Document zdz8qBBMeGE31eDgO4X2ErMjz

LB I PROJECT 20771 FINAL REPORT EFFECTS OF PCB ON THE REPRODUCTIVE PERFORMANCE OF THE RHESUS MONKEY (Macaca aulatta) APRIL , 1977 - DECEMBER 11, 1978 SUBMITTED TO MONSANTO COMPANY 800 NORTH LINDBERGH BOULEVARO ST. LOUIS, MISSOURI SUBMITTED BY LITTON BIONETICS, INC. 5516 NICHOLSON LANE KENSINGTON, MARYLAND APRIL 30. 1981 BIONETICS L HONS 016378 TABLE OF CONTENTS VOLUME 1 -- TEXT I. OBJECTIVES........................................................................... II. SUMMARY............................................................................... III. MATERIALS ANO METHODS ................................................... A. Adults....................................................................... B. Infants...................................................................... IV. RESULTS............................................................................... A. Adults....................................................................... B. Infants....................................................................... C. Intarcurrant D*aths--C11n1cal Raportj .... 0. Protocol Olscrapanclas................:................... E. Pathology.................................................................... V. CONCLUSIONS....................................................................... A. Adults........................................................................ 8. Infants......................................................................... VI. KEY PERSONNEL................................................................... VII. QUALITY ASSURANCE ........................................................... VIII. RECORDS RETENTION........................................................... IX. REFERENCES........................................................................... APPENDICES VOLUME II - APPENOIX A Tost Schodula Btrth/Daath Schadul* Compound Racalpt Vltaaln A Sarua Lavals Infant Oata Fag* No. 1 2 3 3 10 12 12 17 19 23 24 32 32 33 34 35 36 37 MONETICS HONS 016379 TABLE OF CONTENTS (Continued) APPENDIX 8 Correspondence/Meieoranda APPENOIX C Protocol Changes APPENOIX 0 Urinalysis APPENOIX E Sanaa Analysis VOLUME III - APPENOIX F Haaato1ogy/Cyto1ogy Blochaalstry Body Weights VOLUME IV -- APPENOIX 0 Pathology Results BK0NET1CS HONS 016380 I. OBJECTIVES The primary objective of this program was to evaluate the effects of three dose levels (5, 25, and 100 pg/kg/day) of a polychlorinated biphenyl (PCS), Aroclor 1254, supplied by the Monsanto Company (MC) on the reproductive performance of the rhesus eonkey (Macaca wulatta). The pathologic effects of these three dose levels were to be evaluated through gross and micro scopic postmortem examinations. A further objective of the project was defined as monitoring hesMtology and clinical chemistry values during the experimental period as well as periodic examinations to survey organ systems which were known to be affected by PCB Intoxication. Ourlng the course of the study, further objectives were defined as follows: Evaluation of the pathologic effects of PCB's by the gross and microscopic postmortem evaluation of offspring of the fasMles to which PC8's had been administered; Evaluation of the effects of PCB's on the hematological values of these offspring; Evaluation of the clinical effects of PCB's on these offspring. BIONET1C3 MOWS 016381 1 II. SUMMARY Thirty-two TmmIo and slxtoon imIo adult rhesus monkeys were equally ; divided Into four groups which received either a placebo, or 5, 25 or 100 tig/kg/day of Aroclor 1254 for 14 months. After 6 months of compound administration, the animals were bred to non-study counterparts of the opposite sex In order to determine the effects of the test compound on ; fertility. Infants of test females were allowed to nurse for two months and ware than euthanatized If they were clinically normal or nursery ' reared If signs compatible with PCS toxicity were present. Body weights, ( serum chemistry, hematology, urine, and semen samples were obtained at various Intervals for analysis. All adult test animals and Infants of test females were subjected to gross and microscopic postmortem examina tion. No effect upon fertility occurred among the adult males In any dose group. A significant effect upon fertility was observed In the females receiving . 100 pg/kg/day of the test compound, but no effect upon fertility was observed In the lower dose groups. A depression In red blood cell values (PCV, RBC and Hgb) and platelets, probably the result of test compound effect on the bone narrow, occurred In the medium and high dose adult animals. A decrease In serum cholesterol values In high dose faswle animals and an Increase in serum Iron values In high dose males and females and In medium dose females was considered to be treatment related. \. Alterations In albumin, globulin, and A/S ratio were considered to have a causal relationship to test compound administration In adults of both sexes. A dose-dependent effect upon physical changes In the adults and Infants was noted with the majority of all effects to be upon the skin ' and Its adnexa. Body weight loss In the treated adult animals was con sidered to be dose dependent. . The incidence of histologic morphologic effects and mortality was depen dent upon dose, sex, and age. Infants were more sensitive to the test compound than were the adults ranging from severe changes In the only offspring of high-dose level festales to a minimal morphologic effect In the mandible of one of six infants bom to low dose mothers (five live, one stillborn). No effects were seen in the control or low dose adults; however demonstrable morphologic effects were present at a higher average ` Incidence In the high dose animals and among the females only in the medium dote group. BIONETICS HONS 016382 2 III. MATERIALS AMO METHODS A. Adults 1. Animals , Thirty-two female and 16 malt, adult rhasus monkeys (Macaca mulatta) wara salactad from thm Litton Blonatlcs, Inc., (LBI) commarclal rhasus breeding colony for usa In this study. Tha animals had baan all wildcaught and had baan at LBI for a period ranging from 2 to 9 years. At i ' tha beginning of tha study, tha animals ranged In weight from 4.4 to 13.4 kg (Females: 4.4 to 8.1 kg; Males: S.7 to 13.4 kg). Tha animals wara Identified according to a unique numbering system by a tattoo on . tha skin of tha thorax. All of tha animals selected for this study had ' an established average or above-average reproductive history. Since the animals were wild-caught, no exact way of determining their age exists. Because of their reproductive history, all were probably at least 6 years of age. 2. Experimental Oesign a. Introduction The animals were divided Into four groups, each consisting of eight females ` and four males: a control group receiving a placebo (Group I), a "low dose" group In which each animal received 5 ug/kg of test compound per ' day (Group II), a "medium dose" group In which each animal received 25 pg/kg ; of test compound per day (Group III), and a "high dose" group In which each animal received 100 pg/kg of test compound per day (Group IV). The animals were randomly divided Into groups which were then adjusted so as to minimize differences between groups: I. The criterion for males was body weight. , 2. The primary criterion for females was the minimization of differences In the number of live births, abortions, and stillbirths, with minimization of body weight differences ^ secondary. Test animals were dosed for a period of 6 months before they were mated. Mating began according to the menstrual cycles of the females on or after . October 1, 1977. This time period coincided with the expected period of highest conception for the LBI rhesus colony; that Is, September through February. (Even though LBI has maintained colonies of rhesus monkeys for well over a decade In constant temperature rooms with 12-hour light ' cycles, and births occur during all months of the year, a definite breeding season continues to exist.) Mating was stopped as of May 31, 1978, after 8 months (Table 1, Appendix A, Protocol Change (PC) 20771-13]. (All protocol :. changes are presented In Appendix C.) BIONETICS HONS 016383 3 b. Frt111ty testing 1) Males The reproductive function of the adult, male experimental animals was evaluated through breeding and semen analysis. The 16 males were to be mated to female non-test animals for a 4-month period which was estimated would Involve 16 mating periods. The females used for these matings were from IBI breeding stock with average or above-average breeding records. The timed breeding technique utilized was that In which the female Is placed In the cage of the nale only during the 72 hours of the menstrual cycle when ovulation Is expected to occur. The diagnosis of conception or lack of conception was made based on alterations In the menstrual cycle and a digital examination of the reproductive tract of the female. Based upon LBI colony records, each nale would be expected to produce at least two pregnancies from 16 mating periods. Examinations of semen obtained from electroejaculation of the test males were made to measure sperm concentration, total sperm, sperm motility, percent of abnormal cells, and llve/dead ratios. [1] Baseline semen parameters from the experimental animals are available for comparison, but since a seasonal variation exists In parameters, comparison of test groups to the control group would provide more meaningful results. The semen of the experimental males was to be evaluated monthly throughout the experimental period. Analysis was done monthly from just before the beginning of the administration of test compound until April 1978 (14 months) when It was discontinued (PC 20771-11). A final semen analysis was performed In June 1978. 