 116 ( 13 ""(- - -:.,:••"" """ • -•'',•"' "" ""' . ."" " "=, •; .""• ,•= -•.s• "' .. .""-•:; . ". ". ' ' ' ."." ,",. ",,".•"..'..",,' - '- '...."".- "'_'_'' " .. ", I. INTRODUCTION * Command Development The U.S. Army Medical Research and to evaluate the potential hazards (USAMRDC) has been directed from munitions to living systems of wastewater discharges to facilities. Of primary concern are the toxicologic effects of hexahydro-1,3,5-trinitro-1,3,5-triazine mammalian systems No. 121-82-4). This high explosive is (RDX; CAS Reg. In filling shells and bombs. Wastewaters routinely used from the loading of this explosive Into shells are resulting treatment discharged into the environment without significant to limitations imposed by governmental and are subject hazards of Evaluation of the potential regulatory agencies. health Is therefore a necessary these wastewaters to human establish comprehensive portion of the data-base required to environmental criteria. * evaluation The present study was conducted to aid In this and carcinogenicity of ROX and assessed the chronic toxicity when administered In the diet for at least in Fischer 344 rats derived from this Information ultimately 104 weeks. USAMRDC In comprehensive long-term toxicology study will aid of effluent for the establishment developing criteria levels of treatment for its standards and In defining pollution abatement program. in accordance The study reported herein was conducted with the IITRI Quality Assurance Program designed to comply Thus, all with FDA Good Laboratory Practice Regulations (1). test article, raw data, used in this report, e.g. terms set specimens, etc., are In agreement with the definitions forth In the aforementioned document. II. hA•IE•LSJ A. A&W METHIOiD Ig,_J Artic]J SHexahydro-1,3,5-trinitro-1,3,5-triazine (RDX: CAS Reg. HOL 435-37, 100 pounds, was made No. 121-82-4), batch No. the IITRI Kingsbury study from stocks at available for this La Porte, In. The Ordnance Plant (KOP) Explosive Facility, at the facility at ambient room test article was stored and in the dark. Upon temperature, relative humidity, phase of the initiation and at termination of the treatment study, 30 g samples were taken and stored under conditions similar to those for the batches. ( determined The purity of the test article was with annlytical performance liquid chromatography as. described in Appendix provided by the Sponsor by high standards I. RDX 15 The this study. three times during was analyzed purity May 1982 + 2.9%), May 1981 (91.0 results were as follows: The maln + 2.0%). (98.7 1983 April + 8.0% and (89.2 contaminant was HMX and represented approximately 3-10% of the concentration on the basis of percent area sample (estimated Integration). The other Impurities were not determined. and 1979 In November "Particle size analyses were done 1981 by the Fine Particles Research Section of the November of IIT Research Chemistry and Chemical Engineering Division "Institute. The results were as follows: November- IM9 Date• "£Lze U.mI <22 22-44 44-66 66-110 110-220 220-330 330-440 >440 Number 355 184 75 38 22 10 2 1 Nov ember 19811 I umnuJ.• 51.7 26.8 10.9 5.5 3.2 1.5 0.3 0.1 51.7 78.5 89.4 94.9 98.1 99.6 99.9 100.0 Ii ume IN 105 216 92 41 38 8 0 0 21.0 43.2 18.4 8.2 7.6 1.6 ul I 21.0 64.2 82.6 90.8 98.4 100.0 10% In (approximately article, the test Premixes of 5002, Ralston Purina Co., Rodent Chow No. Certified Purina hereafter referred to as 5002), were prepared St. Louis, MO., on a monthly basis in 4 kg quantities at the KOP facility by Chemistry Division personnel. Undiluted RDX was handled in for explosive and fire hazards. accordance with procedures The test article was ball milled with equal parts of 5002 and additional 5002 in a twin shell diluted with subsequently 10% premixes. yield approximate blender to Each RDX premix was routinely tested for homogeneity, RDX 10% premixes of the test article. and recovery potency had previously been shown to be homogeneous and stable for at Homogeneity testing consisted of analyzing least 7 weeks (2). taken premix for test article concentration of each batch of Premix stability random locations of Its container. from 6 later for a was establ ished for a period of seven weeks and nine weeks by conducting homogeneity tests at the period of "Initial and the terminal point of the 7 or 9 week period. eight test diets (2 diets/sampl ing week) used in Test Twenty 100 and Weeks 2, 15, 30, 45, 52, 58, 64, 70, 76, 82, 88, 94, In addition, 104 were analyzed for accuracy and homogeneity. "one control and two test diets were monitored for stability 16 .-. . . . . sampled They were under animal cage conditions for one week. they were placed in the animal's cages and also one the day week later from the uneaten portion of the diet. a of adding Premix and diet recovery studies consisted to a weighted quantity of of test article known quantity (the of acetonitrile volume untreated 5002 In a measured to achieve the procedure) used in the extraction solvent samples The spiked calculated premix or diet concentration. subsequently underwent the identical procedures as the actual diets. articles Toxicology Section personnel received the test These premixes posed in 5002. 10% premixes as approximate (2). described explosive or fire hazard as previously little Following chemical analysis of the premixes to determine test article concentration (Appendix 1), sufficient quantities were a twin shell blender by with 5002 In diluted subsequently toxicology personnel to achieve the concentrations of the test necessary to administer the required dose levels on a article food body weight and The previous weeks' mg/kg/day basis. by sex were used for each test group measurements consumption and food consumption to calculate the projected body weight the desired dietary on these values, values. Based and Twenty concentration of the test article was calculated. later 16 kg of each test diet (in two batches) was routinely prepared on a weekly basis. G, was analyzed 24 82 One sample of 5002, lot March Park the course of the study by Trace Elements, Inc. during in the 5002 listed Ridge IL. (TEl) for those contaminants certification profile as shown in Appendix II. The references Ill. On the Appendix to the procedures used by TEl are In chlortetracycl ine content, the analytical results for basis of reserve samples of al iquots from this and three additional In addition, al iquots sent to TEl for analysis. 5002 were sent to Scientific reserve samples were from these four Louis, MO, Woodson-Tenent Laboratories, Associates, Inc., St. Inc., Lincoln, Laboratories, Inc., Memphis, TN, and Harris Samples of each 5002 analysis. for chlortetracycl ine Neb. nitrite lot used in the study were also analyzed for nitrate, and mercury content. C. *a An Jma Is Fischer 344 (F344) rats, obtained from Harlan Four WI, were used for this study. Sprague-Dawley, Madison, in hundred and thirty six males and 447 females were received good condition on November 3, 1980. They were 3 to 4 weeks 17 .::•.•:,••..•._. ,..-j- ,• ,-•',:: .' ... • ,•'-./ .;. •]• .,.,'.: • ,,:,,- ,.,,., ... ,:-,- .-' -'y..,:::..,,.,... , .. /.2: old upon arrival three days (females). of and random body receipt were weight means 37+ 8 g recorded (males) within and 35± 7 g The shipment was housed In two quarantine rooms, one for each sex. The animal room conditions during quarantine, pretest and test periods were as follows: 20-25 degrees centigrade, ambient relative humidity (30-70%), and 12 hour light/12 hour dark cycle. There were no other test animals in the rooms. The animals were housed three per polycarbonate x 8"; 8" height) with Ab-sorb-drl bedding cage (16.5" (Ab-sorb-dri Inc., Rochelle Park, N.J.) from arrival until their termination. Animals were transferred to clean cages twice weekly. Some of the male rats at the 40.0 mg/kg/day dose level were Individually housed at the onset of Test Week 30. By Test Week 40 all males at this dose level were housed separately. This was done to avoid Injuries by cagemates and to avoid additional stress as RDX appeared to effect the nervous system. Each animal was Identified during the quarantine period by a combination of cage number and ear punch. Test animal selection was done at the onset of Test Week -2 (2 weeks prior to initiation of treatment). Animals placed on test received a study-unique test animal number (N=750) which appeared as a tall tattoo. Subsequently they were identified by an ear tag which was Included with necropsy specimens. Upon arrival at the IITRI animal facility, the animals were held In quarantine for 9 days. During this period, they were observed for signs of disease, general unthriftiness, poor coat, discharge from body openings, abnormal feces, etc. Any animals found to be unhealthy were eliminated from the test animal selection process. At the end of the quarantine period five animals of each sex were sacrificed. Extensive gross necropsles were performed under the supervision of the pathologist. Blood samples were collected for measurments of hematology and cl incal chemistry parameters (see section I I.D) and serum antibody titer determination for the fol lowing: GD-VII, H-1, Kilham Rat virus, Adenovirus, Sendal virus, Rat Coronavirus-Sialodacryadenitis, Reovirus 3, Pneumonia virus of mice and Lymphocytic Choriomeningitis. These antibody titers were measured by Microbiological Associates, Bethesda MD. The Sendai virus antibody titer was elevated for some of these animals. Blood samples were subsequently collected from five males and five females from the control treatment group and serum was sent for additional testing. All ten of these samples were found to be within normal limits for Sendal virus antibody titer. their 18 Animals received 5002 rodent chow from arrival until termination, except during a 17 to 19 hour fast prior to The food was blood collection and/or scheduled termination. from powdered diet feeders (Model HB-69B, Hoeltge, available AA libtur Tap water was available Inc. Cincinati, Ohio). bottles. from glass or plastic D. Ex&Lerlmntal Dlescli test-eligible animals period, Following the quarantine were assigned to five treatment groups by a stratified weight). Fol lowing randomization procedure (blocked by body assignment to treatment groups, all animals were randomly Mean body weight animal numbers as shown below. assigned test at randomization were 58.7 ± 10.0 g (males) and 59.1 ± values This procedure was performed at the onset of 8.6 g (females). The animals were approximately 6-7 weeks old Test Week -2. weight mean values of treatment and body upon initiation Test Week -1 (the most recent data prior to recorded during Initiation of treatment) were 111 + 11 g (males) and 93 ±+ 8 g article was day of exposure to the test (females). The first December 1, 1980. Tr..a±wea± fzQLj2 Allocation:a Treatment Group Animals Dose Level per Sex (mg/kg/day) I. II. III. IV. V. 0 75 75 75 75 75 0.0 0.3 1.5 8.0 40.0 Test Animal No. (males) Test Animal No.(females) 1- 75 151-225 301-375 451-525 601-675 76-150 226-300 376-450 526-600 675-750 available to the test The appropriate test diets were termination animals AAd IlLiLb±m from Test Day 1 until their prior to either blood hour fast except during a 17 to 19 53 and 78 or scheduled Test Weeks 13, 26, collection In Thus, all animals termination In Weeks 27, 52 and 105-106. diet until approximately one day received the appropriate test were Weekly test diets prior to their scheduled sacrifice. group by sex on the basis of prepared for each treatment predicted body weight and food consumption data. termination, all Commencing with Test Week -1 until their morning for any observed once daily in the animals were Afternoon mortality pharmacologic and/or toxicologic signs. Physical examinations were initiated on Test Day 1. checks masses were palpations for which Included body weights and conducted weekly from Test Week -1 through Test Week 14, then Food consumption was measured biweekly through Test Week 104. weekly for each cage of test animals commencing with Test Week 104. Week -2 through Test Week 14,then biweekly through Test 19 • '.°. •.". o . °.". . .. 'o% , ' _. %.. .. ,... . .