2) Females The reproductive status of the adult female test animals was evaluated through breeding. Fifteen male rhesus monkeys not receiving test compound and from the LSI breeding colony were mated to the test females In the seen manner as described above for the test males; that Is, for 72-hour periods according to menstrual cycles. Breeding was to continue for a maximum of 12 months or until a conception was diagnosed by digital examination of the uterus and alterations In the menstrual cycle of the female. In light of the number of conceptions and the relative infer tility normally occurring In the summer months, breeding was discontinued after May 31, 1978 (8 months) (PC 20771-13). Although the result of each mating was recorded, for the purpose of statistical evaluation, each female was considered to have conceived or not conceived over the 8-month period of breeding regardless of the number of matings. If no affect upon fertility existed, each group of eight fasMles would be expected to produce six live Infants. The decision as to the disposition of the Infants was made during the study (PC 20771-11 and PC 20771-13). Also, the decision to attempt collection of milk/ colostrum from the mother as soon as practicable after the birth was made at the seme time (PC 20771-11). These samples were frozen and sent to MC. MOMS 016384 BtONETlCS 4 Any nonexperlmental female impregnated by a test Mlt was allowed to continue the pregnancy for Its natural coursa. Body weight and eeasuremnts wara taken froa thasa Infants at birth and ara avallabla in LSI records (PC 20771-11). Offspring of thasa pragnanclas wara not studiad furthar at a part of this experimental dasign. 3. Tast Coapound Shipping Plan In ordar to ansura tha availability of adapuata suppllas of tast coapound which wara racalvad pre-mixed by MC, tha following system was davlsad: Bafora tha start of study: 2-aonth supply sant to LSI (control and thraa dosa lavals, tach supplltd In S-gallon contalnars for aach month). First aonth of study: Usa supply for tha first month (that is, half of original shipment); to racaiva supply for tha third aonth by and of aonth. Sacond aonth of study: Usa supply for sacond aonth (that Is, sacond half of original shlpaant); to racaiva supply for fourth aonth by and of month. In this aannar LBI would always hava a back-up supply 1 aonth In advance of actual usa. Onca aach now supply had boon racalvad, tha coapound raaalnlng froa tha prior aonth was to ba returned to MC where It would ba analyzed for contant (saa letter dated March 8, 1979, Appendix B). Qua to tha nature of tha tast coapound, there is no reason to ballava that any instability existed In the tast compound which would result in loss of strength under tha conditions of this study. 4. Tast Compound Administration Tha tast coapound (Aroclor 1254) supplied by MC was administered orally onca per day to each animal. To assure the proper quantity of tast compound for Its body weight, each animal was weighed every 2 weeks. Animals wara weighed at approxlaataly the same time aach day to minimize the effects of feeding. The test coapound was racalvad froa MC preaeasured for aach dosa level In a corn oil base so that 0.5 ml of the material was tha proper amount for each kilogram of body weight. MC performed all analyses on the tast material both before and after usa. Tha tast coapound was added to apple sauce and offered to the animals in a shallow bowl prior to the day's feeding. A pal stability test was conducted in cull animals prior to the start of the tast period. Tha control animals received 0.5 ml of corn oll/kg In apple sauce aach day. The original protocol was written so that treatment of the females that became pregnant was to be discontinued after parturition. In fact, tha protocol was changed (PC 20771-13) so that tast compound administration to all animals began April 4, 1977, and stopped on May 31, 1978, a total time period of 14 months. HONS 016385 M0NETIC3 5 5. Statistical Analysis a. Studant's t-tast Tha computation of tha Studant's t-tast during the coursa of tha study for tha quartarly raport* was Mda using a "dagraas of freedom" nuabar which was basad upon tha size of tha tast groups at tha baginning of tha study. No affort was made during tha study by tha LBI statistician to adjust tha dagraas of fraadoa for tha raductlon In group size that oceurrad as a rasult of daaths or othar daltod dataralnatlons. As a rasult, tha analysas of data using tha Student's t-tast, presented during tha quartarly reports, ware unduly conservative In the coaparlson of groups. By taking Into account tha animal daaths, the final raport uti lizes reduced dagraas of fraadoa In soaa categories, and thus tha level of significance aay differ from tha Interim reports. b. Effects of pregnancy A potentially confounding variable occurred during tha conduct of tha study In that tha majority of tha faaala animals became pregnant during tha study at various times. Slnca this variable could affect many of tha aaasurad physiological paraaatars as well as physical .onas such as body weight, wa Identified Iseaatology and biochemistry tast results which wara obtained during tha first 3 months of pregnancy, tha last 2 months of pregnancy, and tha 2 months following pregnancy (whan tha body would still be adjusting to tha changes of pregnancy). Wa than compared tha values obtained during each of these months In tha control animals against the values of nonpregnant control animals to sea (f, during tha period of pregnancy or tha 2 months following It, these values ware significantly different from tha nonpregnant controls. For body weight values, all periods of pregnancy wars compared against nonpregnant animals. If tha values of any period wara statistically significantly different, a value judgsient was made as to whether the difference was real or artlfactual based upon the range of the values, how tha value differed from the control value (above, below, within the range), and reported changes In that value In human pregnancy for which the only large body of comparable data exists. All values thought to represent real changes wara delated from all faawle animals for the affected time period of pregnancy prior to statistical analysis of tha data. To affect consistency, females which experienced fatal wastage had their values analyzed as though the pregnancy had continued to term except for the body weight values, which wara used only when the animal was actually pregnant. 6. Determination of PC8 Levels a. Tissue (fat biopsies) A biopsy of subcutaneous fat from tha ventral abdominal region was ob tained before any test compound was fed. For this and all subsequent biopsy samples on adults, when Insufficient fat was available In the subcutaneous location, a laparotomy was done and omental fat obtained. An attempt was made to obtain between 0.5 and 1.0 gm In all adult animals. When biopsies were Bade of the Infants, they consisted of a segment of ventral abdominal skin and the underlying fat, since sufficiently large BIONEDCS HONS 016386 samples of subcutaneous fat wart not aval labia, fat biopsies of the adults were taken according to tha schadula prasantad In Tabic 1, Appendix A; that Is, prior to drug administration and during Months 2 (May 1977), (July 1977), 5 "(August 1977), 6 (SapUnbar 1977), 8 (November 1977), 10 (January 1978), 12 (March 1978), 14 (May 1978), 15 (June 1978), 17 (August 1978), 19 (October 1978), and at necropsy. Once tha decision was aada to allow tha Infants to nurse on the aothers, tha fat biopsy schadula was amended (PC 20771*10) so that fat biopsies of tha females wara not performed on females of more than 100 days gasta* tlon or on aothers that ware raarlng their Infants during tha first aonth of tha Infant's Ufa. This change was made to avoid Inurfaranca with the lets gastatlonal and tarly postnatal periods. Fat/skln blopslas of tha Infants wara takan at birth, at 1 aonth, and at 2 aonths of age, as well as monthly tharaaftar If tha Infant was allowed to live. In all casa* tha detarmlnatlon of whether or not to taka a fat or fat/skln biopsy was aada by tha clinical veterinarian basad upon tha apparent health of the anlaal. The samples ware placed Into Individually aarkad plastic bags, which ware than placed into larger bags by sax and treatment groups. The saaplas wara frozen and sent In an Insulated carton containing dry lea to MC for analysis of PCS levels. b. Olet Feed sampIds wara obtained prior to compound administration and at Months 10 and 19, frozan, and sant to MC (Section IV.0.). 7. Clinical Tasting Certain clinical tests wars performed on each anlaal throughout tha experimental period. These heiMtologlc and clinical chemistry tests on tha blood wara scheduled to be performed prior to Ust compound adminis tration, at 2, 4, S, and 6 aonths, and blaonthly tharaaftar from tha start of tast compound a<hrin1strat1on. These tests Included tha following; Red Blood Count PCV Hemoglobin W8C and Differential BUN Creatinine Uric Acid Cholastarol SCOT S6PT Total Protaln Albumin Calcium Phosphorus A/G Ratio Globulin Total Bilirubin Alkaline Phosphausa lactic Dehydrogenase Total Iron Triglycerides Creatinine Phosphokinasa Serum Potassium Sarum Sodium Serum Chloride Carbon Dioxide Glucose Fasting Ourlng a routine cytologic examination at L3I, the technician subjectively notes the occurrence of platelets In the field, and this Is reported as "adequate" or otherwise. Beginning April 1978 (PC 20771*7), an actual count of platelets and reticulocytes was made and recorded. The frequency of hematologic examinations was changed to monthly In the smee protocol change. The frequency of the clinical chemistry testing was changed to monthly In May 1978 (PC 20771-13). lotteries MONS 016387 7 Urine was scheduled to bo collected for urinalysis boforo coapound adalnlstratlon mi begun and during tha 3d, 6th, 9th, 12th, 15th, and 18th oonths. This was axtandad to locluda Month 21 whan tha and of tha study was flnallzad (PC 20771-13). To avoid conflict with tha schadulsd necropsy of tha anlaals, tha 18* and 21-aonth collections wars cancallad and raplacad with ona during Month 19 (PC 20771-17). Tha following paraMtars wars aaasurad: Total VoIusm Color Appearance Spaelflc Gravity pH Protaln Glucosa Katona Bilirubin Blood Bilirubin, qual. 17-Katostarolds Urobilinogen, Randoa White Blood Calls Rad Blood Calls Epithelial Calls Aaorphous Crystals Triple Phosphate Crystals Calclua Oxalata Crystals Mucous Threads Casts Bacteria Other In aach casa urlna for tha urinary katosterold output was collactad for a 24-hour parlod batwaon Oay 22 and Oay 26 of tha aanstrual eyela of tha anlaal, with tha first day countad as Oay 1. Tha collactlon of tha first urlna saapla was tlaad to occur during tha aonth prior to tha baginning of coapound adalnlstratlon. In aach succeeding aonth of collactlon, tha urlna was collactad during tha tins fratM aentloned above which first fall In th^ specified aonth; that Is, tha collection parlods ware as follows Month of StlflL 0 3d 6th 9th 12th 15th 19th Tlae of Urlna Collection Prior to adalnlstratlon of coapound. Ftrst appropriate ties fraaa after 2 aonths of coapound adalnlstratlon. First appropriate tlae fraaa aftar 5 aonths of coapound adalnlstratlon. First appropriate tlaa fraaa aftar 8 aonths of coapound adalnlstratlon. Ftrst appropriate tlaa fraaa aftar 11 aonths of coapound adalnlstratlon. First appropriate tlaa fraaa aftar 14 aonths of coapound adalnlstratlon. First appropriate tlaa frasw aftar 18 aonths of coapound adalnlstratlon. BIONCTICS HONS 016388 Because pregnancy nay affect the outcome of the 17-hydroxysteroid tests and because the pregnancy status of an animal might not be known when the urine was obtained, results were not used and the animal no longer tested for this paramter If It was determined to have been pregnant at the tine of urine collection. In order to account for the effects of pregnancy on the urinary 17-ketosteroid levels, these were not to be done until the second menstrual cycle following birth. If this second cycle fell within 60 days of the next regularly scheduled determination, tasting was postponed until the sched uled month (PC 20771-14). A decision was made at the conclusion of the study to analyze sen* for vitamin A levels just prior to necropsy (PC 20771-22). 8. Necropsy The 48 experimental adult animals were necropsled either as they died or at the beginning of the twenty-first month of study (PC 20771-13). This was a modification of the original schedule. In which the males were to be necropsled Immediately following the period of fertility testing and the female animals after parturition or after 12 unsuccessful matings. Following Is a list of tissues histologically evaluated on all 48 experi mental animals. Brain, 3 sections Pituitary (1) Eyelids (2) Lips (2) Mandible (1) Salivary glands (2) parotid with duct (1) and submaxillary (1) Thyroid (2) Lymph nodes (2), axillary (1) and mesenteric (1) Heart (3) Thymus (1), If present lungs (4) right and left of both apical and diaphragmatic Tongue (1) Esophagus Stomach (1) **[add 1 section to Include both pyloric and fundic regions] Duodenum (1) Liver (1) Gallbladder (X) Pancreas (1) with duct Jejunum (X) Ileum (X) Colon (1) Kidneys (2) Adrenals (2) Urinary bladder (1) Ovaries (2) Uterus (1) Vagina (1) Cervix (1) Testes (2) IONET1CS HONS 016389 9 Epididymis (2) ItsIons (1) *Bon marrow **[$terum (1) and femur (1)] Parathyroids, If prosent Spleen Extrahepetlc bile duct Prostate Cecum left nipple Foreskin labium Skeletal muscle Sciatic nerve Olstal tlblal nerve Spinal cord, lumber region Skin, left hip Skin, left thigh Fat biopsy to be sent to MC Note: Those Items not marked with an asterisk nor In brackets appeared on the original protocol. Additional tissues authorized per memorandum of June 21, 1978; Steve Johnson to David Martin, IB1. Sections of liver were to be stored for ultramlcroscoplc evaluation. Further additions made after final consultation In early December 1978 with Ors. P. Berteeu and W. Rlbelln, MC, just prior to necropsy of remaining adult animals. See PC-20771-22 to 24. Also at that same meeting the decision was made to obtain 30 ce of clotted blood from each adult and as much as possible from each Infant just prior to euthanasia, from which determinations of vitamin A levels were to be made (PC 20771-22). A 5-gram sample of liver was also to be ob tained at necropsy and frozen In a plastic bag for future use In determining vitamin A levels (PC 20771-22). B. Infants The question of the disposition of Infants born to experimental females was to be made as the study progressed. On April 13, 1978, the following protocol wet Instituted by Inter-office memorandum (see Appendix B): 1. A C8C with platelet and reticulocyte counts was performed on a monthly basis on each Infant born to a study female. 2. Birth weights and somatic measurements of all offspring born of both study males and females were taken and recorded. 3. Offspring of study males were not to be retained cs part of the study beyond No. 2 above. BIONETICS HONS 016390 10 4. Offspring of study results were to resiain nursing on.the other. A skln/fat biopsy was to be taken of the Infant if the health of the anlisal allowed it. This was reiterated by a protocol change sheet (PC 20771-11). A further modification followed (PC 20771-12) in which body weights and soaetlc measurements at 1 and 2 months of age were to be taken of off spring bom to feiMles assigned to the study. A physical examination of the Infants at birth, 1 month, and 2 months of age was added, and signs of PCS toxicity In the infants were to be recorded (PC 20771-13). This same protocol change dictated that infants not showing PCS toxicity signs would be euthanatized at 2 months of age and those showing signs would be weaned from the mother and observed for the reversal of signs.* A further protocol change (PC 20771-15) Indicated that Infants showing signs would be given a physical examination at 2-week Intervals with observa tions as necessary. This was to continue In those Infants transferred to the Nursery at 2 months. A clarification that all procedures (CSC with reticulocyte and platelet counts, body weight, somatic measurements, and fat [skin] biopsies) be done at monthly Intervals In animals kept alive beyond 2 months was added (PC 20771-16). The use of antibiotics In Infants, with prior sponsor approval, was de clared (PC 20771-18). Antibiotics had been in use when Indicated prior to the asslgnaent of Dr. Peter Berteau to the contract by MC, and, there fore, continuation of this policy was allowed. Infants which died or were euthanatized prior to the revision of the necropsy protocol In December 1978 (PC 20771-24) were subjected to the same protocol as outlined for the adults In the original protocol (Section III.A.8.). "infants were to be given a physical examination 3 to 5 days prior to the scheduled euthanasia to determine if signs were present (memorandum of June 7, 1978). BIONETIC3 HONS 01639) a IV. RESULTS A. Adults 1. Ftrtllity Tasting a. Malts Each aula had baan axpactad to product at laast two pragnanclts fro* 16 aatlng periods If thara was no affact of test coapound upon fertility and the animals performed according to colony history. None of the nales was able to ba scheduled for the predicted 16 mating periods due to a lack of available females as a result of pregnancy. Extending the breeding period to May 31, 1978, to attempt to get more study females pregnant still did not allow 16 mating periods to occur for the study nales. Never* theless, the males exceeded the predicted conception rate, and each male In each group produced at least two pregnancies. No statistically significant effect upon fertility was observed when any of the three groups receiving test compound was compared to the control group. b. Females If no effect upon fertility was seen, each group of eight females would hava been expected to produce six live births. Two groups (the control and medium dose) met or exceeded that prediction with the production of six and seven live births each, respectively (Table 2, Appendix A). One additional feaMle in the control group died during pregnancy after carry ing an apparently nonaal fetus to 94 days of gestation. The low dose group produced five live births, one stillbirth, and one pregnancy which ended in fetal wastage. The high dose group produced one live birth and two pregnancies which ended in fetal wastage. The low dose feeiale and one of the high doee fasMles that experienced fetal wastage resumed men strual cycles, which allowed further breeding but with no known pregnancy resulting. Three of the females In the high dose group died during the breeding phase of the program without being diagnosed as pregnant. Because pregnancy was diagnosed In this study by digital palpation of the uterus at approximately 28 gestational days (and later by alteretions In the menstrual cycle), It Is possible that some other pregnancies occurred which terminated prior to 28 days or gestation. Statistical analysis of the data Indicated that there was a significant difference In fertility between the control females and the high dose females only. 2. Test Compound Receipt Tha test coapound received, the dates of receipt, and the dates of return to MC are presented In Table 3, Appendix A. As a result of late receipt of the shipment of test coapound to be admin istered In July 1977, the animals received tast compound meant for June 1977 In the first week of July until the July test compound arrived July 6, 1977 (Section [V.O.). BIONET1CS HONS 016392 12 3. Test Compound Administration The tast compound was succassfully administered In the apple sauce vehicle to tha majority of animals. In tha three animals In which refusal was a problem (569-H, control female; *32-J, low dose male; 427-0, high dose male), a crushed orange quarter, white bread, or a banana proved a suc cessful substitute vehicle. The two male animals were given the tast compound by stomach tube for 1 day early In tha study. 