- " " "l "" ' °°'""'" oI " " % "° %" " % '* ""%" " "" "Z %"• .2. ,° l•%, ,*. Mean daily these data. food consumption per animal was calculated from ophthalmic to subjected were animals surviving AlI The 25, 51, 76 and 103. during Test Weeks -2, examinations and ophthalmoscopy indirect of consisted examination found to be free of clinically Only animals blomicroscopy. In the examination were used apparent lesions In the pretest study. randomly 10 were performed for Serial blood collections animals/sex/dose level during Test Weeks 13, 26, 52, selected and chemistry cl inical of measurments for 104 and 78 its to prior died animal an If parameters. hematology scheduled blood collection it was replaced by another randomly Approximately the same sex and dose level. of rat selected the via animal each 1.5-2.0 ml of blood was collected from 3 a over collected were samples The sinus. orbital consecutive day period and analyzed in a randomized order. The fol lowing parameters were measured: Hematocr it Hemog I ob i n Mean corpuscular Mean corpuscular Mean corpuscular Erythrocyte count Leukocyte count, count Platelet £L lJinLcaf. Cb itr volume (MCV) hemoglobin (MCH) hemoglobin concentration (RBCs) total and differential (MCHC) " "Glucose "Blood urea nitrogen (BUN) Serum glutamic-pyruvlc transaminase total and direct Billrubln, Lactic dehydrogenase Creatinine phosphokinase Alkal ine phosphatase Triglycerides Total cholesterol Total protein Albumin Globulin (calculated value) (calculated value) A/G ratio (SGPT;ALT) 20 - *=K « x~*-* , Sod I umr Potassiumrn Chloride Calcium Methods used to measure the above parameters are listed In Appendix IV (hematology) and Appendix V (cl inical chemistry). All animals which were sacrificed In a moribund state or died on test were necropsled regardless of autolytic state. Ten randomly selected animals/sex/dose level, after exclusion of animals designated for blood collection, were sacrificed during Test Weeks 27 and 53. Three hundred and forty six surviving test animals were sacrificed and necropsled in random order during Test Weeks 105 and 106. Terminal body weights were recorded immediately prior to sacrifice. Euthanasia was accomplished with carbon dioxide anesthesia followed by exsanguination from the abdominal aorta. The necropsy procedure was a thorough and systematic examination of the animal viscera and carcass with collection and fixation of the following tissues: ro *Adrenals Bone marrow smear *Brain I. Cecum Colon Costochondral junction, Duodenum EpIdidymes Esophagus Eyes and optic nerve Gross lesions *Heart Ileum rib Jejunum 6 *Kidneys Larynx *Liver Lungs and mainstem bronchi Lymph nodes (mandibular and mesenteric) Mammary gland Muscle Nasal turbinates *Ovaries Pancreas Pituitary gland Prostate Rectum Salivary gland Sciatic nerve 21 . . ... ..--.. .- .-.---. .-.---- v --..--...-.-- -. - - .- - . -- - - . - , .- .- --,--. , . .- -, -- . . . -- .-..- . . . .. Seminal vesicles "Skin, abdominal Spinal cord (cervical, thoracic, *Spleen Sternum, Including bone marrow Stomach *Testes Thymus Thyroids (parathyroids) Tissue masses Trachea Urinary bladder Uterus *• lumbar) *These organs were weighed during scheduled necropsles. All tissues, except eyes, testes and bone marrow smears, were fixed at a thickness not exceeding 0.5 cm In 10$ neutral buffered formal in (NBF) which was changed 24 hours later. Eyes and testes were fixed In 3$ aqueous glutaraldehyde and Bouin's Solution, respectively, for 24 hours. They were transferred to 50% ethanol for 24 hours, then placed in 70% ethanol. Bone marrow smears were prepared from the femur using the "paint brush technique". They were air-dried and fixed In absolute methanol. Lungs and urinary bladder were Inflated with NBF prior to immersion in this fixative. The stomach was opened and flattened on paper prior to fixation. All tissues examined microscopically were cut at a thickness of 4 to 6 microns and stained with hematoxylin and eosin. Tissues from all animals receiving 0.0 and 40.0 mg/kg/day were subjected to comprehensive histopathologic examination, defined as microscopic examination of the following tissues and/or organs: Adrenals *Brain (3 sections) Cecum Colon Duodenum Esophagus Epididymes Eyes and optic nerves Gonads Gross lesions Heart Ileum Jejunum Kidneys LLiver Lungs and mainstem bronchi 22 Mammary gland Mesenteric lymph node Pancreas Pituitary gland Prostate Rectum Seminal vesicles Skin, abdominal Spinal cord (cervical, thoracic and lumbar) Spleen Sternum Including bone marrow Stomach Tissue masses Thyroids (parathyroids) Trachea Urinary bladder Uterus * *(I) frontal and thalmus; C cortex and basal and (3) ganglia; (2) parietal cortex cerebellum and pons. Tissues from all animals receiving 0.3, 1.5 and 8.0 mg/kg/day were subjected to limited histopathologic examination defined as microscopic examination of at least the following tissues and/or organs: *Bra i n Gonads Heart Liver Kidneys Spleen Spinal cord (cervical, thoracic and lumbar) *(I) frontal cortex and basal ganglia; (2) and thalmus; and (3) cerebellum and pons. E. parietal cortex SaatLstIcaJL Analys Those variables that were repeatedly measured, e.g. body weight, food consumption, and cl inical pathology parameters were statistically analyzed using a multivariate analysis of variance for repeated measurements model. Variables that were measured a single time, e.g. organ weights, were analyzed using both univariate and multivariate analysis of variance procedures. In the presence of significnt ANOVA results, a series of post-hoc analyses were conducted. Individual between group comparisons at each time-point were performed using Tukey's b test for multiple comparisons. Frequency data, such as Incidence of mortality, gross necropsy observations and histopathologic lesions were compared using 23 . . .... . * . '* . .* .. .. Time to appropriate. where log linear analysis techniques death data were analyzed using Kaplan-Meler and Cox regression Chi-square analyzed by were lesions analyses. Ophthalmic test. Individual animal data can be found in Appendix VI. IIII. RE..JLIL A. Tes-t IDiets body on their animals based by test Doses received weights and food consumption, and theoretical concentration of in the diet are shown In Tables 1 and 2. test article test in of RDX Analytically determined concentrations intended very close to their were found to be diets for the ± S.D. mean The overall percent concentrations. diets were When test ratio was 97.0 ± 9.8%. actual/intended room, a in the animal being placed sampled one week after The decrease in RDX concentration may have occurred. slight negligible of RDX may have accounted for this known volatility change (Table 3). Fooad .0nd Water fgont mJI nantf B. for those contaminants 5002 sample The analysis of a profile Is shown In Appendix listed In the 5002 certification for of 5002 samples The results of the repeat testing II. The Is contained in Appendix VII. content chlortetracycl ne three reference laboratories which reanalyzed the 5002 samples of negligible quantities reported TEl generally following chlortetracycl mne. for nitrate, was analyzed A sample from each 5002 lot shown results are content. The and mercury nitrite sample obtained from a Analytical results Appendix VIII. Chicago water are contained In Appendix IX. in of MortaLItyLfLInlcaLL OQrvatiLQoas C. and males RDX at 40 mg/kg/day was lethal to most of the during the two year treatment period. many of the females 14.6 months Mean survival time for these high dose males was For these high 22.3 months for control males. compared with dose females, a 20.6 months mean survival time was seen versus females. Both of these reductions for control 22.0 months Figures 1 and 2). (Table 4; significant were statistically *T first convulsions, death, tremors and/or Prior to their observed around Test Week 25, were often seen for 40 mg/kg/day of the males and some the In addition, many of animals. 24 • =• .•• . - • Q. .-. •=- . - • " -• • . - - - • o • ° • . - .*.*.**.*.o .• -*. • *• .. females were on occasion hyperreactive to approach. This was first apparent by Test Week 9, and this Increased sensitivity to stimuli apparently resulted In fighting among many of the co-habited males. As a result, high dose males which were fighting were singly housed during Test Week 30. Subsequently in Test Week 40, the remaining high dose males were placed in separate cages. 0 Discolored and/or opaque eyes were seen in an increased frequency for high dose females. This was first observed approximately midway through the •+udy and continued until termination. D. aQ4~ "Jgi2± Dose-related reductions in body weight gain were seen throughout the study for males receiving 8 or 40 mg/kg/day. At this latter dose, a 20-30% reduction was apparent for most of the study. For males administered 8 mg/kg/day, an approximate 5% reduction was observed. Body weights of female rats were less affected than males. Although females receiving 40 mg/kg/day often showed 10-15% reductions in body weight gain, there were several points In time when their body weights were actually higher than control females. Occasional slight but statistically significant reductions In body weight gain, about 5%, were also seen for 8 mg/kg/day-treated females (Tables 5-8, Figures 3-4). E. Food Consumption Food intake was slightly but significantly reduced for male rats receiving 40 mg/kg/day. For females at this dose, slight decreases but more often slight increases were observed. Occasional Increases or decreases were also seen for other dose levels, however they were sporadic and were not considered to be treatment-related (Tables 9 and 10). tHg atoI DU F. Reductions in hematocrit, hemoglobin, and RBCts were seen throughout the study for rats administered 40 mg/kg/day. Males were In general more affected than females. The observed anemic state was slight and physiologic compensatory responses, I.e., reticulocytosis, macrocytosis, etc., were not in evidence. Thrombocytosis was seen throughout the both sexes administered 40 mg/kg/day. receiving 8 mg/kg/day demonstrated elevated during Test Weeks 13 and 26. Total white were sporadically Increased for the 8 and 40 study for rats of In addition, males platelet counts blood cell counts mg/kg/day-treated 25 . . . . . . . . . . ... ...... pattern was not evident. a dose-related rats, however, apparently altered by other hematology parameters were treatment (Tables 11-20, Figures 5 and 6). G. C _Ln I a c No RDX htnjjjEY_ "Hypoglycemia was observed throughout the study for rats Whereas males were more sensliive 40 mg/kg/day. administered opposite year of the study, the than females during the first the second year. seen during was, In general, (40 mg/kg/day) for high dose Hypochloesterolemla was seen time points tested except for Test Week 104. at all animals showing only females with affected, were more Males in serum, cholesterol levels at Test reductions significant Reductions In serum triglyceride levels occurred for Week 52. This was seen, females administered 40 mg/kg/day. males and being both sexes at all time points testod with in general, similarly affected. during Serum GPT (ALT) levels were s~go'.flcantly lowered Weeks 26 and 52 for male rat/V receiving either 8 or 40 Test demonstrated the latter dose Females receiving mg/kg/day. For females administered 8 this effect only at Test Week 26. were serum protein and/or 40 mg/kg/day, reductions In total Slight increases In alkaline phosphatase were seen "observed. clinical No other 40 mg/kg/day. females receiving for by RDX to be altered appeared chemistry parameters treatment(Tables 21-30, Figures 7-11). H. "hthaImoID." "The ophthalmology Appendix X is contained in report In the Increases significant ll)*. Statistically "incidence of cataracts were seen during Test Weeks 78 and 104 The administered 40 mg/kg/day. but not males for females period was evaluation incidence of cataracts at this latter All significantly higher than that observed for Test Week 78. random In seen occurred abnormalities opthamologlc other to be treatment-related not considered fashion, and were (Table 31). (Volume Effects Requests for Volume II should be directed to Health Research Bloengineering U.S. Army Medical Division, Research and Development Laboratory, Fort Detrick, Frederick, Maryland, 21701-5012. * 26 I. e T.gA Relative liver and kidney weights were elevated at Test Weeks 27, 52 and 104 for male and female rats administered 40 mg/kg/day. During Test Week 52, females at the 8 mg/kg/day dose level also showed these relative organ weight Increases. Slight Increases for relative adrenal weights were seen during Test Week 27 for 40 mg/kg/day males and at Test Weeks 52 and 104 for 40 mg/kg/day females. Statistically significant reductions in testes weights were observed at Test Week 52 for 40 mg/kg/day males. Due to testicular masses for nearly all of the males (including the controls) at the terminal not weights were (Test Weeks 105 and 106), testes sacrifice evaluated at that time (Tables 32-43). J. athology The Pathology Report appears In Appendix Xl (Volume III)*. Histopathologic lesions observed for rats administered RDX for up to six months were confined to the 40 mg/kg/day dose level. Splenic extramedullary hematopolesis and spermatic granuloma of the prostate were present in these animals although gross morphologic changes were not in evidence. * By twelve months of ROX treatment, pathologic lesions of the urinary bladder, kidneys, testes and spleen were considered to be treatment-related for 40 mg/kg/day males. Only splenic lesions were seen for females receiving this dose. Urinary system lesions consisted of distended urinary bladder containing dark-red fluid, red-brown fluid in the abdominal cavity, and dark-brown kidneys with renal pelvis dilatation. Corresponding histologic observations were luminal distention and cystitis of the urinary bladder, and renal medullary papillary necrosis. Small testes with germinal cell degeneration, enlarged red-brown seminal vesicles, and enlarged prostate distended with red-brown fluid were also seen. Splenic lesions observed for both males and females consisted of enlarged dark-red spleens with histologic evidence of sinusoldal congestion. Requests for Volume III should be directed to Health Effects Research Division, U.S. Army Medical Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, Maryland, 21701-5012. * 27 -= -•o -° .'