4. Statistical Analysis a. Mathematical vs. biological significance Although the tast group sizes wera theoretically large enough to allow statistical analysis, several factors must be kept In alnd. (1) The group sizes were small, especially the groups of males which had only four animals at maximum. (2) The death(s) of anlaal(s) In groups of this size could profoundly altar tha statistical analysis of data, both by decreasing the group size and by removing the most seriously affected animals from future analyses. Statistical analysis of the data showed levels of significance In numerous categories. Examination of these instances revealed that tha deviations fell Into one of four categories; (1) A statistically significant change occurred, but tha change was toward a more normal level than the control or baseline data. (2) A statistically significant change occurred, but the tast results were still within the range considered normal for that pareawter. (3) A statistically significant change occurred but was judged to be biologically Inconsequential. (4) A statistically significant change occurred but did not fall Into any of the above categories. Only those changes which fall Into category 4 were considered further. b. Analysis of variance Although an analysis of variance was planned in order to determine changes within groups over time, this was not done due to the shifting of both control and test results which occurred, making such an analysis meaning less. BtONETICS HONS 016393 13 5. PCS Lav*Is Samples of subcutaneous fat and/or skin, placenta, milk, and liver (post mortem), as wall as dlat, war* collected and submitted to MC. Analysis was carried out by MC and will be presented as a separate report. 6. Body Weights a. Significance of values during pregnancy Body weight results were adjusted to account for pregnancy, but only weights actually obtained during pregnancy were checked for significance. As expected, body weights of pregnant control animals when compared to nonpregnant controls were higher and these values were deleted froe the analysis of body weight data. b. Actual results Analysis of body weight data showed a variably significant difference between the high dose females and the control females at nuaarous tin* periods. Because the study groups were chosen primarily based upon breeding history, the weights of the high dose females varied signifi cantly at the 95X confidence level from the contrai females prior to drug administration. Visual analysis of the data and observation of individual animal weights, however, leaves little doubt that the loss of weight in the high dose females was truly significant. 7. Clinical Testing a. Significance of values during pregnancy Values obtained during pregnancy were analyzed according to the methods outlined in Section IU.A.S.b. An explanation of those values considered to be related to pregnancy is presented in Appendix F. b. hematology A depression in red cell values (PCV, RBC, and Hgb) which was statistically significant, was observed in many collection periods when the medium and high dose anlsMls were compared to the control animals. Even though the significance is not present for all values and for all collection periods, one should note that the most profoundly affected animals died or were euthanatized, thus rasMving their results from all future analyses. Con founding the interpretation of this data is the fact that, due to the selection process, both the medium and high dose animals differed signifi cantly from the control anlsMls prior to test compound administration. Observation of individual animal data, however, shows that an effect on red cell vaiues of certain high dose animals most certainly did occur. Platelet values which were only done by actual count for a portion of the study showed a significant change between high dose and control values in only a few time periods. Observation of the data, however, showed a profound depletion of platelets In certain high dose animals. Often these latter animals died, removing these values from future calcula- MONET1CS HONS 0L0394 14 lions. Based upon the postmortem findings, an efftct of the ttst compound on the bon* marrow scans to b* th< most Ktcly mechanism of action. c. Biochemistry Occasional, and apparently random, statistically significant results were t observed In many parameters In numerous time periods. Only the following were thought to be of biological significance. 1) Serum cholesterol ( Serum cholesterol values were reduced significantly In the females of the high dose group during seven blood collection periods. Five of these I tlmo periods clustered around the time period just after compound adminis tration stopped. Values were lowest In the time frame when the animals could be expected to be under the maximum Influence of the compound. Although significantly lower levels were only observed In the females of l the medium and low dose groups on two occasions each, visual observation of the data demonstratad a general lowering of values for both sexes following the end of compound administration and a slow return towards I normal during the remainder of the study. This lowering of serum choles terol values has been previously reported [3]. 2) Serum Iron Serum Iron levels were significantly higher for the high dose female ani mals during nine blood collection periods (seven for males) and for the 1 medium dose females during seven blood collection periods (zero for males). This finding Is consistent with alterations in hematology parasieters. 3) Albumin, globulin, and A/G ratios Alterations In these parameters were Inconsistently present In all treat ment groups, usually resulting In a slightly decreased A/G ratio. Although V no pattern of change was apparent, the frequency with which significant change occurred was such that a causal relationship to compound administra tion seesM likely. d. Vitamin A Analysis of vitamin A levels In the serum of animals just prior to necropsy ( showed a significant difference between the medium dose females and the control fesHles. Because of the wide range of results In all dose levels of both saxes and because the high dose females are not significantly different, this difference Is thought to be of no biological significance (Table 4, Appendix A). *. Urinalysis Urinalysis results are presented In Appendix D. No statistical analysis of these data has been done due to the nature of the data; however, examination of tha data has revealed no dosa-related trends. BIONET1CS HONS 016395 15 8. SMn Evaluations Siatn avaluatIons ara presented fn Apptndlx E. No statistical analysis of tnasa data has baan dona dua to tha natura of the values and the fact that no semen was obtained by electroejaculation from various anlaals (n som tlaa periods. Examination of tha data has revealed no dose-related trends. Analysis of pregnancy data of females bred to these Mies substan tiates the lack of effect since Mies of all dose groups Impregnated fcMles satisfactorily and with no significant difference between groups. 9. Physical Exaailnatlons--Adults No toxic effects of PC8 administration ware observed until the fourth quarter of test compound administration when two of four males and three of five feMles in tha high dose group were observed to have edeM and wrinkling of tha eyelids. One male was observed to have swelling of the i . lips as wall. Redness and/or edema and wrinkling (puffiness) of the eyelids continued and becaM more apparent In the fifth quarter when two of four Mies and t four of five of the reMlnlng females in the high dose group were observed to have this condition. Two Mies and one feMle were observed to have swelling of the lips during this quarter. During the sixth quarter of the study, redness and/or edeM and wrinkling of the eyelids continued In Mny high dose adult anlMls but becmae less severe In Mny anlMls and disappeared In the two Mies. All high dose anlMls and one medium dose feMle Mnlfested swelling of the lips; how ever, this condition appeared to subside In severity In som anlMls during the quarter. During the seventh quarter of study, tha signs noted In the eyelids appeared to subside but did not disappear completely. All high dose anlMls and one medium dose feMle continued to Mnifest swelling of the lips, but the condition was reported as slight in many anlMls. One medium dosa Mle was observed to have slightly swollen lips towards the end of the report period. t. Alopecia Is difficult to quantify and occurs occasionally In rhesus mon keys not receiving any treatment. Because dally changes are almost impos sible to observe, the condition was noted only at the time of the monthly l physical examination. Alopecia In varying degrees was observed more frequently and to a greater extent than previously in the medium and high dose anlMls during the fifth quarter. Ourlng the sixth quarter, alopecia continued to be more severe In the medium and high dosa anlMls of both sexes, but som anlMls appeared to experience a regrowth of hair. Regrowth of hair continued during the seventh quarter among the medium and high dose anlMls which had been observed to have alopecia. BIONET1C3 HONS 016396 16 During the sixth quarter, two high dose mala animals war* observed to have a black coloration to the calculus present on their teeth. A high dose female was observed to have this sane condition early in the seventh and final quarter of study.- Slack coloration of the teeth continued In the seventh quarter In the two surviving anlaals which had manifested this condition. In-the single male which survived with this condition, the coloration became somewhat browner than black by the end Of the report period. 8oth of these animals were observed to bleed easily from the gums: the female while eating and the male near the lower Incisors, when restrained for a physical examination. Frequent bleeding from the gums could account for the stain ing of the calculus of the teeth. One high dose male experienced a dysenteric syndrome at the close of the sixth quarter of study from which Shigella was isolated. The animal was treated with supportive therapy In the fora of fluids, but no antibiotics were used. The animal recovered early In the seventh quarter, and no relapse occurred. Several medium and high dose group animals were observed to have an abnormal growth pattern of the fingernails and/or toenails. This had the appearance of an outward growth of the nail away from the nail bed with a seeming accuaulatlon of debris under the nail. Sparseness of the eyelashes of the high dose animals continued In the seventh querter, but some regrowth was observed. 8. infants 1. Analysis of Data Ho statistical analysis has been performed on any values obtained from Infants. Because the mothers became pregnant after receiving the test compound for verlous lengths of time and because the exposure of the offspring to PCS's, which only occurred via the mother during pregnancy or while nursing, was variable and unmeasurable, and because only one offspring was born to a high dose mother, statistical analysis of data would be meaningless. 2. PCS Tissue Levels The analysis for PCS levels was carried out by MC and will be presented es a separate report. 3. Body Weights Observation of Infant body weights at birth and at 1 and 2 months of age showed them to be generally within acceptable limits (Table 5, Appendix A). The one high dose Infant which was within an acceptable weight range at birth, however, failed to gain weight as expected during Its 3-month life span. Tha one medium dose Infant which survived, showing signs compatible with PCS toxicity, for 7 months gained weight beyond what wes expected. BIONET1CS HONS 016397 17 4. Clinical Tasting a. Hawto logy-cytology Obsarvatlon of the valuas of tha formed alamnts of tha blood ara ganarally within normal Halts for all dosa groups for tha parlods for which samples ara avallabla. Blood was not taken whan. In tha opinion of tha clinical vatarlnarlan. It was potantlally dangarous to tha haalth of tha Infant. Infant valuas ara prasantad in Tabla 6, Appendix A. b. Vitamin A strum lavals Only thraa vitamin A strum ltval valuas ara avallablt (Tabla 7, Appandlx A) eolncldant with tha thraa Infants allva at tha tlma tha saaplas wara takan. Thaso valuas hava a ranga slallar to thosa of tha adults. 5. Physical Examinations Births of offspring from study ftmalts bagan to occur In tha fifth quartar of study with abnormal physical findings obsarvad as follows: Tha off spring of tha ono high dosa ftmala (312-1) to glva birth to a llva Infant (B-8542) was obsarvad to hava swalllng of tha ayallds and Kps at birth and at 30 days of ago during tha fifth quartar of study. During tha sixth quartar, thraa infants (8-8S46 from nadlum dosa mothar 757-1, 8-8560 from madlum dosa mothar 540-H, and B-8542 from high dosa mothar 312-1) wara aovad to tha Nursary dua to poor haalth (8*8546) and/ I. or signs eampatlbla with PCS toxicity (8-8560 and B-8542). All wara movad aftar 2 months of ago, which was a protocol discrepancy (Section IV.0.). t All thraa animals ware born with or davalopad lesions while nursing on tha mother compatible with PCS toxicity; that is, swollan lips and/or eyelids and/or scanty ayolashas. The lesions wara more pronounced in the Infant from the high dose mother (B-8542), which also failed to gain i weight satisfactorily In the Nursary. The high dose animal and one of the medium dose animals (B-8546) died while In tha Nursary, while the remaining Infant (8-8560) manifested swollan lips and ayallds, scanty eyelashes, scaly skin, and a chronic respiratory wheezing. This latter Infant remained alive and continued to demonstrate signs compatible with PCB toxicity until tha and of tha study. It had a weight gain which was 1. greater than expected when compared to non-study animals. Some evidence of eyelash regrowth occurred toward tha and of tha study. Tha animal continued to show tha chronic respiratory wheezing until tha end of the study. Although the deciduous teeth of this animal wars observed to be brown, the new taeth which erupted in tha seventh month of life were normal In color. A subjective observation was made that this animal became more excited when approached by personnel than similar, non-study animals. One Infant (B-8815 bom to a low dosa mother, 483-F) was aovad to tha Nursary early In the seventh quartar for haalth reasons. All other Infants ware clinically normal. BIONET1CS HONS 016398 18 c. Intarcurrent Deaths--CHnlcal Reports Adults a. Animal 296-1, femala (low dose) This animal was found dead on April 12, 1977. The death was caused by acuta gastric dilatation which was not related to tha tast compound adalnlstratlon. Because of the short time on study and the conclusive ness of the gross necropsy, a hlstopathologlcal examination was not performed on this animal. Acute gastric dilatation Is a recognized clinical entity In many species of animals Including the rhesus monkey. It occurs sporadically In many colonies, and although much studied. Its etiology In the nonhuaan primate remains obscure. An animal may recover If treated early In the course of the syndroaN; however, the outcome depends on the quick relief of the gastric tympany and the ability to reverse the shock present when the condition Is discovered. Because the death occurred only 8 days after the start of the study, a substitute animal, 483-F, was assigned to the low dose group as a replace ment. Death'was probably not related to the test compound. b. Animal 152-F, female (high dose) The clinical changes that were noted In this animal during the study were moderately elevated SGPT values In April through August 1977, alopecia of the chest and abdomen In June 1977, and generalized alopecia In January 1978. Slight reddening of both eyelids was observed In September 1977, and although this continued. It was thought to be within normal limits. On the morning of January 22, 1978, this animal was found unable to stand In Its cage. The left aye was swollen shut, and numerous petechias were noted In both eyes. Eplstaxls was present, mucous membranes were white, and the animal was markadty dehydrated. It was treated with 500 ml of Ambex* Intravenously, 100 mg of Solu-Cortef*. and 1 million units of potassium penicillin. The animal died at 11:15 a.a., January 22. Death was attributed to an Indirect effect of the test com pound. c. Animal 486-H, female (high dose) At the time of the regularly scheduled fat biopsy of this animal on March 7, 1978, It was noted to be extremely pale. Because the blood sample for hematology had been taken that morning, the results were available and Indicated that the animal had a PCV of 14.5, a platelet count of 10,000, and a WC of 1,400 with a differential count of 17 segeented neutrophils (sag), 82 lymphocytes (lym), and 1 monocyte (non). The lymphocytes were noted by the reader as "very small.1' BIONETICS HONS 016399 19 A transfusion of whole blood and on* of red blood colls was given to the antaia). A hemogram taken the next morning Indicated a PCV of 23.5 and a platelet count of 95,000. Over the next 2 weeks, the animal responded briefly to transfusions given on March 17 and 19, but its course was generally "downhill.1' The fat biopsy incision did not heal properly, with bleeding noted on March 19 and dehiscence noted the following day; the eyelids appearing swollen on both of these days. Also on March 20 the animal was noted to have petechial hemorrhages on the ventrum and gums with a tendency toward bruising easily. Bruises were noted over the arms, legs, and face on March 21 and 22, as was weakness. On March 22 the animal appeared dehydrated and Inactive, and on that day euthanasia was performed. The condition of the animal was attributed to the effect of the test compound. Test Date me S.R. Cell Hgb Plate W8C Differential (X) Ow*xlO*) (mm/hr) VoUX) (gmX) (mm3) (we3) Ban sag Lym Won 3/07/78 3/08/78 3/10/78 3/17/78 3/20/78 3/22/78* 1.73 2.68 2.42 1.75 1.97 0.73 3.9 - 42.0 61.0 - >78.0 14.5 23.5 19.0 15.0 12.5 4.5 6.0 10,000 1,400 7.9 95,000 1,400 6.5 84,000 1,200 4.7 5,000 1,500 i 4.3 4,000 1,600 . 1.7 6,000 500 17 82 1 25 74 1 13 87 - 1 98 - 1 99 - 8 88 4 `Terminal sample. d. Animal 87-F, female (control) This animal was In apparent good health and 94 days pregnant. It was observed to have had profuse vomiting late on the afternoon of March 29, 1978, followed almost Immediately by severe weakness and apparent anemia. Physical examination revealed a distended and very painful abdomen and pale gums and skin. An exploratory 1aparotomy was performed that evening. The uterus was found to be very pale and turgid and much larger than expected for the number of gestation days. Whan opened, the placenta was found to have separated from the endometrium with a large amount of both clotted and unclotted blood between the placenta and myometrium. Some separation of each of the two placental discs was evident. The apparently normal fetus was dead at the time of surgery and was removed with the placenta. Uterine contraction was good. ' Although the surgery was uneventful other than the findings noted, the animal did not respond to treatment and died on March 31. At necropsy, the findings were related to the postoperative condition of the uterus, severe cecltls and colitis, and aspiration. Although relating the physical findings to their cause Is speculative, we think the animal probably suffered an attack of acute gastric dilata tion on the afternoon of March 29. This condition occurs with some frequency In singly caged, nonhuman primates, particularly macaques, and may end with relief by emesis or death from respiratory embarrassment BIONETICS HONS 016400 20 and/or andotoxic shock. Judging fro* the amount of vomited matarial, tha dlltansion oust hava baan considtrabie and perhaps anough to cause placental separation by pressure. Another possibility is that tha placental separation was the result of violent abdominal muscle contractions during emesis. Since this was a control animal, death was not related to tha test compound. a. Animal 770-1, female (high dose) The results of the scheduled hematology testing on March 7. 1978, indicated that this animal had a low PCV (28%), slightly decreased number of platelets, and a low WBC (4,100) with an absolute decrease in segmented neutrophils. After the scheduled fat biopsy that day, the animal was observed to be lethargic and dahydratad with soon bleeding rrom the Incision site. Transfusions ware given on March 8 and 31; however, the animal did not respond and was found In a terminal state on April 2. Fluids were admin istered, but the animal died. Tha death of the animal was attributed to the effect of the test compound. Test Date R8C (mm^xlO*) PCV iil (aHmgbX) P(mlma3te) WBC (mm3) Differential (X) II SI fl II 3/07/78 3/08/78 3/09/78 3/10/78 3.81 3.03 3.62 . 