°.-, - .*=.' - •-...,'.." ." = •= .........................................................-..................... ... . -• . . Urogenital for lesions observed male rats during the treatment period were similar to those described month 12-24 lesions treatment-related significant above. Statistically dose level, except for mg/kg/day the 40 to were confined 1.5 and suppurative Inflammation of the prostate observed at the 24 month scheduled Splenic lesions at 8.0 mg/kg/day. Males "sacrifice were seen for both males and females. reciving 1.5, 8 or 40 mg/kg/day demonstrated Increased levels whereas extramedullary hemosiderin-lIke pigment of a females. in evidence for 40 mg/kg/day hematopolesis was dose this Lenticular cataracts were also seen for females at level (Tables 44 and 45). Iv. DISCUSSION female F344 rats The administration of RDX to male and In a reduction in the survival rate for both sexes resulted often died that animals Those 40 mg/kg/day. given Surviving animals at demonstrated convulsions prior to death. this dose were hyperreactive to approach and appeared to fight seen for with their cagemates to a greater extent than that failed to doses. Histologic evaluation animals at lower nervous lesions of the central detect treatment-related system. and hemoglobin Anemia consisting of reduced hematocrit, RBC's was seen for males and females receiving 40 mg/kg/day. of the usual physiologic was mild and none The effect macrocytosis, responses, i.e. reticulocytosis, compensatory peripheral The anemia appeared to be etc., were in evidence. as bone marrow appeared within normal limits and in origin extramedullary lesions including secondary splenic hematopolesis, sinusoidal congestion, and increased quantities gross were seen. Although pigment of a hemosiderin-like necropsy observations suggested enlarged spleens, organ weight analysis failed to substantiate this. Liver injury, primarily at 40 mg/kg/day, was evidenced by observations. Hepatomegaly was seen at 40 mg/kg/day several for females at 8 mg/kg/day and to a much lesser extent Hepatotoxicity although histologic changes were not apparent. by hypocholesterolemia, also suggested was albumin/total protein reduced serum hypotriglycerldemia, alkal ine phosphatase levels, and possibly by Increased act iv ity. primarily at the 40 RDX-Induced renal damage occurred Kidney weights were elevated at this mg/kg/day dose level. Lesions of this dose and possibly for 8 mg/kg/day females. kidneys with medullary papillary included dark brown organ six necrosis for males receiving 40 mg/kg/day for longer than 28 ........................................... . . . ".. . . . . . . . . . . . .. - on the urogenital system, Additional toxic effects months. bladder Included urinary at 40 mg/kg/day, primarily seen testicular luminal distention and cystitis, distention with enlarged seminal atrophy with germinal cell degeneration and by prostate accompanied In addition, enlarged vesicles. for spermatic granuloma and suppurative Inflammation occurred administered 1.5 mg/kg/day or greater following 24 male rats months of treatment. 40 mg/kg/day seen primarily at Additional toxic effects only), hypoglycemia, (females Included cataracts microscopic although adrenals and enlarged thrombocytosis RDX was not found to be carcinogenic not seen. were changes under the conditions of the present study. 0 In summary, the major toxic effects observed during the to 24 months F344 rats for up of RDX to administration splenic lesions, with secondary Included anemia and CNS Involvement, cataracts possible hepatotoxicity, observance of Increased Based on the urogenital lesions. levels of a hemosiderin-like pigment deposited in the spleen and a hemolytic-type anemia) secondary response to (a prostate seen for rats inflammation of the suppurative no-effect level greater, the administered 1.5 mg/kg/day or under the conditions of the present study is 0.3 mg/kg/day. •V v, EFERE.bLCE S Fed. Reg. 21 1. Good Laboratory Practice Regulations. CFR Part 38. 60013-60020, 1978. 2. Gordon, D.E., Burns, J.M, Levine, B.S., Furedi, E.M., study and Lish, P.M. Thirteen Week oral (diet) toxicity (TNT), hexahydro-1,3,5-trinitro-1, of trinitrotoluene 3,5-triazine (RDX) and TNT/RDX mixtures in the Fischer Study No. 1. 344 rat. Final Report No. L6116/L6121, 29 0 * TABLES (7 ( lIT RESEARCH INSTITUTE 31 --.--- --..-- . . .• ,,-,m ,• , ,b ,i ,, S.-...., ,'i ' " ' ...... ". , . . " ..' ~. . ". -= ~- C- '~~~~ 0 C.x iN 11 tm C.) 0u r- - C- m 0 0 iN fN Cf' t- - - , in) v N 0 m iN - iN - m 0 M' M' C') m' m' (N (N N CN CNy () r) +1 +1 +1 +1 +; +1 + +1 +1 + 0) ?- 0D + 0 C'- U) 0D a) 00 M. 0 0 0' _0 4 W, i - 0 - C- + 0 - D i W . 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OD o -0 0- gE LLt o0I' >, 6--ocooo~o66oooo66ooo00o00oooo6666oo U LL Z -W 0 -- 0 U' 0 >4 Ix 0 M r, 0 ,0a.r ro-o o - mo 0 -o6 ,N ,0Ma ,00 :MX o c ,61 tC ,C )0C oý t;o 6 F- 6 b I w c a-a~zu 6, o IOOOOOOOOOOOOOOOOOOOOOCIC'CIC.CCcOOGc'I 1i ioooooo6ooooo66oo6o6oo6ccc-ooooooc, 0r I. I.-I z a,-I-t ' N. 0 oRo-oF -- U w u LIsU in 4 0. - )m ~ C E 0 E - LrKrrLL E M. CA -- EnEr 6-L 0I o-oo 40 ,M- LL rrXrZL 1 LLD 0 'U I >1j0~E z m, *1le 6 C. 41cl 5U 4 +1. 400 0 I 0 TABLE 4 TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE IN THE FISCHER 344 RAT MEAN SURVIVAL TIME DOSE * Significantly MEAN SURVIVAL 0.0 M F 22.3 ± 0.2 22.0 + 0.4 0.3 M F 22.0 + 0.3 22.6 ± 0.2 1.5 M F 21.0 + 0.5* 22.2 ± 0.3 8.0 M F 22.2 ± 0.2 22.4 + 0.2 40.0 M F 14.6 + 0.7* 20.6 + 0.6* different from control group, p< 0.05. 41 a +I +1 ÷1 +1 +1 "I . Nl, 1 +1 1 . . . +I 4 . +1 + I( C4 N ... 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L C.,2 0 +1I +1 0 7 r-l +1 0 +1 0) C. li. * 0) N . Cl 0 y +1 t ~IIn 0 CO C'7w N(N U, inC, ( C) o 0 - - Z Z3 <2 ol M,1 Z L > -9Z >.4 a CO Cli 0 _ 0 0 101 (N ( N (N N (N 0 0 0~ 0 (Ni (N oy v C, 4 >- ex .1 0 - M 0 0 ( * 07 4 1 .1 4 0 f.-0) I C1 .1 . ~ 0 (N -1 N l 0 - a. 4 - 0 ti 0 4 4 0 1 0 D V N ( 0 0 f- -my C- lU .1 I Z39- 0 4 ) 1 - (7 (0U I. U, z cni9 * 0 0I 0N N N 0 (0 0N 0N ( 1 c . 0 -r N-L -2 9-4 0 c0 7CL,7 N -7 -9 - 0- w. v-. 0 0 0 0 0~N ( 0 0 m -C N le mcc)-0I 0) 0 (13 *c Z - 0 - co 4 0 z ' - - 0 N0 .- - m7 I5. 0 24 V L - L 4 U . . f(Nl 222 0 (rC4 NN 4 4 ~Z-.4 Z 0 Ix0 w- :zi 1 Z) ~~'0 c 1 -l 0 (z 0 0 0 0 2C TABLE 44 TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE IN THE MALE FISCHER 344 RAT * Statistical Evaluation of Histopathologic Lesions Dose (mg/kg/day) * 0.0 Time Lesion ____ _ SS 0.3 SS i SOMS 1.5 SOMS : SS 8.0 SOMS SS 40.0 SDMS SS SDMS _1__ Im Spleen, extramedullary hematopoiesis Pr nIs 6 mos -_ _ Present Ab e t Absent _ 10 ,I- 0 -r Present 12 mosAbnt 10 - I 0, 0' 10 Absent 0 r 24 Present 0 10,,I -- 0 10 --_ _ 0 0 3 6*" --- 10 __i 0 10 4 -- 2 3_ _ 0 18 I 0 0 0 17 I_ I 10 r 0 38: mosAbsent -- -I - I 'I I 0 0 19 36: 25 0 27 0 29 0 26 0 4 27 0 10 05 Spleen, sinusoidal congestion 6 * Present mosAbsent 100 ' Abesent Abet 1 Present 2 t . . 38:17 -- 10 0Smos 10 --0 36 0 -- ~ __0_ 3___ I 19 25:27 :-:8 I r 0 I0 -- 29:26 SS = scheduled sacrifice SOMS = spontaneous death or moribund sacrifice during interval following = last scheduled sacrifice significantly different from control group, p< 0.05 Absent . . . .. . ti. .. 3 . . .. .. . of . . . . 9 .7 3 . . . . . . . . . sacrifice. .. .. . . sacrifice.. . . . . . . . 2 2 1-8__ f 4 time of sacrifice . . - I 10 , = • 24 mos . 10 ,• . Time ( . 0 0 mosAbsent -,0 9I2 0 27 ,2 . . . . . . . . . . . . .. 103 _ TABLE 44 (con't) TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE IN THE MALE FISCHER 344 RAT Statistical Evaluation of Histopathologic Lesions Dose (mg/kg/day) Time Lesion SS 1.5 SS: SDMS 0.3 SS: SDMS 0.0 SDMS III .1 Present increased pigment F 0 O: -- 10 mo Absent 10 Present Absent a _ _ 10 10-__ 3 0 38:14 10: I __ 0 36:18 _ _I __ 01 18: 3 _ _ 4 23 I O' 10 :_I _ 0 18:22 II -- Absent 0 I0 -- 16 " 0 , 101, --- _ _ 0: 0 o I if 2 25 : __ U__ 0 0 o 27 __ last scheduled sacrifice * 1014 = significantly different from control group, p <0.05 9 0 4sr4 6:15 0 4 :27 __ __ Time -- time of scheduled sacrifice = scheduled sacrifice SDMS = spontaneous death or moribund sacrifice during interval following - _ _ 0 o, I 5 _ 7 29 :26 _ __I_ 3: 0' i0ra -- 0 _ 4: 0 ,-- 0 0 36 :19 _ 4 10:,-, -- S 0 0 0m o1m b s :,1--010 0 38 :16 ,: , 15 70 _ -- : 10 38 I il _ 10:5 10 ',10 I Prsn Present *SS _ - 3 10:- unilateral/bilateral 0,' 24msPresent Present = of sch1ule , -2mos Absent 10 * _ O - Testicular germinal cell degeneration, Absent 24 mos - I m -- 1 00 10o: 0........ 10 0O3 -- 10 0 i0 10I0 I 0 Present Aben Present mosAbsent 24mos 40.0 : SDMS SS I Spleen, 2 8.0 SDMS _____._____ II. 12 mos SS _ TABLE 44 (con't) TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE IN THE MALE FISCHER 344 RAT Statistical Evaluation of Histopathologic Lesions Dose (mg/kg/day) Time SS Lesion __ _ __ _ SPresent _ _ _ o Absent -- -- 12 mos 0' ,: 10 -r Present ?4 mosAbsent _ 0 36 0 16 0o 3 -- 6"*, 4 2 3 -- O: _ - _I1 -6* I I 9:-- 0 27 0 29 , 0 10 :19 I 0 25: - ,_L 1i, 9: I 0 19 SDMS _ I I, 9: --- SS.: SDMS _ l I 0 38 SS _ Prostate, spermatic granuloma 2• 2 2: -2: -8 8: -l 0 10 10 SDMS SS I I Peet Present Absent SDMS SS _I 0, 10, Peet 6mos SDMS _ 40.0 8.0 1.5 0.3 0.0 V I_ 0 4 0 26 0 27 Prostate, suppurative inflammation 0 SPresent mosAbsent 10 *, 10 ,II__ Prset..-.a: 0, 6msPresent 6 Asn 1010 0 ' O10 10 , -- I SI Present 124 mosAbsent 0 I -: 2 38:14 _ _ _I 3 35:16 _ 3_ 0- I I * T -- 0 O101 00: __ 10_ I__ I0 _ __I 05 -_ _ __ __ I0 -- _ _ 10 _ __ _ 5 _ _ 0 __I_ _ _ _ I 2:I 7** 23:20 __ _ Il_ 4*: 0 25:18 _ _ _ 0_ 19* 4:8 Time = time of scheduled sacrifice SS = scheduled sacrifice SDMS = spontaneous death or moribund sacrifice during interval following last scheduled sacrifice • = significantly different from control group, p<0.05 ** = significant (p<0.05) at this interval only when both SS and SDMS were combined 105 - ',.a,-i~m--ii--. i =mU~il~imm li .l . . . . .... .... . . .. . . .. TABLE 44 (con't) TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE IN THE MALE FISCHER 344 RAT Statistical Evaluation of Histopathologic Lesions Dose (mg/kg/day) 'Time 0.0 SS :SDMS Lesion 0.3 SS ', SDMS 1.5 SS ,SDMS 8.0 SS , SDMS 40.0 SS SDMS Kidney, medullary papilla necrosis, unilateral/bilateral II Present Absent 6mos __ __ I 0, I0 0-, 10 - __I Present 12rnos Absent -: I _ -- __ I0 1 ---- _ 0 0 1o0 10 124 mos' -- "Present 0 1 0 2 osAbsent 38:17 - - ' - 0 1 TI 36:18 I , -0 I --- 10o 0 0 0 0 3 25:27 o: 1oI0 ----- 0 0 0 29:26 Absent 12 mos 24 mos Time *SS Present IAsn 0: 10 __ 15* 0 18* 4 10 , 0 10 Presentl 0: Absent 38: -10 - 10 -- 10 -- 10 0 36: 181 0 25: 0 17 I0 10 0 0~ 10 -- 0 0 3 270 0 0 10 -- 0: 29: 251 =scheduled sacrifice .05 = significant (pO.05) at this interval only when both SS and SDIMS were combined ,106 -- 0 -7. 9 18" 0--- , 1 1 0 1 5 4 :22 time of scheduled sacrifice z significantly different from control group, p< 4 5 SDMS =spontaneous death or moribund sacrifice during interval following last scheduled sacrifice " _ T 0- 0: 0- 0 5 __ 0 I Aben 10 6m Present 0 1 I0: io: 10:- T T Kidney, pyelitis I I TABLE 44 (con't) TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-l,3,5-TRINITRO-l,3,5-TRIAZINE IN THE MALE FISCHER 344 RAT Statistical Evaluation of Histopathologic Lesions Dose (mg/kg/day) I Time 0. 0.0 Lesion L_ SS __ __ _ __ 0.3 SDMS _ __ _ SS 1.5 SDMS __ _ __ __ _ 0 _!_ Present Absent -- _ __ _ _ 0 17 _ 0 o 0, 10 _ _ _ _ _ I _ 0 36: _ _ _ 0 19 I '._ _ I _ 10 _ _ SS : __ _ :25 _I_ -- 10 _ _ _ _ _ __I __ 0 0 o 3 10 2 1 28: I _ 25 .,___ 0, -- 0 -- 10 0 38 : SDMS SDMS plyelonephritis -- ! __ __ _ 24mos 0 10 _ 0 12msPresent reeto Absent 10'-- SS ( -- i0 SDMS I Kidney, Present Absent SS 40.0 8.0 __II ! "6mos _ _ _ _ _ _ _ ! I __ 0 0 : o 1 5 __ -- -_ _ _ _ _ 1 _ _I _ _ 0 4 : 1 25 I __ _ 0: 10 _ _ 18 _ 1 26 __ _ _ _ Urinary bladder, luminal distention I 0 ~~6 mos Present Asn be-- Absent 0 0'I -- 10 I . :0 -- -- 10: I0 1G : 24mos Aben Present Absent i-. 0 ', 0 0: 38 :16 1 36 0 00: 25 " 0.-- -- 0 10 0 5 -- 10 1 10:-91 02 3 1* 24* 4 10 10 I -- :17 3 0 I l24mos ,1-0 -- I 3 0 26 29 19 3 Time = time of scheduled sacrifice SS = scheduled sacrifice SDMS = spontaneous death or moribund sacrifice during interval following last scheduled sacrifice * = significantly different from control group, p<0.05 107 -~~~~~~~~~~~~.'-......................••'.. -,, . • .{-,-...,.. .. ............ TABLE 44 (con't) TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE IN THE MALE FISCHER 344 RAT "Statistical Evaluation of Histopathologic Lesions Dose (mg/kg/day) 0.0 Time Lesion SS 0.3 SDMS SS 1.5 SDMS SS Urinary bladder, cystitis, hemorrhaoic_- :-- 0o 10:-III 10:-- Presentr:-0 l 2 mosAbsent 10 - o 6'o 0 6 m~Present 1 rset Absent _ _ __ _ _ _ _ _ _ : I__ Present 0 24 nmosAbsent 38: -- 10 I 0 16 ~ -- __ Absent i * 2mos 24 mos Present Absent Present Absent __ SS 40.0 SDMS SS suppurative 0 0.,-10,-- 10i 0 1 : 0 3 0o 10 0: 25 :26 1 1 ,'0 -g 10: __ -I 28 -- __ :22 _ __ _ _ 0 _ _ _ _ 5 _ _ 17* _I 18A 0 4: 9 o __ I I _ 0 : 10:2 _ 0 ', o iI _ SDMS hemorrhagic/suppurative/ iI 6mos SDMS ___ 2 17 0 36: : 8.0 _ , I iI I , I ' II I ' Time = time of scheduled sacrifice SS = scheduled sacrifice SD!-IS = spontaneous death or moribund sacrifice during interval following last scheduled sacrifice * = significantly different from control group, p< 0.05 108 .......................--. -I -I'1-L L" -. ,"...z'L"'.-. ..--.:.- ...- ".' . .. . ,. 111-1. .1. .- 1'1-..-'"'.-.. 1..1.-1 TABLE 45 TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE IN THE FEMALE FISCHER 344 RAT Statistical Evaluation of Histopathologic Lesions Dose (mg/kg/day) Time Lesion __ _ __ _ _ _ : SS _ _ SDMS __ _ _ _ SS SDMS SS ! _ _ __ _ _ _ _ __ _ _ I I __ _ _!_ _ _ __ _ _ __I _ SS SDMS SS SDMS _ 40.0 8.0 1.5 0.3 0.0 __ _ _ _ __.._ _ _ _ __.._ , SDMS J _ _ _ _ Lenticular cataracts * Present 100, -- Present Abt 0' 10' - 6mosAbsent 2 24 Present Present- 0, 10 .io -- 0' - - -- 0 ,-- 10 ,1- 9 ,032: 10 0 1 10 10 0 1 0, 101 4: --1 1sPresent 124msAbsent I 14 10 9 Presen 0: 0 Absen~t 43: 10 4 5 01 i J 45: 90 10 0 I I 42: _ 0 12 09 41 0 -- 0 0 10 I 0'10 0 -- 03 - 12s mAbsent 24 0'SI 0, 10 ' 17 0 25' 0 , 1 29' 10 mos -- 3 8- 10 7 09* 23* 5 5__ 11__ 15 , I 0 :11 lo 128 I 200 See,sacrifice timxe of scheduled SS =scheduled sacrifice ý,XS=spontaneous death or moribund sacrifice during interval following last scheduled sacrifice = significantly different from control group, p. 0.05 SII ............................................................ 6log _ TABLE 45 (con't) TWENTY-FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE IN THE FEMALE FISCHER 344 RAT Statistical Evaluation of Histopathologic Lesions Dose 0.3 0.0 Time Lesion SS (mg/kg/day) , SDMS SS -- 1.5 SDMS SS SDMS t____. __ 8.0 SS I 40.0 SDMS SS SDMS ___________ ___ Spleen, sinusoidal congestion 6ms Present Absent 1 m -- 24 mos , Present Absent 0: 10 L. 0 : 10: "'Present Aben Abte 0 1_ _ 0' -- _ _ _ 0 1 0 10: - 0 o0 0 10 4 - 0 ,' 10: _ _ I__ 45 _ _ _ ', 10 I 0 -I __ _ -_ _ ___ _ ,: 1 _ _ o, 1203,'•2 _ I _ _ _ I - 10 o0 42 -- I ,0 10 "I I _ I 6 1 ' I 4 ,, 128 20 I I I I I I 6osPresent Presenl Asend ____i 12 mos 24 mos ___ : Present Present ' Absent , I I I I ,a , , , I I I •I Time = time of scheduled sacrifice SS 0 I = scheduled sacrifice SDMS = spontaneous death or moribund sacrifice during interval following last scheduled sacrifice * = significantly different from control group, p< 0.05 '110 . -. . . .. . I I CFIGURES 0 111 ( !..o.. . .... °.. . .. .. .. .. ....- .. ,. ..... - .. . . .- . - .. - - .. - .. - - - -. . , . -. . .. , .- , - - ,- -.- . ,- ,, - , ,, ,- 4 .4 WWW occo -aoc W coC14 cc L -~CID w E c 0 m 0L Cj) m 1 *r .o co* LU - W * Lii 0 W Ilm z~w 0J ! 0 4 * C.!c V) m P.-WW 4o 'n.~ 4 0 cc W* CD co IJ I-. w to cc c x 14 Z 0 Do o 0 coIf 4 -r + co* : w 4 WW * ~. co co4W* 4 LL. Os I i~u-~ 0 co *o *co u L0 W W4 4L C) w co c 4in LU qd o cc * I co =3 ~0 ccW -n 112 >- > .0 t-nI - -~ -w 6 . .... ... . . . . . . . . . . . . . . . S4 - . to c 4 4 + . .. 4 ÷ 01 a L 00 4C N ,-,momoowowo I0 4:o 0 u. >.-(A -04 (A"- LJJ - n i-w Li- -- 4h 44 0 _z 0 W 1.. LaJ l" w ! ,4< +4 s V) 0 E X "( < 2 z i-.> -". Z"-' •.-- -I•" .- L.0' cc 4€ .. ,4 : 0 i4 00 c-.IA ~ < • 0 N . .in 44 4 4 4 0 -4 o . "0 In,. JJ 800 ' ' w 3 0 mnS' c:c ot W U1 - 0 0 I0 -,,.- 0 0 0, 9 0 0 0 I w. l 0 if)0 .00 (4 U, 0 0 c03 Go ÷ (- 8 00w toii 4 C34000 c -A ON 0 . 0 4 49 m 44 00 4 C-) CD ~ .c 0 4 . 0j O 4 4 40 + LC JO= " A" (A a ,0 .' "t.-n4 0 aLI0 GJ I 0 1 o 04 I0 c •,•m J, L)CcW . U.. w4 Go 4 + Cr*" 00 0 w0 Dt t 00 0 4 .4 0 ,,: 0o 0 0co : 0-. (/K 200- ] I' ' 10 (0 • ' i . 20 . . . . 30 . i' 40 ' - I' " * " 50 ' • 60 ' -. . 'Ii •:' 70 .. 80 . i . . . 90 . I .. . . 10O0 A 10 ,VEEK LEGEND: -x --- 0 0 MC/KG/DAY i .5 MC/~KG/DAY <--o-e P.O MO/KG/DAY ,-,-- -,,--x,-.-- 0.3 MC/KG/DAv e-a-.-B 40 0 1C/KG/DA~v 117 FIGURE 7 TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE(RDX) IN THE FISCHER RAT MEAN GLUCOSE VALUES (MG/DL) VS TIME MALES AND FEMALES COMBINED -- / 1207\ / 1177 -- \ , 1-14, ..-.. + / - . -- 1 ,. \ EE\ A I1 I ------ L 108 •" U ,'i/ / S 105 * " E E ,V E/,, 6 A 102 \._-'. ,// / \ I,, " , / 99-/ S I.//,/ * M• G96- L\ / 7 ', '/ /\ 77/ 7 93/ / 90- - . . ., , E "* ----- / / /\/" /• ,',' C "S- I ,, - 87 0 "I. 10 I .I 20 30 40 50 60 70 80 90 100 110 WEEK LEGEND: * 113 TX .-.--.- ,-, ----. 0.0 MG/KG/DAY 1.5 MG/KG/DAY 8.0 MG/KG/DAY -*-- B--e 0.3 MG/KG/DAY 40.0 MG/KG/DAY I .:...-.. :...:-'.-... .:-:.. ....... .:-:..-.-....... .... ... -..-- ..-:..-: .... ........ ...... ...-....... .--..-......-.. ...-....- . . . ...-.. .,v.-.--.-...- .... ..-......-.-..-.-.... v.-.. .-. ,-. "FIGURE 8 TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAIiYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE(RDX) IN THE FISCHER RAT MEAN CHOLESTEROL VALUES (MG/DL) VS TIME MALES AND FEMALES COMBINED * E0 Jr / M ' 6 "0 * ! E . . A N H // L / " / C/ E 20 ..' " I0 00 . - - -.-- "<- -2' *L-/ .__ L * " / /L I -- / / --.. - 4 . E I -05 "jo GK /A 3E-9 400M /K /A 707 .. .<-2..... N02 " 04 i0 .. . . 06 7 " 09 11 // (• ,"5 " h"" -,, •• " .- ' , -€i I a .• . • . • ."".", i • ,C ."..' j •• '' . " " ' . "....'''''''''''' L ,.... 73~ ............................................................-•.-'- 7. . .".-"..• ... . ".".I FIGURE 9 TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE(RDX) IN THE FISCHER RAT MEAN TRIGLYCERIDE VALUES (MG/DL) VS TIME MALES AND FEMALES COMBINED ,.... 140 t \ /" ff • ", , 130 -, // / S ,, /! ', ---- - -- A i 20 \ N/ N, T ÷ - - "- R 110I C.. / / , mI l - ', , Y i00 00- \ -..N* -- - . , .,,:' .,' L ,'ft.. ,' x. 1! /; _• - / 5, /. ,' / V A L U 80- / "E 70 S m 60 S L D7 507 40- "*....... * 30-- 0 , ..... 10 I......... I.....I.....t.....I.....I...... 20 30 40 50 60 I......... 80 70 I........ 90 ........ I . 100 110 WEEK LEGEND. TX ---- ,- + .- •--e-~- 0.0 MG/KG/DAY 1 .5 MG/KG/DAY 8.0 MG/KG/DAY •--- 6E---- 0.3 MG/KG/DAY 40.0 MG/KG/DAY 120 . ................... .... ,.. ........ .. FIGURE 10 TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-I,3,5-TRINITRO-1,3,5-TRIAZINE(RDX) IN THE FISCHER RAT MEAN FEMALE TOTAL PROTEIN VALUES (G/DL) VS TIME 07 ,/ ' T4 Ep 7~ p 7 A o N / ,/ G A '•" .3/"" ,,-.. 75 5 _ /, • .' /I II ", S + /f --o/- o 8 I, R 6 -121 S.. .. . .. .. . I . ' . . ' ' 2- . . . . I . . . . . . . I . . . . . . . I . . . . . . . I . . . . . . . . . . . . ..- 1/121 "-''-.-,.- N • ' !d••lll 6-''- '- ': .../'.nl lm l l ln ,,i /- V• ni .,-,W ll ,i • l • • ii• 1• • 1 , •`"• `--• • , , - • E • • • - , •_ • - `` : r . • `` , . FIGURE 11 TWENTY FOUR MONTH CHRONIC TOXICITY/CARCINOGENICITY STUDY OF HEXAHYDRO-I,3,5-TRINITRO-1,3,5-TRIAZINE(RDX) IN THE FISCHER RAT MEAN FEMALE ALBUMIN VALUES (G/DL) VS TIME 49- 4 8 / 47N E/ / E A ÷ A A 7. . 4.60 7-' --- 4- .4- ------ -- -- -/ ,,. --- - --- - - --- - - .. . .. . . - ----------------- L+ . 34 -- c * L 4.2- 4 .0. . 25 . . 35 .. .. .. . .. .. .. 45 55 65 75 85 95 WEEK "LEGEND. TX : "122 '.7. -.-- 0-+--+ MG,' E--B-s 0.,3 MG/KG/DAY 40.0 MG/KG/DAY APPENDIX I STANDARD OPERATING PROCEDURES FOR TEST ARTICLE ANALYSIS, PREPARATION AND SAMPLING 123 S . . . . . . .... . . .. ,. . , h... .. .. , , ,.. . . , .• ., . :. ,; APPENDIX IA ANALYSES OF THE RDX TEST ARTICLE SCOPE 1.1 The procedure describes the analysis of the RDX test article for purity. 1.2 This method is recommended for use only by experienced analysts familiar with High Performance Liquid Chromatography (HPLC) or under close supervision of such qualified persons. INTERFERENCES 2.1 Solvents, reagents, glassware and other sample processing hardware may yield discrete artifacts and/or elevated baselines causing misinterpretation of chromatograms. All of these materials must be shown to be free from interferences under the conditions of the analysis by running method blanks. EQUIPMENT 3.1 Higher Performance Liquid Chromatography . constant flow, isocratic pumping system . reverse phase column, 10 i1- 3.9 mm x 30 cm .i-Bondapak C18 column . ultraviolet detector capable of monitoring A = 254 nm . strip chart recorder and electronic integrator capable of measuring peak areas and performing an internal standard calculation. REAGENTS 4.1 4.2 Propiopnenone, an internal standard, Aldrich Chemical Company (Purity 99%) Methanol, Actronitrile, and Water, HPLC Grade or equivalent 4.3 RDX, S.A.R.M., supplied by the sponsor (Purity 99.8%) CALIBRATION 5.1 124 ... Calibration standards were prepared from stock solutions containing 200 pg RDX, and propiophenone per ml acetonitrile so as to bracket the working range of the chromatographic system. These concentrations were: 2 vg/ml, 10 vg/ml, 20 vg/ml, and 40 pg/ml. - * . I I l i .- - --. . . 5.2 A constant injection volume of 15 pl was employed for all measurements. 5.3 In order to determine the precision of the HPLC system, a series of 6 replicate injections of the 20 pa/ml solution were made. 5.4 Retention times should remain relatively constant (within + 5% day to day) with RDX being 3.7 minutes, and propiophenone 7.3 minutes under the specified conditions. If the retention times are not within + 5,%, supervising chemist should be informed prior to the analysis and corrective action should be taken. * 'UALI•2Y ° CONTROL 6.1 Before processing any samples, the analyst should demonstrate through the analysis of a blank tha all glassware and reagents are interference free. 6.2 In a typical sample set, a minimum of one blank and five samples will be analyzed. The analyst will follow each step in an analytical protocol without deviation or im.rovisions in order to accurately assess the performance of the methoa. Prior to making any changes in the procedure, analyst will consult the supervision chemist and the supervising chemist and Q.A. officer will review and approve all the changes. 6.3 • x..-.-w--w--.-. . . SA2MPLE PREPARATION 7.1 The test article will be spread on a sheet of paper, and five samples will be takin from different areas. Each sample shall have a weight of %150 mg. The samples will be collected in ariber vials and stored at refrigerator temperatures in the dark until analysis. 7.2 A portion of the sample (100 mg) will be weiched and tranF erred to a 100 ml volumetric flask. The internal standard will be added and it will be diluted to volume. It will be further diluted to a concentration of 20 jig/ml and analyzed by high performance liquid chromatography. 7.3 If the sample is not analyzed imrrediately it will be stored at refrigerator temperatures in the dark HIGH PERFORL4NCE LIQUID CHROMATOGRAPR'Y (HPLC) 8.1 Each sample was analyzed by reverse phase HPLC using the conditions described below: Column, 3.9 mm x 30.0 cm p-Bondpak C1 8 ; Solvent System, methanol:water (55%:45%, v/v); Flow Rate, 1.5 ml/min; Detection, UV at 254 nm; Sensitivity, 0.1 AUFS. the retention times 125 of RDX and propiophenone were 3.7 and 7.3 minutes, respectively. The limit of detection was 2 jg RDX/ml acetonitrile and is defined by 5x the background nouse. The representative chromatogram is Figure IA.I. 8.2 The chromatographic system was calibrated daily with a minimum of two injections of one standard representative of chromatographic range. 8.3 An injection volume of 15.0 ul was used for each sample. If the peak area exceed the linear range of a sample it was diluted and reanalyzed. CALCULATIONS 9.1 Determine the concentration fo RDX using the formula: RDX in Sample= (Ax) (Wis) (Fx) x D X 100 Ais (Ws) Where Ax = Area (X) where x is RDX Ais = Area (internal standard) Fx = Area (x) Area (is) x Weight (is) x Weight (Wx) Wis = Weight of the internal standard Ws = Weight of the sample D = The dilution factor 9.2 Wx = Weight of component x is RDX The results should be reported in percent RDX. Where replicate samples are analyzed, all data should be reported. All results were recorded in standard IITRI logbooks and these plus chromatograms and data tapes are retained in the Chemistry Division Q.A. files. 126 ............................... ' 1 Figure tA.1 .2 Chromatogram of RDX (1) Propiophenone (2) Standard, 20 p~g/mR. ,127 127 N.;.,. •...-2... .-..- . .- '•.-.. . '- -. -- .% ? ".--i.-..-...' ... . ... : .... -. •---:-.-.i - -.- 'i-.. - +- -.-. -- + .-.-. APPENDIX IB ANALYSIS OF RDX IN DIET PREMIXES SCOPE AND APPLICATION 1.1 This method covers the determination of RDX in diet premixes. at 10% and 50% levels. 1.2 The sensitivity of this method is usually dependent on the level of "interferences present in the samples, rather than the instrumental I imitations. This method is recommended for use only by experienced analysts familiar with High Performance Liquid Chromatography (HPLC) or under close supervision of such qualified persons. 