3.73 28 9.8 SI Oec 4,100 - 11 87 2 21 7.7 26,000 3,700 1 52 47 28 9.1 91,000 4,000 4 36 60 26 8.5 37,000 3,400 - 36 63 1 f. Animal 706-E, male (high dose) This animal experienced a gradual loss of condition and weight from March 1978. There was a weight loss of nearly 2 kg in this time with savere alopecia. Swelling of the eyelids and lips worsened, and the blood picture deteriorated with a reduction In PCV and platelets. There was non-healing of the biopsy site and the development of petechia. Numerous fingernails and toenails loosened and fell out and the animal sat hunched in Its cage. Euthanasia was performed July 19, 1978. The condition of the animal was attributed to the effect of the test compound. g. Animal 641-H, female (medium dose) This animal was found dead from acute gastric dilatation at approximately 9:30 p.m. on August 30, 1978. Although It had been bled that morning. It had been fed by 9:30 a.m. and had appeared normal through the dey. Death was probably not related to the test compound. h. Animal 338-1, female (high dose) This enlmal had been observed to have red and swollen eyelids from May 25, 1978. Dry, scaly skin had been observed from July 1978. A yellow discoloration of tha skin and missing eyelashes were observed in August 1978. A hemogrem collected on the day of death indicated bone marrow depression with a platelet count of 45,000 me3 and total WBC of BIONET1CS HONS 016401 21 1,700 mm3. It was Inactive, dyspneic, sevarely depressed, and then comatose prior to euthanasia. The condition of the animal was attributed to the effect of the test coepound. 2. Infants a. Animal 8*8546, female, born of 757-1 (itedlum dose) This Infant was observed to have signs compatible with PCB toxicity; that Is, swollen eyelids and possibly swollen lips. It had soaa trouble coming out of anesthesia when a fat biopsy was taken at 2 months. Upon being brought to the Nursery.on July 10, 1978, with a rough chest, it was put on 0.2 ml Erythrocln' tld and 0.2 ml Llxamlnol' tld. On July 17 the eyes ware reddened whereupon ophthalmic Chloromycetin* was administered tld; the condition Improved. On July 19 Llxamlnor was discontinued. In the evening of July 26 and continuing the next day, (t was observed to be depresied-and had a loose, green stool. Four ml of Donnagel* and 0.2 ml Blosol-fr were given by stomach tube, and an anal swab was taken In the evening of July 27. The animal was still very depressed and Inactive and was given S ml 1actated Ringer's solution sub-Q, 0.1 ml Llxamlnol" per os, and 0.25 ml sodium bicarbonate IV. It was found dead at 7:30 a.a., July 28, 1978. b. Animal B-8542, female, born of 312-1 (high dose) Oue to signs compatible with PCB toxicity, this animal was saparated from Its mother and brought to the Nursery on July 17, 1978. It was eawclated and had severely Irritated eyes and a rough chest. Admlnls- . tratlon of 0.2 ml of Erythrocln* per os tld and ophthalmic Chloromycetin* tld was begun. The eyes Improved, but the rough chest continued. On July 27.s clinical diagnosis of yeast Infection was made, and 0.2 ml Hycostatln* tld per os was instltutad. The 3-month biopsy was taken July 31 aftar which the animal had difficulty recovering from anesthesia. It was given 0.1 ml Dopram sublingually, 0.1 ml Atropine" IN, and 0. t a) Aalnophyllln per os. Its condition Improved on August 1. On August 3 the infant was lethargic but demonstrated some periods of activity. The eye condition worsened. It had nasal discharge, and was dehydrated. Lactatad Ringer's solution, 10 ml, was given tld sub-Q. Also on this day the Infant stopped sating and had to be fed via stomach tube. The following day, August 4, It was holding fluids, and the Ringer's was discontinued, but stomach tubing continued throughout the day. On the morning of August 5, the animal was found with a bloated abdomen, was Inactive, and In shock. Ten ml of Lactated Rlngerls was given IV as was a 5-ml soapy water eneaw and 0.2 at of Solu-Cortef" IM. The.stomach was aspirated, and due to shallow respirations, 0.1 ml Llxamlnol' was given per os. The animal was found dead at 12:30 p.m., August 5, 1978. BIONET1CS HONS 016402 22 0. Protocol Discrepancies Tho following deviations from protocol ware known to havo occurred during the course of the study: The project protocol called for the sending of a sample of prlaete diet to MC prior to compound administration, at 6 months, and at other tine Intervals as required by MC. The 6-month sample was Inadvertently omitted and was obtained at 10 months (sent February 13, 1978). This was documented In a memorandum of January 24, 1978 (Appendix 8). In addition, a sample was obtained at 19 months (sent March 7, 1979), as requested. PC 20771-13 (May 31, 1978) stated that Infants demonstrating evidence of PCS toxicity would be weaned from the anther at 2 months of age. Three Infants showing signs were Inadvertently left nursing on the mother for periods longer than 2 months. These were as follows: B-8S46 from Mother No. 757-I, medium dose, 7 days late. B-8S60 from Mather No. 540-H, medium dose, 8 days late. 8-8542 from Mother No. 312-1, high dose, 15 days lata. This set of events was documented by a memorandum of July 18, 1978 (Appendix B). No CPK results were obtained In Month 19 (October 1978) of the study due to this test having been deleted from the SMAC 1000 series by the Blonetlcs Medical Laboratory without the knowledge of the study director. This change In the SMAC 1000 profile was Instituted on September 5, 1978. A protocol change (PC 20771-21) wes Instituted to ensure that the CPK test was ordered as a separate test for the final two bleedings of the study. Final blood samples for hematology and biochemistry analysis were done In the last part of Month 20 (November 1978) In order to allow time to repeat any questionable tests. One female animal. No. 226-F (high dose), was easily stressad, and when obtaining the blood from her presented difficulties, a decision was made to wait and bleed her at the time of necropsy. This was not done due to a change In necropsy scheduling which allowed the animal to be killed while the person In charge of obtaining the blood was busy elsewhere. Serum obtained for a vitamin A level determination was used to outaln biochemistry values, but no hematology values were read. This was documented In a memorandum to the project file dated Oecenber 7, 1978. MONET1CS HONS 016403 23  As a result of late receipt of the shipment of test compound to be administered in July 1977, the animals received test compound meant for June 1977 in the first week of July until the July test compound arrived July 6, 1977. The original protocol called for the collection of a 24-hour urine sample for urinary ketosteroid measurement to occur between Days 22 and 26 of the menstrual cycle of each female animal. For the first 9 months of the study (three collection . periods), urine was collected between Days 20 and 26 of the menstrual cycle. This error was communicated to Or. P. Wright of MC by telephone on January 23, 1978. Observation of data shows that the change In collection periods did not influence results. E. Patholoqv 1. Necropsy Procedures Live monkeys were euthanatized by intravenous injection with either Somlethol (Ned Tech Inc., Elwood, Kansas 66024) or Lethane* (Eli Lilly, Indianapolis, Indiana 46206), as Indicated on the individual necropsy records. The euthanatizing agent was administered intravenously, 3 cc to each adult male, 2.5 cc to each adult female, and 0.5 cc to each infant. Gross anatomic abnormalities were recorded In descriptive or diagnostic terminology as determined by judgment of the pathologist in attendance. Carcasses and live animals submitted for pathologic examination are listed and Identified in text Table A. Monkey 296-1, which was originally placed in Group II females, died of acute gastric dilatation on the eighth day of the experiment and was replaced by 483-F. Due to the timing and the fact that the cause of death (acute gastric dilatation) was unrelated to compound administration, tissues for microscopic examination were not collected. This animal is not included in the tables. TABLE A Animals Submitted for Pathologic Examination Adults Group 1 Group II Group III Group IV 227-G-Male-T 416-J-Male-T 426-G-Male-T 739-F-Male-T 119-H-Male-T 428-J-Male-T 432-J-Male-T 516-G-Male-T 20-I-Male-T 46-I-Male-T 595-I-Male-T 599-I-Male-T 425-J-Male-T 427-J-Male-T 600-I-Male-T 706-E-Male-K 200-F-Female-T 274-F-Female-T 569-H-Female-T 578-H-Female-T 604-I-Female-T 618-I-Female-T 764-E-Female-T 87-F-Female-D 284-I-Female-T 309-I-Female-T 483-F-Female-T 605-F-Female-T 624-I-Female-T 646-E-Female-T 701-E-Female-T 839-H-Female-T 273-I-Female-T 281-G-Female-T 307-I-Female-T 540-H-Female-T 757-I-Female-T 762- E-Female-T 763- E-Female-T 841-H-Female-D 226-F-Female-T 269-I-Female-T 287-1-Female-T 312-I-Female-T 152-F-Female-D 338-I-Female-K 486-H-Female-K 770-I-Female-D BIONETICS Litton HONS 016404 24 Infants Group I 8-8518-Male-T B-8557-Nale-T B-8574-Male-T B-8483-Fanale-FW B-8532-Ftaale-T B-8533-Famala-T B-6621-FimIo-T Group II , B-8604-Male-T B-8796-Male-S B-8527-Female-T 8-8562-Fe* ale-T' B-8754-Fm1o-T' B-881 5-Faaal *-T Group III B-8511-Male-T B-8559-Mala-T B-8603-Mala-T 8-8860-Mala-T B-8546-Feaale-D B-8560-Female-T B-8592-Female-T M- T > Taralnal Euthanasia K Moribund Euthanasia D * Found Oaad S * Stillborn FW Fatal Vastaga Group IV 8-8542-Famale-O Tlssuas for alcroscoplc examination wars prasarvad (fixed) In 10X buffarad formalin solution, pH 7.0, dahydratad In athanol, elaarad In xylana and embedded In Paraplast* brand of paraffin aabaddlng aadlua (Linear Division of Sherwood Madlcal, St. Louis, Missouri 63103). Bona specimens were subjactad to prior dacalclflcatlon (n R00 dacalclfylng agentT (Dupage Klnatlc Laboratorlas, Inc., 25 W 550, North Aurora Road, Naparvllla, Illinois). Ttssua blocks wara routlnaly sactlonad at a range of 5 to 6 alcrons In thlcknass with tha axcaptlon of brain blocks, which wara sactlonad at a ranga of 5 to 10 alcrons in thlcknass. All tlssua factions wara stalnad routlnaly with haaatoxylln and aosln. In savaral Instances spatial stains wara aaployad for hlstochaaical daaonstratlon of Iron containing plgaant (haaosldarln) and for aucln In itucin-sacrating calls. A complete listing of tlssuas for alcroscoplc examination appears In Section III.A.8. Stipulations aada concurrent with additions appearing In PC 20771-22 were: Parotid gland and pancreas should be trlmed to Include duct. Tha two lyaph node specloans should be axillary and aesentarlc lyaph nodes. Two sections of stomach should ba taken to Include both pyloric and fundlc regions. ' Lung specimens should include apical and diaphragmatic lobes of both lungs. BIONETICS MONS 016405 25  After final discussion with MC representatives on selection of tissues for microscopic examination, it was dacidad to oaiit skeletal muscle and cecuai from tha list, but to harvast and prasarva than In formalin should futura rafaranea ba nacassary. Additions to tha list of tlssuas to ba examined microscopically that wara mada in accordanca with approvad modifications of protocol account for diffarancas In tha scopa of microscopic axaainatlon in soae aonkays that dlad or wara authanatlzad Intarcurrantly and thosa that wara authanatlzad at tarmlnation of tha study. 