1.3 SUMM4RY OF THE MIETHOD 2.1 A weighed quantity of the premix was stirred with 50 ml of acetonitrile for 30 minutes. The suspension was filtered through a porous glass filter and the filtrate was transferred with washings to a volumetric flask. Propiophenone, the internal standard was added to the filtrate or a portion, thereof and this solution was diluted to its final volume. The samples were analyzed using reverse phase high performance liquid chromatography. Each was eluted on 3.9 mm x 30.0 cm 1i-Bondapak C1 8 column with methanol :water (55%:45%) and the eluant was monitored with an ultraviolet absorption detector at X = 254 nm. INTERFERENCES 3.1 Solvents, reagents, glassware and other sample processing hardware may yield discrete artifacts and/or elevated baselines causing misinterpretation of chromatograms. All of these materials must be shown to be free from interferences under the conditions of the analysis by running method blanks. 3.2 Interferences coextracted from the samples will vary considerably from source to source, depending on the type of animal feed used in the study. MATERIALS "4.1 Erlenmeyer flasks, 125 ml 4.2 Filtering apparatus, vacuum flask, 125 ml; fritted glass filters, porosity M, ASTM 10-20 microns. 128 ,- . . . - --.. " "t e. le itt' CI 5. te t~n' rrer s,.33C 351 3. t 1c ýriIn' t 55 -ii ;ner 'er * An, ijt r]i' t n -)1et l Je t t2 r ~ r-. n.cnk s~ater' s As sac iates) ~rlo t c)ra~ hy *i ~ 3~- n r adf 'eý drj., 3rer , e- fi r-ir, irn J~f - j r 6.1 Propi ophenone , an i nternd I 6.2 'IethanoI , Aceton itr i Ie, and ýa erYr 6.3 ROX, S.A.R.M., supplied by thie 3~'i K 'r corecktiresacind shouldrbe taken.",," *7.1 - - 1 ~rt i Ltria pertk area S * I ; 1J ~ *re ve r-se ~na se rro. 1-910 ndljtiril i3alance, "'", Calibration standards were prepared from stock solitions cDn'taining 200 výg RDX, and propiophenone --r ml acetonitr-ile so as to Dracket the working range of the chro,-iizwgraphic system. These cconcentrations were: 7.2 7.3 2og/mi, 10 pg/mi, 20 ý.g/ml, and 40 9./71. A constant injection voluee of 15 -1 was employed for all . .---. ............. .easurements. . .. In order to determine the precision of the HPLC system, a series of 6 .:-. 7: Z .','9 replicate injections of the 20 pig/mi solution were made. 7.4 Retention times should remain relatively constant (within + 5) day to day) with ROX being 3.7 minutes, and propiophenone 7.3 minutes under the specified conditions. If the retention times are not within + 5%, supervising chemist should be informed prior to the analysis and %*A. 'TY 8.1 W"T.ROL Before processing any samples, the analyst should demonstrate through the analysis of a blank that all glassware and reagents are interference free. Each time a set of samples is extracted or there is a change in reagents, a method blank should be processed as a safeguard against laboratory contamination. Standard quality assurance practices were used with this method. A minimum of 6 replicate spiked samples were analyzed to validate the accuracy of the method. If doubt should arise concerning the identity of the peak on a chromatogram, confirmatory techniques such as mass spectrometry should be used. 6.2 8.3 In a typical sample set, a minimum of one blank and scheduled samples will be analyzed. A control sample will be prepared by adding a known concentration of RDX to the sample. The concentration will be in the working range of chromatographic system as determined by calibration experiment. 8.4 The analyst will follow each step in an analytical protocol without deviation or improvisions in order to accurately assess the performance of the method. Prior to making any changes in the procedure, analyst will consult the supervising chemist and the supervising chemist and the Q.A. officer will review and approve all the changes. 8.5 The typical analysis will consist of the following samples shown in the diagram. One blank sample, 6 premix samples as is, 3 spiked samples. Blank 6 PrLemix _ Sample Samples Anat.yz ed Anatyzed _AAJ _I• AA IZ SIr 7- , ox!igi Original Extrtact Ext'atct PZls I mt Spike 50 050mg • . _ TA +Z 130 % - . .. -- - "o. - ° ." . o" , . -" . % "- . % - . ° . .•m % ','. . % % ". •. ' , • SAKPLE COLLECTION 9.1 Samples are collected and stored prior to analysis according to SOP 81-sample collection (TNT & RDX Premix) SY'PLE EXTRACTION 10.1 The appropriate amount of sample is weighed into a 125 ml Erlenmeyer flask using standard operating procedures. The sample amount for both the 10 percent and 50 percent premix is one gram. Approximately 50 mls of acetonitrile is added to the flask and it is stoppered. The sample is extracted by stirring for 30 minutes at room temperature. 10.2 Following extraction, the fritted glass filter. In swirled to form a uniform glass funnel. A stirring from the flask. 10.3 The extraction flask was rinsed with three portions of acetonitrile of approximately three mils each, and the rinse is poured into the funnel. procedure is repeated three times, then the vacuum is reapplied and the washing process is completed. 10.4 The filtrate is transferred via a short-stem funnel into a volumetric flask. The filtering flask is rinsed three times, with approximately 6 ml portions of acetonitrile, and the rinses are added to the volumetric flask. The size of the volumetric flask and the subsequent treatment of the sample depend on the initial RDX concentration in the sample. The dilution for sample is shown in Table IB.1. 10.5 An aliquot (approximately 10 ml) is filtered using a Water's Organic Sample Clarification Kit using 0.5 jim filter. The sample is now ready for analysis for HPLC. * 0 )This * sample was filtered through a medium porosity this operation the extraction mixture was suspension and immediately poured into the rod was used to drain the last drop of liquid STORAGE OF SAM4PLES 11.1 All samples including premixes and blank feed will be stored in the dark at refrigerator temperatures. 11.2 If the sample preparation procedure is stopped at.any point during the working day, the samples should be stored in stoppered vessels in the dark at refrigerator temperatures. 131 , ...-....-. ,..,. . . •.. -. .. . .....-.... , ..... . . .. . .• *.-.*..-.w .- ,........'..-... ,..-...- .-.. '•." '..-..-,.- .- • TABLE IB.1 DILUTION SCHEME FOR SAMPLE EXTRACT Premix Concentration Original Extract Volume Secondary Dilution 10% 50% 100 ml 500 ml 1 ml extract plus 1 ml I.S. to volume of 50 ml with acetonitrile 1 ml extract plus 1 ml I.S. to volume of 50 ml with acetonitrile 1. I.S. solution concentration is - 1000 1ig/ml 2. In the case of a sample analyzed by the method of standard addition I ml of the original extract was diluted with 50 ml acetonitrile, and 1 ml of the extract added to 1 ml of the spiking solution of known concentration was diluted with acetonitrile as above. Samples that are ready for HPLC analysis will be stored in theS11.3 dark at refrigeration temperature. 11.4 Similarly, RDX and propiophenone standards and all standard solutions will also be stored in the dark at refrigerator temperatures. HIGH PERFOR1?LVCE LIQUID CHROMATOGR4PHY (HPLC) 12.1 Each sample was analyzed by reverse phase HPLC using the conditions described below: Column, 3.9 mm x 30.0 cm p-Bondpak C18 ; Solvent System, methanol:water (55%:45%, v/v); Flow Rate, 1.5 ml/min; Detection, UV at 254 nm. The retention times of RDX and propiophenone were 3.7 and 7.3 minutes, respectively. The limit of detection was 2 pig RDX/ml acetonitrile and is defined by 5x the background noise. The representative chromatogram is Figure IB.I. 12.2 The chromatographic system was calibrated daily with a minimum of two injections of one standard representative of chromatographic range. 12.3 An injection volume of 15.0 pl was used for each sample. If the peak area exceed the linear range of a sample it was diluted and reanalyzed. 13 132 6l * 12.4 Following the completion of an analysis or set of analyses, a gradient going from initial solvent conditions to 100"%methanol in 15 minutes will be used to elate polar compounds from the column. Elution at 100% methanol will be continued -or at least 1 hour. 13.1 Determine the concentration of RDX using the formula: "%RDX in Sample = (Ax)(Wis) x D x 100 (Fx) Ais Ws Where Ax = Area (X) where x is RDX Ais = Area (internal standard) Fx =Area(x) x Weight(is) Area(is) x Weight(Wx) Wis = Weight of the internal standard G) Ws = Weight of the sample 0 = The dilution factor Ux = tit of component x is RDX. #4 13.2 The results should be reported in percent RDX composite. This is the RDX actually used in the toxicity study. Where replicate samples are analyzed, all data should be reported. All results are recorded in standard IITRI logbooks and these plus chromatograms and data tapes are retained in the Chemistry Division Q.A. files. 14.1 Safety regulations will be followed at all times especially with regard to the handling of toxic materials. When the premix samples are being handled, a lab coat, gloves and a mask will be appropriate attire. When solutions as extracts are being handled, a lab coat and gloves should be worn when there is the change of direct contact with these materials. 133 [L. .=-OD . I .,y'J ° Figure IB.1 1 °" 134 "3 Chromatogram of RDX (1) Propiophenone (2) 20 Ug/mt Standard, 6 APPENDIX IC ANALYSIS OF RDX IN DIETS 2• 9?E A:;D 1Z)FLICATiON 1.1 This method covers the determination of RDX in diet samples at 0.0005% to 0.100% level. 1.2 The sensitivity of this method is dependent on the level of interferences present in the samples, rather than the instrumental limitations. 1.3 This method is recommended for use only by experienced analysis familiar with High Performance Liquid Chromatography (HPLC) or under close supervision of such qualified persons. -A4RY 2.1 0 • )The OF TIE Y.ZTHOD A weighed quantity of the diet was stirred with 50 ml of acetonitrile for 30 minutes. The suspension was filtered through a porour glass filter and the filtrate was transferred with washings to a volumetric flask. Propiophenone, the internal standard was added to the filtrate or a portion, thereof and this solution was diluted to its final volume. samples were analyzed using reverse phase high performance liquid chromatography. Each was eluted on 3.9 mm x 30.0 cm p-Bondapak C1 8 column with methanol: water (55%:45%) and the eluant was monitored with an ultraviolet absorption detector at X = 254 nm. IJ;TE'FERENCES 3.1 Solvents, reagents, glassware and other sample processing hardware may yield discrete artifacts and/or elevated baselines causing misinterpretation of chromatograms. All of these materials must be shown to be free from interferences under the conditions of the analysis by running method blanks. 3.2 Interferences coextracted from the samples will vary considerably from source to source, depending on the type of animal feed used in the study. 6' 1 ,'.:4TERIALS 4.1 Erlenmeyer flasks, 125 ml 4.2 Filtering apparatus, vaccum flask, 125 ml; fritted glass filters, microns. porosity M, ASTM 10-20 ! 135 , -. '.,,• ,- . .' ,. .- .. .-.-- -,. ,,-, , . , S• - .. , ._ • . -, . .. ,. ..- -, -. -9-. S. -. .. - .- ., - S.. EQUIPMENT 5.1 Mettler Grammatic Analytical Balance, No. 1-910 5.2 Corning Hot Plate Stirrers, BC 351 5.3 Buchi Evaporator, Model R 5.4 5.5 Sample Clarification Kit, Organic (Water's Associates) Higher Performance Liquid Chromatography . constant flow, isocratic pumping system * reverse phase column, 10 11- 3.9 mm x 30 cm p-Bondapak C18 column . ultraviolet detector, capable of monitoring X 254 nm 0 strip chart recorder and electronic integrator capable of measuring peak areas and performing an internal standard calculation. * ~REAGCENTS * 6.1 Propiophenone, an internal standard, Aldrich Chemical Company (Purity 99%) D136 6.2 6.3 Methanol, Acetonitrile, Water HPLC grade or equivalent RDX, S.A.R.M. Supplied by sponsor (Purity 99.8%/) •.'...........-. CALIBRATION * *7.1 Calibration standards were prepared from stock solutions containing 200 pg RDX, and propiophenone per ml acetonitrile so as to bracket the working range of the chromatographic system. These concentrations were: 0.5 ijg/ml, 2 pg/ml, 10 vig/ml, 20 iig/ml, and 40 pig/ml. *7.2 A constant injection volume of 15 Vil was employed for all measurements. 7.3 In order to determine the precision of the HPLC system, a series of 6 replicate injections of the 20 v~g/ml solution were made. 7.4 Retention times should remain relatively constant (within + 5% day to day) with RDX being 3.7 minutes, and propiophenone 7.3 minutes. If the retention times are not within + 5%1; supervising chemist should be informed prior to the analysis and correcti-e action should be taken. 1 _5•LITY CONTROL 8.1 Before processing any samples, the analyst should demonstrate through the analysis of a blank that all glassware and reagents are interference free. Each time a set of samples is extracted or there is a change in reagents, a method blank should be processed as a safeguard against laboratory contamination. 8.2 Standard quality assurance practices were used with this method. A minimum of six replicate spiked samples were analyzed to validate the accuracy of the method. If doubt should arise concerning the identity of the peak on a chromatogram, confirmatory techniques such as mass spectrometry should be used. 8.3 In a typical sample set, a minimum of one blank and scheduled samples will be analyzed. A control sample will be prepared by adding a known concentration of RDX to the sample. The concentration will be in the working range of chromatographic system as determined by calibration experiment. 8.4 The analyst will follow each step in an analytical protocol without deviation or improvisions in order to accurately assess the perforof the method. Prior to making any changes in the procedure, analyst will consult the supervising chemist and the supervising chemist and Q.A. officer will review and approve all the changes. 8.5 The typical analysis will consist of the following samples, one blank sample, 6 diet samples as is, 3 feed samples spiked for the recovery determination at the diet concentration. * * 2mance * * C 137 -,-7. 7%7- -.. TABLE IC.1 Diet "Level % Extract Volume (ml) - - r - C DILUTION SCHEME FOR RDX DIET SAMPLES Extract Diluted (ml) Propiophenone (IS) Added Final Volume (ml) 1 ml, 50 pg/ml 100 - 1 ml, 500 pg/ml 100 100 - 1 ml, 1000 pg/ml 100 0.0500 100 10 1 ml, 500 pg/ml 25 0.1000 100 10 1 ml, 1000 pig/ml 50 "0.0005 100 0.0050 100 "0.0100 1 )>138 ............................. S2AMLE COLLECTION 9.1 Sampler are collected and stored prior to annalysis according to SOP 81sample collection (TNT and ROX diet samples). SAMLE EXTRACTION 10.1 The appropriate amount of sample is weighed into a 125 ml Erlenmeyer flask using standard operating procedures. The sample amount for the diet mixture is ten grams. Approximately 50 mls of acetonitrile is added to the flask and it is stoppered. The sample is extracted by stirring for only 30 minutes at room temperature. 10.2 Following extraction, the fritted glass filter. In swirled to form a uniform glass funnel. A stirring from the flask. 10.3 The extraction flask was rinsed with three portions of acetonitrile of approximately 5 mls each and the rinses are poured into the funnel. The vacuum is reapplied and the washing process is completed. 10.4 The filtrate is transferred via a short-stem funnel into a volumetric flask. The filtering flask is rinsed three times, with approximately 5 ml portions of acetonitrile and the rinses are added to the volumetric flask. The size of the volumetric flask and the subsequent treatment of the sample depend on the initial RDX concentration in the sample. The dilution for various sample levels is shown in Table IC.1. Diet samples will be diluted to a volume that places them in the working range of the chromatographic system. 10.5 An aliquot (approximately 10 ml) is filtered using a Water's Organic Sample Clarification Kit using 0.5 ;m filter. The sample is now ready for analysis for HPLC. • U" * AI * sample was filtered through a medium porosity this operation the extraction mixture was suspension and immediately poured into the rod was used to drain the last drop of liquid ( ) 139 "..........................................................."........."......... ..... - "-"."". ".- ." STORAGE OF SAMPLES All samples including diet and blank feed will be stored in the dark 11.1 at refrigerator temperatures. * 11.2 If the sample preparation procedure is stopped at any point during the working day, the samples should be stored in stoppered vessels in the dark at refrigerator temperatures. * 11.3 Samples that are ready for HPLC analysis will be stored in the dark at refrigerator temperature. i 11.4 RDX and propiophenone standards and all standard solutions will be stored in the dark at refrigerator temperatures. HIGH PERFORMNNCE LIQUID CHROMATOGRAPHY (HPLC) 12.1 Each sample was analyzed by reverse phase HPLC using the Column, 3.9mm x 30.0cm conditions described below: v/v); u-Bondpak C 8 ; Solvent Systein, Methanol:Water (55%:h5%, The retention 5 ml/min; Detection,UV at 254 nm. Flow Rate, times of RDX and propiophenone were 3.7 and 7.3 minutes, The limit of detection was 0.2 ug RDX/ml respectively. and is defined as 5x the background noise. acetonitrile "The representative chromatogran is Figure lC.1. For levels at and below 0.005% RDX, the chromatographic conditions have to be changed, since UV absorbing compounds interfere The eluting solvent in these cases with the RDX quantitation. v/v) at a flow rate of 1.5 ml/min. is Methanol:Water (45%:55%, "12.2 The chromatographic system was calibrated minimum of two injections of one standard of the chromatographic range. daily with a representative of 15.0 ul was used for each sample, except ator An injection volume S12.3 The injection volume at 5 & 10 ppm "below 0.0010% level. If the peak exceeds the linear range of a sample "was 25.0 ul. it was diluted and reanalyzed. S" * 12.4 For levels of 0.005% and below the retr' ion times are 4.8 and 12.9 minutes for PDX and propiophenone respectively. 12.5 Following the completion of an analysis or a set of'analyses, solvent conditions to 100% a gradient doing from the initial methanol in 15 minutes will be run and the column will be eluted one hour. with 100% methanol for at least 140 -. --• -" " -.- ". .-" ."." ."................................................................".......................•',r•.t • L••t • - •- C.A • ULA TrcS 13.1 Determine the concentration of RDX using the formula: x D x 100 RDX in Sample = (Ax)(Wis) (Fx) Ais (Ws) Where Ax = Area (X) where x is RDX Ais = Area (internal standard) * Fx - Area(x) x Weight (Wx) -Area (is) x Weight (Wx) Wis = Weight of the internal standard C-) 13.2 Ws = Weight of the sample D = Dilution factor Wx = Wt of component x is RDX The results should be reported in percent RDX composite. This is the RDX actually used in the toxicity study. Where replicate samples are analyzed, all data should be reported. All results are recorded in standard IITRI logbooks and these plus chromatograms and data tapes are retained in the Chemistry Division Q.A. files. SAFETY 14.1 Safety regulations will be followed at all times, especially with regard to the handling of toxic materials. When the diet samples are being handled, a lab coat, and gloves will be appropriate attire. When solutions or extracts are being handled, a lab coat and gloves should be worn when there is the chance of direct contact with these materials. ( 141 ( "-' ,".' :.- . .'.".'.---- i i.,.- '- ,.,."- :- t .''%"-'-''''t ~~~~~..................' "e''__'*•••_,_ ' •._'*L.'-..••..•............. - ",[, --- '-''i . ::::•:W ;ii ; •• :","-- :' --- ' ••"•.• "• •" •• :r : : ." ?va-I?rvat1112 Li APPENDIX ID STANDARD OPERATING PROCEDURES FOR THE PREPARATION OF TNT AND RDX DIET PRE-MIXES * 1. OBJECT The object of this Standard Operating Procedure (SOP) is to set down procedures which, when followed, will assure quality from lot to lot of the * subject pre-mixes. It also provides a guide to safe practices in the handling of these explosives materials. 2. HEALTH AND SAFETY The materials (TNT and RDX) being handled in these feed preparations are not only explosive but are also toxic. It would appear that the greatest *• risk is incurred by the covert inhalation of the aerosolized finely divided powders produced by filling and emptying the ball mill and the V blender. Accordingly the following rules are hereby promulgated. 2.1 0 When charging or emptying the ball mill or "V" blender a respirator mask, and gloves must be worn. The 3M "Dust and Mist Respirator," 09910 is available to the Chemistry Storeroom and is recommended. Surgeons gloves are recommended for use when handling these materials. They are also available in the storeroom. The above are considered disposable and will therefore be discarded: the respirator mask at the end of the day and the gloves immediately after they are removed. They %.,ill be incinerated along with all other expendable materials at the end of the work day. This will be done by the resident staff at KOP. These people should be made aware of the nature of the material to be burned. C ,1) 2.2 Safety glasses, with side shields supplied by IITRI will be worn at all times in the operating areas. 2.3 Cleanup of contaminated surfaces will be accomplished with damp wipers, by washing or wet mop. Dry sweeping is not permitted. 143 Spills will be cleaned up immediately. The spilled material should be discarded and not returned to the processes. Spilled material should be placed in an appropriate container, given to the KOP staff, with explanation of its nature, for disposal. General cleanup will be performed between mixes, i.e., say after preparing a TNT pre-mix and before starting on an RDX pre-mix. All work surfaces, the ball mill, "V" blender, and balance will be cleaned at the end of each work day. 2.4 Only a sufficient amount of explosives material for immediate (one or two days operation), will be removed from the explosives magazines to the work area. This will be done by the approved resident KOP staff. Overnight storage of explosives is permitted only in designated areas within the facility. Explosives material will be stored and transported in appropriate "Velostat" containers. Explosives in proper containers should be kept on the bench top, not stored in drawers. 2.5 After the pre-mixes have been prepared and further work is not planned for the following day, residual bulk explosives will be returned to the storage area. This will be done by KOP resident staff. 2.6 Hands, forearms, work area. 2.7 Eating and drinking in the work area is strictly prohibited. * 3. and face will be washed upon leaving the GENERAL 3. I Logbooks All activity and supporting data will be recorded in the approved IITRI laboratory logbook. 144 0 4 -- 3.2 Sample and Lot Designation Samples or lots of pre-mix are designated by a number relating it to a logbook and page as follows: NNN-nn.j where "NNN" is the last three digits of logbook registration number, "-nn" is the page number in that logbook and ".j" is the sample number. For example: Sample 838-14.5 would be described on page 14 of IITRI Logbook 24838 and would be the fifth sample on that page. "PRE-MIXES" "PRE-MIX" 3.3 • prepared for the feeding program will be designated as using a similar code. For example: TNT PRE-MIX 838-13.1. Data Format The data regarding the preparation of the pre-mixes shall be kept in the logbook in a prescribed manner. The format proposed is shown in Figure 1. 3.4 4 Labeling TNT pre-mixes, contained in "Velostat" bags will be identified with yellow tape and with a label as: 10% TNT PRE-MIX Wt Kg Lot No. (see 3.2) Date: mm/dd/yy Initials of the preparer 4 RDX pre-mixes contained in "Velostat" bags will be identified with blue tape and with a label as: 10% RDX PRE-MIX •) Wt Kg Lot No. (see 3.2) Date: mm/dd/yy Initials of the preparer 145 -....... . .................... ..... ....... .......................... ,. -.. *.o-.N ..-. -.-..- ..- 4. PREPARATION OF TNT OR RDX/FEED PRE-MIXES 4.1 Introduction Animals are to be fed the test materials (TNT or ROX) at very low doses. This requires that gram quantities of these materials be dispersed as uniformly as possible among large quantities of feed. In order to accomplish this the test materials are first dispersed at a concentration of 10% in feed. This is known as the PRE-MIX. Appropriate quantities of the PRE-MIX are then blended into large quantities of feed to attain the required dosage level. The correct preparation of the PRE-MIX is the subject of this SOP. ) TNT is a soft waxy substance with a low melting point which is difficult to grind. The starting material is TNT flake which must be reduced in particle size to meet the needs if this program. This cannot be done by ball-milling the TNT without a "grinding aid". The aid used in this study is the certified feed material; an animal feed in the form of a finely divided meal. Equal portions, i.e., 50/50 by weight of TNT and feed, are ground in a ball mill to form a TNT CONCENTRATE. This 50/50 CONCENTRATE is subsequently blended with more feed in a P-K "V" blender for form the 10'a PRE-MIX. The same procedure is followed for the preparation of the ROX PRE-MIX. While it was not necessary to ball mill the ROX with feed as a grinding aid this procedure was followed to avoid introducing another variable in the feeding programs. Thus the preparative procedure is the same for the TNT and the RDX PRE-MIXES. 4.2 Preparation of TNT Pre-Mix The required amount of materials are shown in Table ID.1. The following stepwise procedure is to be followed for the preparation of a 10% TNT PRE-MIX. 146 Table ID.1 IWATERIALS REQUIRED PPE-_'![: FEED CONCENTRATE For Pre-mix Wt (kg) Blend ".ith Ball Nill TN'T (g) + Feed () >- Feed (g) 1 100 Cog 200 2 200 200 1600 3 300 300 2400 4 400 400 3200 5 500 500 4000 6 600 600 4200 ) 147 IL ..."