2. Microscopic Examination Compound (polychlorlnatad biphenyls)-related altaration In Maibomian glands of nonhuman prlmatas has baan daslgnatad "squamous aataplasia with eonvarslon of tha glands to a row of squamous cysts___ "[4, 5] Compound-ratatad altaration in sabacaous glands of tha skin has also baan daslgnatad "squamous metaplasia."[4] Tha microscopic eharactar of sabacaous gland altaration obsarvad In tha prasant study posad questions of Intarpratatlon and nomenclature. The long ducts of tha Maibomian glands and tha short ducts of tha sabacaous glands in tha skin are lined by stratified squamous epithelium. Tha secretory alveoli ara rounded sacs consisting of epithelial calls under going breakdown to fatty detritus which constitutes tha holocrine-type oily secretion.[6] Thera Is disagreement concerning replacement of cells that dlslntegrata Into oily secretion. It has baan stated that tha regenerative activity occurs [4] In tha basal layer of epithelial calls lining tha secretory alveoli and [5] In "calls closa to tha walls of tha ducts, whara tha new calls nova Into the secretory regions."[6,7] In this report terminology was salectad to indicate tha morphologic appearances of compound-related alterations without regard to kinetics of their development. In a majority of animals with Maibomian gland alterations, there was total absence of recognizable secretory alveoli. This was daslgnatad atrophy-sebaceous elements. In two Instances with lass than total atrophy of tha sebaceous elements (daslgnatad subtotal atrophy-sabacaous alamants), an appearance of squaswus metaplasia also was recognized, and tha designation squamous aataplasia was used. Round and elongated structures with thick epithelial walls and larga central areas packed with epithelial squames were generally present. The round structures had the appearencs of squamous cysts, but a majority were elongated and could be Identified as Meibomian gland ducts impacted with squamous debris. The designation squamous Impaction-ducts was used. Another Meibomian gland alteration, not reported in literature that was reviewed, was tha presence of large mucinous vacuoles in calls lining soma of the ducts. Tha mucinous character of the vacuoles was confirmed by staining with Alclan blue and Periodic acld-Schiff techniques. Ducts so affected contained a mixture of squamous and mucinous materials. This alteration was designated mucinous metaplasia-ducts. BIONET1C3 HONS 016406 26 Changes In sebaceous glands of the skin wara designated according to Microscopic appearance as either atrophy-sabaceous glands or subtotal atrophy-sabacaous glands. Slnca only snail araas of skin wara routlnaly examined, a conservative Intarpratatlon of tna Microscopic appaaranca was aada to avoid ovarstataaant of tha affects of the coapound. Distention of hair follicles with karatinizad Material was designated follicular keratosis. Squamous cysts In tha derals (without denonstrable connection with overlying eptdermls and without definite Indication of origin) wara designated squanous cysts. Coapound-related atrophy of tha thymus In Infants was designated "accidental Involution" to differentiate It froa the physiologic Involution that was Invariably present In adults to a greater or lesser degree. The systaa of noaenclature used In publications on the effects of poly chlorinated biphenyls In nonhuaan primates was used for coapound-related lesions observed In the biliary tract, liver, gastric mucosa, and aandlble. [3.4,5,8,9,10,11] Descriptive designations were given to unique, apparently coapound-related lesions teen In the pancreas of two Infants and kidneys of one Infant. Tha pancreatic lesion was characterized by deficiency or diminution of acinar epithelial cells and Increase of centroaclnar cells and saell ducts. It was designated ductal hyperplasia-acinar atrophy since both tha ductal and acinar elements were Involved. The lesions In the kidneys had the microscopic appearance of areas In tha cortex where there was retarded cortical maturation and were so designated. 3. Results A list of all pathologic alterations recognized on microscopic examination Is given In the tables presented In Appendix G and Identified as follows: Table 3A: Summary of Histologic Findings - Adult Males - Terminal Kill. Table 38: Summary of Histologic Findings - Adult Females - Terminal Kill. Table 3C: Summary of Histologic Findings - Adults - Males and Females - Intercurrent Oeath. Table 30: Summary of Histologic Findings - Infants - Males and Females. The pathologic alterations can be grouped into the following categories: Pathologic alterations incident to euthanasia and environment; for example, terminal hemorrhages and minor mucosal lesions, etc. Spontaneous lesions of nonhuaan primates. Pathologic alterations representing direct morphologic effects of the test compound. MONET1CS MOMS 016407 27 I'.... r In this study the pathologic alterations roprosentlng dlroct morphologic offsets of ths tost compound could bo Identified because of their unique character and their similarity to lesions described in lltersture on the effects of polychlorinated biphenyls. . These alterations Include the foil owing: Characteristic changes associated with loss of secretory epithelium of the Meibomian glands In the eyelids and the sebaceous glands In the skin. There also were Instances of mucinous metaplasia In the Meibomian gland ducts (previously unreported). Follicular keratosis--skin. Squaawus cysts--skin. Subgingival squamous cysts (microcysts) and Intraosseous squamous cysts In mandible. Squamous metaplasia In ducts of oral mucosal glands and In such unusual places as thyroid, eplcardlum, and mammary tissue. Hyperplastic epithelial changes Involving the biliary duct system and the main pancreatic duct. . Nonsquamous metaplastic changes In gastric glands and Brunner's glands. Non-specific bone narrow alterations that were compound related on the basis of experimental conditions. Previously unreported lesions of Infants. (a) Areas of retarded cortical maturation--kidneys. (b) Acinar atrophy and ductal hyperplasia (Increase of centroaclnar cel Is)--pancreas. Accordingly, the set of tables presented In Appendix G was prepared listing only the pathologic alterations representing direct morphologic effects of the compound: Table 2A: Summary of Olrect Morphologic Effects of Compound - Adult Males - Terminal Kill. Table 2B: Summary of Direct Morphologic Effects of Compound - Adult Feawles - Terminal Kill. Table 2C: Summary of Direct Morphologic Effects of Compound - Adults Males and Feawles - Intercurrent Oeaths. Table 20: Summary of Olrect Morphologic Effects of Compound Infants - Males and Feawles. BIONETICS HONS 016408 28 At the end of each table, a numerical score of th number of pathologic alterations llstad for aach anInal was entered. Each pathologic altaratlon was glvan a value of 1 regardless of whathar or not tha Itaai was recorded as prasant (x) or was racordad by a numerical grading symbol (1 through 5). A final tabla was praparad (Tabla I) antftlad "Survay of Olract Morphologic Effacts of Compound." This tabla Idantlflas aach animal In ralatlon to tha tast and fumlshos Individual and avaraga group nusarlcal scoras of pathologic altaratlon* for usa In final (ntarpratatlon of tha pathologic axaailnatlon. Incldanca of dlract aorphologlc affects of tha coapound was dosa ralatad, balng highest In Group IV animals and substantially lower In Group III animals. Thera was a traca morphologic affact In one Group II Infant and absanca of affects In Group I animals (controls). Only 1 of tha 10 Group II animals showed dlract morphologic affact of tha coapound; It was an Infant with minimal pathologic altaratlon In tha mandible. In Groups IV and III, where comparisons could be made, tha incidence of direct morphologic effacts of the coapound was greatast In tha Infants and least In tha adult males. Avaraga scoras and Incidences of dlract morphologic affects of tha compound are summarized In taxt Tables B and C, respectively. TABLE 8 Average Score of Pathologic Alterations Representing Direct Morphologic Effects of the Compound Group IV Group III Group II Group I Adult males Adult faaHlas Infants* 7.25 9.13 20.00 0.75 0.88 3.14 0 0 0.33 0 0 0 This score has apparent validity because tha single Infant on which It Is basad represents the only conceptus In Group IV adult resales that developed to Infancy. TABLE C Adult* Infants Group IV h~ t 3/4 8/0 VI Group III tr--r 1/4 3/8 2/4 2/3 Group II wf 0/4 0/8 0/2 V4 Group I fM r 0/4 0/8 0/3 0/4 BIONETICS MOWS 016409 29 Intercurrent deaths, Including animals killed In extremis, In adults occurrad In a single Group I female, a single Group III feaale, a single Group IV Mle, and four Group IV females. The Group I female died of hemorrhage end the effects of surgical treatment following placental separation. The Group III faawle died of acute gastric dilatation, a relatively common but poorly understood spontaneous affliction of maca ques. There was no apparent connection between the cause of death and the test compound In either of these two cases. Two of the Group IV animals, a male and a female, developed Isolated cates of severe Infectious disease. The male had extensive gangrenous stosMtltls and was euthanatized when moribund; the female died of systemic herpesvirus Infection. Under the prevailing status of spontaneous diseases in the primate colony, one might speculate that the unusual saverlty of Infection In these two animals Is a function of compound effect on the Imaunologlc competence of the animals. A second high dose female died of Intestinal bleeding and a third was euthanatized when moribund with clinical signs of anemia. In both of these cases, haiMtologlc effects of the compound were apparently respon sible. The fourth high dosa adult female that died had severe biliary tract lesions related to the compound with secondary hepatic necrosis, abscess formation, and periportal Inflammation. 