-,-" " •...".."..-............ """ ""' " '" "" ,l•Ir~ • ""•""""emn l••' " ""• ' "-""• """"""'I ""-• ", ••• """'- -. ,•" "i•'" 4.2.1 4.2.2 Determine the appropriate quantities of feed and TNT (from Table 1) to be weighed out to prepare the 50/50 TNT/feed concentrate. Weigh out the appropriate amount of TNT and feed. 4.2.3 Examine the ball mill jar and balls to be sure they are clean. The ball charge should weigh approximately 3.83.9 kg and is a mixture of 3/4" and 1/2" balls. (See logbook C24838 pg. 3). 4.2.4 Place the balls in the jar, pour in the weighed feed and then the TNT. Firmly fasten the jar cover and place on the mill. Turn the mill on and record the time. 4.2.5 Ball mill for 40 minutes. Record the exact time in the logbook. Remove and open the ball mill jar. Sift the powdered feed/TNT from the balls onto a large piece of paper. (Use the large hardware cloth basket provided to separate the balls and powder.) Weigh this feed concentrations and record in the logbook. 4.2.6 Weigh out the larger amount of feed into an appropriate bucket. Record the weight. 4.2.7 Open the blender top on each leg of the "V". Put about 1/3 of the concentrate into the blender (i.e., at the point of the "V"). Pour about 1/2 of the feed into the blender. Put another 1/3 of the feed, in the blender, dividing it between the two legs of the "V". Pour the rest of the feed into the blender (1/2 into each leg) and divide the rest of the concentrate between the two legs of the "V". Close the blender. 4.2.8 Run the blender for 15 minutes without the intensifier bars on. Turn the intensifier bars on for 15 min. Turn off the blender. Note and record the total blender time. 4.2.9 Open the cover at the apex of the V and dump the content of the blender into a Velostat bag. Make sure the blender is completely empty. Seal the bag and label (see 3.4). 4.2.10 Clean the blender by running with about 2 kg. of feed material only. Repeat this twice. Discard the feed material. Disconnect the blender from the wall outlet, clean the blender with damp wipers. 148 S. . . . . . . . . .... . . .. . . -. . .- ' '. .,- -. , ."-"-'. .• -"".% -'.% ' ' ' " " ' Clean the ball mill by a similar procedure, i.e., milling feed only, repeated twice followed by a damp wiping. * 4.2.11 Notify the proper individual that the PRE-MIX is available and expected date of shipment to the Chicago laboratory. 4.2.13 The PRE-MIX procedures will be witnessed by second indiviual and second individual will sign the logbook as witnessed. * 4.3 Preparation of RDX PRE-MIX The required amounts of materials are shown in Table ID.2. (stepwise The following procedures are to be followed for the preparation of a 10% RDX PRE-MIX. 4.3.1 "• about one kg of alcohol wet RDX is placed in a wide mouth gallon jar containing ½ gal of distilled water "• the jar is agitated for five minutes by rolling on the ball-mill or by shaking "• the RDX water-slurry is decanted through a 60 mesh or finer seive muslin cloth. The crystal cake is drained to remove excess water * the water wet crystals are then returned to the jar and the above procedure repeated two more times "• after the final wash the crystals are spread on paper toweling and let dry overnight or dried in oven at approximately 100°F overnight * ) • .. "'"""".-... ...... 4.3.2 Determine the aDpropriate quantities of feed and the washed and dried RDX (from Table 2) to be weighed out to prepare the 50/50 RDX/feed concentrate. 4.3.3 Weigh out the appropriate amount of ROX and feed. 4.3.4 Examine the ball mill jar and balls to be sure they are clean. The ball charge should weigh approximately 3.83.9 kg and is a mixture of 3/4" and 1/2" balls. (See logbook C24838 pg. 3) 4.3.5 Place the balls in the jar, pour in the weighed feed and then the RDX. Firmly fasten the jar cover and place on the mill. Turn the mill on and record the time. ''"'.-"".''"."."."'".... -• w.............""-. "" .. """"""" .. Table ID.2 ,.ATERIALS REQUIRED For Pre-nix ',wt (',g) FEED COETP.I.TE Ball Nill RDX (+ Feed (g) PPE-.IX Blend With Feed (g) 100 1 9 0 ,2u 200 1500 300 3.] 2400 400 400 3200 5 500 500 4000 6 600 6CO _300 3 150 L-I * ) k 150 -----------------------' "- .",",' " ." -",', N"" :": -", : •••--- -; -"-" - ------ ,. Y "i * 4.3.6 3all mill for 40 minutes. Record the exact time in the logbook. Remove and open the ball mill jar. Sift the powdered feed/RDX from the balls onto a large piece of paper. (Use the large hardware cloth basket provided to separate the balls and powder.) Weigh this feed concentrate and record in the logbook. 4.3.7 Weigh out the larger amount of feed into an appropriate bucket. Record the weight. Open the blender top on each leg of the "V". Put about 1/3 of the concentrate into the blender (i.e., at the point of the "V"). Pour about 1/2 of the feed into the blender. Put another 1/3 of the feed, in the blender, dividing it between the two legs of the "V". Pour the rest of the feed into the blender (1/2 into each leg) and divide the rest of the concentrate between the two legs of the "V". Close the blender. 4.3.8 I 4.3.9 Run the blender for 15 minutes without the intensifier bars on. Turn the intensifier bars on for 15 min. Turn off the blender. Note and record the total blender time. 4.3.10 Open the cover at the apex of the V and dump the contents of the blender into a Velostat bag. Make sure the blender is completely empty. Seal the bag and label (see 3.4). • 4.3.11 Clean the blender by running with about 2 kg of feed material only. Repeat this twice. Discard the feed material. Disconnect the blender from the wall outlet, clean the blender with damp wipers. Clean the ball mill by a similar procedure, i.e., milling feed only, repeated twice followed by a damp wiping. 4.3.12 Notify the proper individual that the PRE-MIX is available and expected date of shipment to the Chicago laboratory. 4.3.13 In the first four months of this program 8 kg of the RDX PRE-MIX will be required each month. This exceeds the capacity of the available ball mill and blender. The following procedure is to be used. 4.3.13.1 Prepare two (2) 4 kg pre-mixes by following steps 4.3.1 through 4.3.10 above. This will result in two sub-batches for the final lot, identified as sub-batch 1 and 2. * 151 S. "-. -,'i ,-',-L. -" . ." -. >.-. .:- -. ,i " - ""17 4.3.13.2 Blend one-half of sub-batch 1 with one half of sub-batch 2 in the "V" blender for 10 minutes with the intesifier bar "on". Repeat with the remaining halves of the sub-batches. These become two new sub-batches. Repeat the procedure. 4.3.13.3 Combine the results by alternately dumping portions of the resulting sub-batches into the large Velostat bag. Periodically shaking the bag and mixing the contents. Proceed with steps 4.3.11 and 4.3.12. 4.3.13.4 The premix procedure will be witnessed by second individual and second individual will sign the logbooks as witnessed. 4.4 Any deviation from this procedure must be first cleared with the project leader and must be recorded S) as an addendum or revision to the SOP. Record any unusual occurrence in the logbook and advise the project leader immediately. ,. "In cases of uncertainty contact Robert Remaly, ext. 4309 or Barry Levine, ext. 4901 before proceeding. "4.5 Transmittal Record "Transmittal record will be initiated by the person who is preparing the premix. * All the pertinent information must be filled. record must accompany the premix. -* obtained from the Principal The test article premix Copies of the transmittal record can be Investigator. lj .. ,...........-.. -.................. ... '..'....'....-...... ....-.. ,-*..- .'••,--- --- --- • ,-, -•• APPENDIX IE * SAMPLE COLLECTION AND STORAGE (TNT AND/OR RDX PREMIX SAMPLES) Scope. * 1.1 This procedure covers the coilection and storage of TNT and RDX premix samples prior to analysis. FMaterials and Equipment * 2.1 Small scoop 2.2 Powder funnel 2.3 Amber vials with plastic screw cap Sample Collection 3.1 Personnel of the Life Sciences Division will inform the supervising chemist and the analyst when they receive TNT or RDX premixes. The analyst will collect 6 samples from the'Velostat bag container, one from each of four corners and two from the middle. At least 5.0 gram quantities of premix will be collected in order to permit the extraction and analysis steps to be perAll samples will be identified according to the Chemistry Division identification system. All detailed information will formed in duplicate. be placed in the sample identification logbook inmediately. The sampling procedure for the premix will be performed as follows: One sample is removed from the center of the storage bag with a small scoop which will permit the removal of a 5.Og quantity. The second sample will also be removed from the center of the container in the same manner as the first sample but at a deeper level. g 153 IL After center sampling, the surface of the premix is restored by leveling and four additional samples will be removed with a small scoop from each of the four corners of the bag at gradually increasing depths by lifting the corners of the bag. The 6 samples will be labeled and placed in amber vials with plastic screw caps. Number, The label will contain Date Sampled, Sample Premix Identification, Lot Number and Sampled by Initials. Sample Storage 4.1 All samples will be stored at refrigerator temperatures in the dark prior to analysis. and control samples. -) will be changed. This includes feed that will be used for blanks Every three months (from manufacturing date) feed This manufacturing date will be supplied by Life Science Transmittal Record 5.1 Transmitted record will be completed by responsible personnel. "Acopy of Test Article Premix (T.A.P.) and/or T.A.P. Sample Transmittal (or custody) record i5 attached (Figure IE.1). Sample Disposal 6.1 Samples or parts of samples will be returned to the Safety Officer for disposal. 154 FIGURE IE.1 TEST ARTICLE PREMIX (T.A.P.) AND/OR T.A.P. SAMPLE TRANSMITTAL (OR CUSTODY) RECORD Project No. - Study No(s). T.A.P. Lot No. * T.A.P. Intended Concentration: Prepared T.A.P. Received (L.S.R.) Date/By: % Quantity (kg): Logbook No./Page No. * (K.O.P.) 5002 Lot No.: Storage Conditions of T.A.P. Date/By: (KO.P.): Logbook No./Page No.:__ Storage Conditions of T.A.P. in L.S.R.: T.A.P. SAMPLING AND ANALYSIS * T.A.P. Sampled Date/By: Witnessed By/Date: * S, " * Logbook No./Page No.: Storage Conditions of T.A.P. Sample by Chemistry Personnel: Extraction Performed By/Date: Logbook No./Page No.: Analysis Performed By/Date: Logbook No./Page No.: Data Reviewed & Approved By/Date: Analytical Report Prepared By/Date: Checked By/Date: Quality Assurance Check By/Date: Analytical Report Received (L.S.R. T.A.P. First Used By/Date: Excess T.A.P. Supervisor) By/Date: T.A.P. Last Used By/Date: Submitted to K.O.P. Personnel for Disposal by Burning By/Date: Quantity (kg) Excess T.A.P. )• Received By/Date: Key K.O.P. = Kingsbury Ordinance Plant, La Porte, IN. 5002 = Purina Certified Rodent Chow 5002 155 APPENDIX IF SAMPLE COLLECTION AND STORAGE (TNT AND/OR RDX DIET SAMIPLES) Scopre 1.1 diet This procedure covers the collection and storage of TNT and RDX samples prior to analysis. Materials and Equipment * • 2.1 Small scoop 2.2 Large scoop 2.3 Powder funnel 2.4 Amber vials with plastic screwcap Sample Collection 3.1 -• Personnel of the Life Sciences Division will inform the supervising chemist and the analyst when the TNT or RDX diets are available. Tne analyst will collect 6 samples from the plastic tub container, one from each of four corners and two from the middle. - The tubs receiving the rat diets are The tubs receiving the mouse rectangular with a capacity of 42 liters. At least 30.0 gram quantities diets are square with a capacity of 27 liters. of diet will be collected in order to permit the extraction and analysis steps to be performed in duplicate. All samples will be identified according to * the Chemistry Division identification system. All detailed information will "be placed in the sample identification logbook immediately. ., The sampling procedure for the diets will be performed as follows: One sample is removed from the center of the storage container at the surface of the diet. This sample will be removed with a small scoop which will permit the removal of a 30.Og quantity. The second sample will also be "removed from the center of the container about half the distance to the bottom after toxicology personnel have exposed the sampling site by shifting the 156 !~~~~~~~~. .•--.-" . .' .•::''......-.... . .... n - ,,,, n -W3 diet toward the side of the container using a large scoop. sampling, After this the surface of the diet will be restored by leveling. Four additional samples will then be removed with the small scoop, one from each of the four corners of the container at gradually increasing depths within the container, again using the large scoop to expose the sampling sites. The 6 samples will be labeled and placed in amber vials with plastic screw caps. The label will contain Date Sampled, Sample Number, Diet Identification and Lot Number and Sampled by Initials. Sample Storage 4.1 All samples will be stored at refrigerator temperatures in the dark prior to analysis. and control samples. .. will be changed. This includes feed that will be used for blanks Every three months (from manufacturing date) feed This manufacturing date will be supplied by Life Science Personnel. Transmittal Record 5.1 Transmittal record will be completed by responsible personnel. A copy of transmittal record for diet sample analysis is attached (Figure IF.0). Sample Disposal 6.1 Samples or parts of samples will be returned to the Safety Officer for disposal. 157 0 FIGURE IF.1 TRANSMITTAL RECORD FOR DIET SAMPLE ANALYSIS Study No. Species Test Week Test Article Lot. No. % Conc. of Premix Diets calculated by: Date: Premix weighed by: Date; Diet prepared by: Date: 5002 Lot No.(s) Dose level: mg/kg/day sex: T.W. intended conc. mg/g Dose level: mg/kg/day sex: TT.W. intended conc. mg/g Diets Stored in the Refrigerator (4°C) From: To: First Cay of Test Animals Exposure to the Diet: Dict Samples Taken By/Date Loghook No./Page No. Witnessed By/Date: Storage Conditions of Diet Samples in Chemistry Division Extraction Performed By/Date: Logbook No./Page No. Analysis Performed By/Date: Logbook No./Page No. Results Logbook No./Page No. Calculated By/Date: Data Reviewed and Approved By/Date: Analytical Report Prepared By/Date: Quality Assurance Check By/Date: Results Reveived By: Date: KEY: -) T.W.= TEST WEEK 5002 = Purina Certified Rodent Chow 5002 158 :::.-......•..•..•,,..,..•-......:.•..--.,.-......-..,-~~............. . . . . ...-.......-... .. ,... ............ _..-............ ,............... .. , -:,..-.. ., S 0 APPENDIX * II 5002 CERTIFICATION PROFILE/ANALYSIS 1 '59 ,4 ,. a,• .:,-:--:- -: .'•,@ ,:,-.* •,•a.• " L:J ' "L-' a,,j, " " " "* ",*". "..-- -- "-" ".V "- " " " " " .." " * " '" - TEl ANALYTICAL, INC. 460 soUTH NoRwwTEST .NOWAY • PARK RIDGE. ILINOIS 9 W October 29, 1982 0 SlITT •12/•1•1-201) LABORATORY REPORT L R Page 1 of 2 paces #9166 Dr. Marianna Furedi P.O. #16092 Research Institute 10 West 35th Street Chicago, Illinois 60616 Sample received June 9, 1982 [TEI-14080J Rodent Chow #5002 - March 24-822G Result in ppr.n * Method Nitrate Nitrogen Nitrite Nitrogen < 0.05 0.014 < 0.05 hiercury Arseni c 0.61 < 10 Lead Penicillin * < 1.0 BHT BHA Total Estrogen Chlortetracycline 4 Aflatoxin C, fflatoxin G2 Dieldrin < < < < 0.005 0.02 0.005 0.005 0.001 Endrin Aldrin Heptachlor Epoxide BHC < < < < 0.001 0.001' 0.001 0.001 PAflatoxin B2 .,? .1.'1.' 0.01 ...' " ' ',6.. 25.103 JAOAC 60.813 25.02C SCadmium 25.058 Snell & Snell, Colorimetric Methods of Analysis Vol IVAAA. p. 221 JOOAC 60,505 JAOAC 60,505 < 1.0 not detected 39.000 to be reported at a later date fflatoxin B1 4 7.030 7.030 19.0 0.24 j 26.003 26.003 26.003 26.003 29.000 29.000 29.000 29.000 29.000 . 71 - L- 7- . TEl ANALYTICAL, INC. 40 SOUTH NORTHWEST HIGHWAY PARK RIDGE. ILUNOIS 9 SOM October 29, 1982 312/INC9-2070 LABORATORY REPORT #9166 .Pae 2 of 2 pages Dr. Marianna Furedi ""IT Research Institute 10 West 35th Street Chicago. Illinois 60616 ""TEI-14080J Rodent Chow #5002 P.O. #16092 Sample received June 9, 1982 - March 24-822G Result in ppm Lindane * Method < 0.001 < 0.001 29.000 < < < < < 0.001 0.001 0.001 0.001 0.001 29.000 29.000 29.000 29.000 29.000 29.000 Polychlorinated Dioxins < 0.001 < 0.001 < 0.006 Parathion [lethyl Parathion < 0.001 < 0.001 29.000 29.000 Enthion Carbophenothion < 0.001 < 0.001 29.000 29.000 .ialathion konnel < 0.001 < O.D01 29.000 29.000 Dtazinor, < 0.001 29.000 Vi sul feton Phorate < 0.001 < 0.001 29.000 29.000 DDT Total Nethoxychlor Chl ordane Mi rex Toxaphene Strobane HCb PCE 29.000 29.000 28.128 *Official Methods of Analysis of the Association of Official Analytical Chen ists. 162 7 , ~~-/a~ Irma.-. -. ?Aw .w .Www r- ?rm-17.h?nn APPENDIX TEI ANALYTICAL CHEMISTRY METHODS ANALYTICAL PROCEDURES USED BY TEl ANALYTICAL, INC. PARK RIDGE, IL TO ANALYZE PURINA CERTIFIED RODENT CHOW NO. 5002 FOR ItPURITIES "Limit of Chlorinated i PetciduDee Screena1 pt Chlorinated Pesticide Screen Phosphated Pesticide Screen 10 ppb 50 ppb Polychlorinated Eiphenyls (PCBs) Hexa-, hepta-, octachlorodibenzo-p-dioxin -eavy t*etals Arsenic Cadi urn Lead 1,iercury Nitrates Nitrites Aflatoxins Penicillin 100 <100 Chlortetracycl ine 10.0 ppm A.O.A.C. 1.0 10 10 <1 pyruvate Henry, R.J., Berkman, S. technique Chiamori, N., Golub, O.J. and Am. J. Clin. Path. 34, 381, 1960. Modified Bessey-Lowry technique Neumann, H. and Van Vreedendaal, Acta. 11, 183, 1967. M. Clin. Silver chlcride precipitation method Chloride Meter (Corning Medical Co.) Catlove, E., Trantham, V. and Bowman, Lab. Clin. Mled. 5-j, 358, 1958. Chem. R.L. J. Biuret technique Centrifichem Centrifugal Analyzer system Failing, I.F., Jr., Buckley, M.W. and Zak, D. Flame Flame photometry Klina Flame Photometer (Beckman) photometry Klina Flame Photometer (Beckman) Am. J. Clin. Path. •, 83, 1960. 170 -'-- -' -'. '-•<' ---- -"• •" '• -"' -.- <-'<' ," " '1 "<""- -'.' " ""% "- ""," "• '' ,.-'--<-%V'-. -,%,', .. Bromocresol green method Centrifichem Centrifugal Analyzer System Rodkey, I.L. Clin. Chem. 11, 478, 1965. reduction method Tetrazolium salt Centrifichem Centrifugal Analyzer System R. Klotzsch, S., Serricchio, M. and Furedi, Advances in automated Analysis 1, '-Iediad Inc., Tarrytown, N.Y. P. 111, Vol. 0 1973. fModified Oliver method Centrifichem Centrifugal Analyzer System Oliver, l.T. Biochem. J. al, 116, 1955. Cholesterol esterase-cholesterol oxidase method Centrifichem Centrifugal Analyzer System P., Bernt, E. and Gruber, V. Z. Roseschlaw, Lin. Che. u. Klin. Blochem. 12, 226, 1974. F. Alizarin method Centrifichem Centrifugal Analyzer System Clin. Chem. Connerty, H.V. and Briggs, A.R. 11, 716, 1965. Modified Walters and Gerarde method Centrifichem Centrifugal Analyzer System Microchem. J. Walters, M. and Gerarde, H. 1970. 15, 231, Modified Walters and Gerarde method Centrifichem Centrifugal Analyzer System Walters, M. and Gerarde, H. Microchem. J. 15., 231, 1970. 171 - a APPENDIX VI INDIVIDUAL ANIMAL DATA 173 Li •~, ~~ ~ ~ ~ ~ ,~uj>L • - -.. -.0,-•-.• ---••~~.., •i-•-.-• •, - N>NZIN > z 0 - - - - -.- .-- .- - - - • .. •, N 0o--o0-N - .. - -u D " z x * -cZ (D : w coU - x L F- - U) -• I- 0 (x z 0- o.. 0 ý Z~NN CNC' 2-NC dJN' IICC . 17k - 0 ý,ý -I LL CUý ý LC JCNL I U ý I I L -Z= >Z 0------------------- 0- ------------- foo~0 1714 00L r -- .w LcmN0w r--rTNmm - - - 0mt - W ---- ---- -o er - - LL L & I -- - - --- -LL LýL -- 0 0- -0 - O O ---0 --- 0 -- - - -- 0 W-n I ýL -W -IL.L --f W~--------D ~ n t-- -- 0' W Z *Z >-L I-in 0---c U u -I - I UIe >,, 4L t 0 ~I I V)L CL In. -jw 0 0Z=' -CCN CCCN. - C-4~ N ----- NC ~ - NC CN4 NC4CN CN NNCN CN CV~ N CU Cl- Cz C ->~ C> -I C ZCI C~---------- CI ý wC U. 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Nw 0r - U ~L~t0 ~ MNw- M N - O vv ~0W M-iflt v I mot- o m c' r w m I N v~ ) 1 0 0 1 mmI0m 0 I 00W(-( mm o r-- Ow r- I I)v00 Ni W to nIVIf-- m 0nA r -. CD IC1 1 I v w fr- M cc w0 0I- I~IC 4-)rl0 O C z) ~ D I C -llI0 C V ~ 0 zI N M 1C)0 I- 00v 3t m v 0) I m~N( r- V)0 c Nf m r- U) n : v r-0O)0qOI mvmI I NC v ~m i -Or-r v 0 C ) C)V ~ ) ') )~ I I C) I I N C 0 (N(N I C )I cLru 0 CLLW uz > NC D C D 0 N ) Iý 0 C0mf ) ( C -- N N - L ( Iu-0Lr) Cc~ cc CY (z > M 0 -r-mwO-mwf W-MN z~L 4o- M 0: - * rWMMWN00r -w0r 100mmmwmr-mwr 1I - - - - - - - - - - - - - - - - - - - CLA~-------------------------(NC)C-------W - - -- - - - -i CC 182o z I NC) nWr Dm0-cif -- - O 0 <-X - I~ . I - LrIOInO w 0 -MI n0r 0 oww -r-MMr ( om0-C4MN ( r, n in mw w w w-w ,0r w--c MOO -f CC ) w w.~ MC M0 MD~lC~ r- M M T X Nc nWI wwC, ~ NN~ 0WU WN---0 -- 0I4 i I -M-I0W N 0 Omw- mmMO•O NN N N-4W N 0 NO --- g W 0 i-No, INC(NINININ(NIN- 0:0 MO•O0 SO ~ O -O• OM M O--O0mm-WNWO-- MMO MOO N- -- - - •0 0M N -0.40- . (Z cl - I mmo - .4-1 0~WI W 0 c- = P :O (NIxt 0OMMOMM-O --- mO-OOmM-MmOMMmOMMMM ---------0 0----- MOMMO - W W w - ~ w 000000 t-Or- -0- O .- LLtI- 0- W >LL ý- N -- f D - >0O 0 P w w r~W~ - C - I - - C N - - - I- ~ W0rW~ -- - - - - - -- - - - w w w ~(w 0In-W -0 -- - - - - - OW W W -W P W W 4 ---- - - 0 WWWuW 4IW 0-N- w w~-0 t- - N• - - Ce( LlW 8 - W UPLl cc 0-0 -~ -3 C14 Mb C, C- C14 (NO - mf m N)0 m m 07- D (NP-em N- NM 0 ( N P-P M N~ M - M~ NM(qNI- N 000 a) -0 - L- o w 0W000))O w0 w - 000000 0 700 -------- 3.42. ---- - ----- ---- ---- ---- --- N)L 0•~~ O-- :••0 0• N0• -0--- W00-OC VIN. wf00n0 -0 WI 0~~~~ SI(NO OD~~f l O00;MMNNN0W(N '•N 000N InN I M NI (NI I- N 471 04 -O 0IN n-N MO-(N-0 I•O (N-C NN 0 N 0•0 "N --- 00o- O ( N N 'OI N-t- O M O OWO • (N W0 0 ~ 0 0 INN041C4 I 0-0 -O-O OCO- m0 • WWoICC )I -I --- 00-000W o0 N N -t•00•0 0- V O)O -mW• 0 I( .V) ) W 0W • 00 0l . ) N-0n o1 w -O -c 06 IX IN( "-l > > . -- • O c0 I----0------------•0 -- N O WO • - 00- - > O00--0OW•OOO0LtI N NN - -------- 0( --- 1-J -u -- -r - N ýw .,0. ) 00 11f .)r -Mr n M I p0 ur M r--- -- -- I -- --- -- - -- - fI a -2 0W f- NW~Nt 0 I -OW- W Q ~~DI * ~ -- - I _3 U - n 0- 0 M4 - 31 o0- C) I D W 0 N 0 O WW W LO f C C. 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U00*- . . 0' ' 0-eo~o CN0W 0' -O0 0 33 I 0000,00000000.00000000 0 00000000000000NO00 Ix LlW I -0W-00O 000000000000000000000000000000 I-d ~~ * 1.IL 0000000000000000000000000000000000000000 *~2Z 3L 4-) 0u z0t- L)ZJJ-/ u m I-( 0 N 0 Nw -- O N -0-N d~ 0 -- Mr 0 N 0t- M00 M O ~I(M N W W N ~ L)Ix ~ j - .Le c > LL4 .0 c 0 * . . . . . . '00000000000-000 1-. > . . :,:.-0000000-,00-00-, 0 C--. x 6-. -- 338 wi IC-(N WN M LL 6WLW L 4 & . &i MN(D CC- (N VN ID MNCe (n W (N-) (N LLu. 6U. -N (N r- QN (N -N LL r- (N W CON(n u (N (N W ýL () ýL .ý LwL ) I- (CN ) f(N (N ýýU (N (N ) CWO 0 LD 00000 0OZ400000000 0 -- ::00000000 0C)I Y) rU W )L)M0N0 0 r.o 0*r) ' 0C V) f) c 0 00000000000O 4CMWZ4.jo 6-L.J 1000000000000 U-L& w -..- a 0.u U* I Z> I-J x I (D 4 6-a a O . ICX I;n0 - 0 ) l qmwwom" O wMWO >6- MJ oo C14 M N) N0V r- r- 4~o)O 0z 0 m CW-co0 wO e eqIf M -0-r I, r f,33 APPENDIX ViI CHLORTETRACYCLINE CONTENT OF 5002 - .. ~%-.-*. .- : '~-.-~~~~cx. % ~ . ~-:-:~ ~ 341 . -4 CHRLORTETRACYCLINE ANALYTICAL *' RESULTS .•QURJ;i OE A1•ALY•IS TEl ANALYTICAL* TEl ANALYTICAL* CONTENT OF 5002 (ppm) A B 9.9 12 9.9 7.7 10.2 "SCIENTIFIC ASSOCIATES** 1.76 1.72 1.20 1.64 WOODSEN-TENENT N. N. N. N. HARRIS LABS, LABS, INC.** INC.** Sample A = Lot Sample B = Lot Sample C = Lot Sample D = Lot *Method: Snell Vol. IVAAA, pg. D. <0.05 No No No No and 184 Sept.18.81 Dec.10.81 March.24.82 Sept.10.82 Snell, <0.05 (Original Colorimetric "**Method: AOAC, XIII, pg.722-723, Detection limit > 9.1 ppm N. D. = None Detected D. D. <0.05 D. <0.05 lot) method of paragraph analysis. 42.211-42.214; 342 . . . .. . . . - . . . . . . . . . . . . . . . ; .-. ;:- APPENDIX VIII NITRITE AND MERCURY CONTENT OF 5002 NITRATE, *0 ( ( 343 . . . . . . .. . • ' "! . . •" • ' 4 b v • " " " " b " ° %. S%"% . . * '...° *." .. NITRATE, LOT NUMBER , " SEPT 17-80IG OCT 29-801N NOV 19-802K DEC 02-801G JAN 08-811J JAN 15-812E FEB 03-811B JAN 21-811N MARCH 05-811A MARCH 17-811M APRIL 30-811D MAY 13-812K JUNE 01-812D AUG 04-811T SEPT 18-811A OCT 07-811J V 12-811G 10-811A 22-821K 2Eu 09-821C MARCH 24-822G MAY 12-822F JUNE 04-821K JULY 29-821G SEPT 10-822J OCT 20-822L NOV 23-821M .................... 344~ NITRITE, AND MERCURY NITRATES(ug/g) 7.8 5.6 3.4 1.1 14 32 9.2 32 13 <3 <3 15.3 <2.0 28 <2.0 6.3 16 12 14 7.2 19.0 16.4 17.0 11.8 5.0 4.7 15.4 CONTENT OF 5002 NITRITES(ug/g) 0.04 0.4 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 0.3 0.2 0.6 0.5 <0.1 0.2 0.4 <0.2 <0.2 0.4 0.24 0.1 0.1 0.1 0.1 0.1 0.2 MERCURY(ug/g) 0.03 0.06 0.05 0.04 0.04 0.02 0.04 0.11 0.14 0.02 0.01 <0.06 <0.1 0.03 0.05 0.15 <0.02 0.09 <0.05 0.05 <0.05 <0.05 <0.05 0.06 0.2 0.2 0.05 I , , , I1 . U, U . .. . . .*.. . ,. ** '*.,uI . in U I, .. " m . .'- ., W•~ , •. •,. * .b - 0 APPENDIX IX CHICAGO WATER CHEMICAL ANALYSIS !* 3 S 4! 4 345 . - w•- •• \ 0~ 4n4 z'IL 0'0 0 z O a we a a- i Is ~C4 dat wdc.~r. -g 000 I.-- z.a -Im ! ju ID' § 0 0 £l 00 bi -i :; u w 346L 0.*,.p. > w c6 US4.,.Kw .*~-~*. m*--GCd~ .... sV..-v *...* - - - a-* -; V; 9;*** DISTRIBUTION LIST * 25 copies Commander ATTN: SGRD-UBG U.S. Army Medical Bloengineering Rest ,r,.h and Development Laboratory Fort Detrick, Frederick, MD 21701-5010 * 4 copies Commander U.S. Army Medical Research and Development Command ATTN: SGRD-RMS Fort Detrick, Frederick, MD 21701-5012 * 12 copies 1 copy * 1 copy 1 copy * Defense Technical Information Center (DTIC) ATTN: DTIC-DDAC Cameron Station Alexandria, VA 22304-6145 Dean School of Medicine Uniformed Services University Sciences 4301 Jones Bridge Road Bethesda, MD 20814-4799 of the Health Commandant Academy of Health Sciences, U.S. ATTN: AHS-CDM Fort Sam Houston, TX 78234-6100 Army Commander U.S. Army Medical Bioengineering Research and Development Laboratory ATTN: SGRD-UBD-A/LIbrarian Fort Detrick, Frederick, MD 21701-5010 lIT RESEARCH INSTITUTE 347 ;--,. . -.•. --. -.j,,-. ... .- .-...-. ..- -... .. . - .- .- .: ..- . . ,-. ,-,,. -. -. . . . . . .. - .. . . . . .. -,- .. - . .. - . -.. .. . .. .. . . -. .. . . . .