4. Summary Compound-related morphologic alterations were identified in adult males, adult females, and In Infants of either sex that were bom to the adult females. The effects of the compound were greatest in Group IV animals and were slightly more evident in adult females than adult males. They were most evident In the single animal representing the only conceptus In Group IV adult females that developed to infancy. Four intareurrent deaths (n Group IV adult females (50S) and one In Group IV males (25X) could be attributed to lesions that were either direct or indirect effects of the compound. The method for scoring lesions which was used Indicated a considerably lassar amount of morphologic effect of the compound In Group III animals as compared with Group IV animals. Here again, the compound-related morphologic effects were slightly more evident In adult feieales than adult males and most evident In the Infants. An adult fmeate (Group III) that died Intercurrently had acute gestrlc dilatation, apparently unrelated to compound administration. The Group III Infant that died Intercurrently had bacterial meningitis and septicemia. In this animal the score of pathologic alterations representing direct morphologic effects of the compound was approximately four times greater than the average score of Group III Infants, Indicating a probable relationship between death and administration of the compound. Group II animals (adults and infants) were entirely free from morphologic effects of the compound, except one Infant which had a single intraosseous squaanus cyst In the mandible and several minute subgingival squamous cysts. BIONETICS HONS 016410 30 5. Conclusion Pathologic axial nation rivalled dosi*rtlttid and, to a ltssar dtgraa. ax-railtad and aga-ralatid laorphologic ifficts of thi compound In high and Mdlua dosa anlaals. Sactlon Itl.E. , Pathology, Subulttid by: ri*\ 3 / J , Haman R. Salbold. V.M.O. Data Vatarlnary Pathologist Rivliwid by: ititnaru n. iary, v.v.rw Olractor OipartiMnt of Pathology BI0NET1C3 HONS 016411 31 V. CONCLUSIONS A. Adult* 1. Fertility No effect upon fortuity occurred among the nolo* In any dose group. A significant effect upon fertility was observed In the feaales receiving 100 Mg/kg of the test compound, but no effect upon fertility wes observed In the lower dose groups. 2. Pathologic Effects Pathologic effects were determined by Identification and tabulation of the direct morphologic effects of the compound. The Incidence of these morphologic effects and mortality was dependent on dose and sex. Group IV monkeys had a higher average score of morphologic effects then Group III monkeys. No effects were seen In Group II and Group I monkeys. In both ?roups that had demonstrable morphologic effects of the compound, the ncldenc* of these effects was higher In females than males. Four Inter current deaths In Group IV adult fesMlas (SOX) and one In Group IV males (2SX) could be attributed to lesions that ware either direct or Indirect effects of the compound. 3. Hematology-Cytology A statistically significant depression In red cell values (PCV, RBC, and Hgb) and platelets, was observed In variable numbers of collection periods when the medium and high dose animals ware compared to the control animals. Although all values were not depressed significantly for all collection periods, and the Interpretation of these observations Is confounded by the fact that the medium and high dose animals varied significantly from the controls In the pre-test period one should note that the most profoundly affected animals died or were euthanatized, thus removing their results from all future analyses. The postmortem findings suggest that this was a compound-related effect on the bone narrow. 4. Biochemistry Statistical changes thought to be of biological significance were observed In the following three parameters: A decrease In cholesterol values appeared to be dose related and sax related with the females significantly different from controls more often than the males. A slow return towards nonaal values was observed after the cessation of compound adalnlstrati on. An Increase In scrum Iron values appeared to be dose related and sex related, with the feaule animals signif icantly different from controls more often than the male animals. MONETtCS HONS 016412 32  Alterations In albumin, globulin, and A/G ratio, usually resulting In a slightly dacrtasad A/G ratio, war* prasant In all treatment groups. Although tha changas wars not conslstant and thara was no apparant pattarn of changa, tha fraquancy with which significant changas occurrtd was such that a causal ralatlonshlp to compound administration seam 11kaly. S. Physical Effacts Physical changas attrlbutabla to tast compound administration war* obsarvad In all animals racatvlng tha high dosa of tha tast coapound. Changas of a lass savara natura wara obsarvad In soma aadlum.dosa animals. Tha majority of affacts obsarvad upon physical axamlnatlon wara attributad to tha affact of tha tast coapound upon tha skin and Its adnexa. Signif icant walght loss was raportad avan In tha faca of a good appatlta. B. Infants 1. Pathologic Effacts Pathologic affacts wara datarmlnad by Idantlflcatlon and tabulation of dlract morphologic affacts of tha compound. Tha slngla high dosa Infant had tha hlghast scora of dlract morphologic affacts. This scora also was hlghar than tha avaraga scora of medium dosa Infants. This comparison appears to ba valid because the slngla high dose Infant that was examined represents tha only conceptus of tha high dosa adult females that developed to Infancy. Minimal morphologic affect was seen In tha mandible of one low dose Irffant. Thus a dose-depandent affect on Infants was apparant. Tha hlghar scora of dlract morphologic effects among infants, as compared with adult females and adult males In tha corresponding groups, Indicates that tha Infants wara more sensitive to tha compound than wara tha . adults. 2. Hematology No statistical evaluation of the values of tha Infants was attaaptad due to the variable and unknown exposure of tha Infants to PC8's, which occurred only via tha mother during pregnancy or while nursing. Tha birth of only one anlswl In tha high dosa group also made statistical comparisons meaninglass. 3. Physical Effacts Physical effacts obsarvad In tha one Infant born to a high dose mother and In several of the Infants born to medium dosa anthers were dosa depen dant. These affacts wara similar In natura to those in the adult tast anlMls but wara more profound. BIONETIC3 MOWS 016413 33 vi. KEY PERSONNEL Kay personnel assigned to this program were David P. Martin, V.M.O. Study Olractor; John l. Clemanee, D.V.M., Study Veterinarian; and Harman R. Selbold, V.M.O., Pathologist. BIONETICS M0A|S OI6414 34 VII. QUALITY ASSURANCE q.A. Inspection Statement PROJECT 20771 LBI COMPOUND NO. Aroclor 1254 TYPE OF STUDY PCB EFFECTS ON RHESUS REPRODUCTION This study was inspected by the LBI Quality Assurance Unit and findings were reported to the study director and management. In-process audits were performed on two occasions - 9/21/78 and 11/14/78. The final pathology report was audited in detail on 5/11-5/15, 1979 and the entire final report was audited on 5/16/-5/22, 1979 and a follow-up audit was done on 8/7/80. Study records were inspected on 9/12-9/29, 1978, 10/20/78, 11/14/78 and 5/16/79. ' The protocol was reviewed early in the study and all protocol changes were reviewed as they were implemented. This study was perfoneed according to acceptable laboratory practices. Those discrepancies noted were minor in nature and would in no way invalidate the study. qA Manager m BIONETICS Litton VIII. RECORDS RETENTION All original data. Including this report, will ba stored at tha IBI Archives at 1330 Piccard Orive, Rockville, Maryland. Submitted by: flivicTf.W: r\t\--ln.\,iV' \V. .H.V. ."Study Director Director Laboratory Anloal Medicine and Science Date BtONETlCS MOHS Olb^lb 36 IX. REFERENCES [1] Valerio, D.A., leverage, W.E., and Munster, J.H.: Semen evaluation In macaques. Lab. An(. Cara, 20:734-740, 1970. [2] Switzer, J.W., Valarlo, D.A., Martin. O.P., Valerio, M.G.,. and Rlninger, B.: Hematologic changes associated with pregnancy and parturition In Hacaca aulatta. Lab. Anl. Cara, 20:930, 1970. [3] Btrsottl, D.A., Marlar, R.J., and Allan, J.R.: Reproductive dysfunction In rhesus aonkeys exposed to low levels of poly chlorinated biphenyls (Aroclor 1248). Food Cosisat. Toxicol., 14:99-103, 1976. [4] McNulty, W.P.: PCS poisoning In rhasus monkeys. Primate News. Publication of Oregon Regional Primate Canter, 15(11:2-7. 1977. [5] McNulty, W.P. and Griffin, D.A.: Possfbla polychlorinated biphenyl poisoning In rhesus monkeys. J. Med. Prlmatol., 5:237-246, 1976. C6] Bloom, W. and Fawcett, D.W.: A Textbook of Histology, W.B. Saunders Company, (Ninth Ed.), p. 497, T95ff! [7] Ham, A.W.: Histology. J.8. Llpplncott Company, (Seventh Ed.), p. 609, 1974. [8] Allen, J.R.: Response of the nonhuman primate to polychlorinated biphenyl exposure. Research Activities at Regional Primate Centers. Fed. Proc., 34(8). 1975. [9] Allen, J.R., Carstans, L.A., and Barsottl, D.A.: Residual effects of short-term, low-level exposure of nonhuman primates to polychlorinated biphenyls. Toxicol. Appl. Pharmacol., 30:440-451, 1974. [10] Allen, J.R. and Barsottl, O.A.: The effects of transplacental and mammary movement of PCBs on Infant rhesus monkeys. Toxicology, 6:331-340, 1976. [11] Becker, G.M., McNulty, W.P., and Bell, M.: Polychlorinated b 1 phony1Induced morphologic changes in the gastric mucosa of the rhesus monkey. Lab. Invest., 40:373-383, 1979. BIOMETICS HONS 016417 37