F. Bryce

Toxicological consequences of aroclor 1254 ingestion by female rhesus (macaca mulatta) monkeys. Part 1B. Prebreeding phase: Clinical and analytical laboratory findings

A group of 80 menstruating rhesus (Macaca mulatta) monkeys, with an average estimated age of 11.1 +/- 4.1 yr SD were first randomly allocated to four similar test rooms (20 monkeys/room), and then randomly allocated to one of five dose groups (four females/dose group/room). Each day, the monkeys self-ingested capsules containing doses of 0, 5, 20, 40 or 80 micrograms Aroclor 1254/kg body weight. After 25 months of daily dosing, approximately 90% of the treated females attained a qualitative pharmacokinetic steady state with respect to the concentration of polychlorinated biphenyl (PCB) in their adipose tissue. Subsequently, oestrogen and progesterone concentrations in serum were determined for one complete oestrous cycle and various immunological tests were conducted, while the monkeys continued to receive their daily dose of PCB. During the prebreeding phase of the study, blood for clinical and analytical monitoring including haematology, serum biochemistry, serum hydrocortisone, serum proteins (alpha 1, alpha 2, beta and gamma-globulins), serum immunoglobulins (A, G and M) and thyroid variables (thyroxine/triiodothyronine (T3) uptake ratio, percentage T3 uptake and free thyroxine index), were obtained monthly, as were specimens to ascertain the concentration of PCB in the blood, adipose tissue and faeces. Major findings among treated monkeys included the following: changes in haematology (decreased erythrocyte count, haematocrit, reticulocyte count, and mean platelet volume), serum biochemistry (decreased cholesterol and total bilirubin), immunotoxicity (decreased antibody production to sheep red blood cells and alterations in the percentage of T helper and T suppressor cells) and pathology (the number of regions of sebaceous gland lobules per unit of histological length was significantly reduced). These effects were observed at PCB doses lower than those previously reported for non-human primates.

Toxicological consequences of aroclor 1254 ingestion by female rhesus (macaca mulatta) monkeys. Part 1A. Prebreeding phase: Clinical health findings

A group of 80 menstruating rhesus (Macaca mulatta) monkeys, with an average estimated age of 11.1 +/- 4.1 yr SD, were first randomly allocated to four similar test rooms (20 monkeys/room) and then randomly allocated to one of the five dose groups (four females/dose group/room). Each day, the females self-ingested capsules containing doses of 0, 5, 20, 40 or 80 micrograms Aroclor 1254/kg body weight. After 25 months of daily dosing, approximately 90% of the treated females attained a qualitative pharmacokinetic steady state with respect to the concentration of polychlorinated biphenyl in their adipose tissue. The test monkeys were monitored daily for health and menstrual status, as well as feed and water consumption. On a weekly basis, each females body weight was determined and a detailed clinical examination was conducted. Minor treatment effects included a slight, but not statistically significant, decrease in feed and water consumption as well as a decreased feed conversion ratio and a slight increase in the duration of menses. Statistically significant, dose-related treatment effects included inflammation and/or prominence of the tarsal (Meibomian) glands, eye exudate, and various finger and toe nail changes. These results were found at doses lower than those previously reported for non-human primates.

Toxicological consequences of feeding PCB congeners to infant rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) monkeys

In a study designed to minimize interspecies extrapolation of toxicological data, nine rhesus (Macaca mulatta) and 15 cynomolgus (M. fascicularis) day-old infant monkeys were separated from their dams following parturition and hand-reared using a liquid non-human primate formulation. The infants were randomly divided into a control and a treated group which received a mixture of polychlorinated biphenyl (PCB) congeners analogous to those found in breast milk from Canadian women. The concentration of congeners in the dosing media resulted in each infant receiving a total of 7.5 microg PCB congeners/kg body weight/day. The congeners were added either to the liquid formulation or to corn oil and administered to the back of the monkeys mouth for 20 weeks. Monthly blood and adipose specimens were obtained during the dosing period and then periodically until the monkey was necropsied or taken off test (minimum of 66 weeks on test) for congener analysis. Parameters such as body weight, formula consumption, tooth eruption, somatic measurements, haematology and serum biochemistry were monitored throughout the study. In addition, a qualitative evaluation of the absorption and depletion of the various congeners was undertaken as was an immunological evaluation. For the monitored parameters, very few differences were found to be statistically significant. For the immunological parameters, the only statistically differences found were a reduction over time for immunoglobulins M and G antibodies to sheep red blood cells (cyno, P = 0.025; rhesus, P = 0.002) and a treatment-related reduction in the levels of the HLA-DR cell surface marker (mean percent, P = 0.016; absolute levels, P = 0.027). There were some qualitative differences regarding absorption and depletion rates for the various congeners, but it could not be definitely ascertained whether these differences were due to species differences or dosing mode. However, statistically significant differences were found for treatment (P = 0.0293) as well as for species and vehicle regarding the concentration of PCB in blood (species;--P = 0.0399; treatment--P = 0.0001) and adipose tissue (species--P = 0.0489; treatment--P = 0.0001).

Effects of toxaphene on the immune system of cynomolgus (Macaca fascicularis) monkeys. A pilot study

Toxaphene in glycerol/corn oil was administered at 1mg/kg body weight/day, 7 days/week in gelatin capsules to four healthy young adult cynomolgus (Macaca fascicularis) (two male and two female) monkeys for 52 weeks. Control monkeys ingested glycerol/corn oil only. Testing for immune effects was initiated at 34 weeks of treatment. Results included: reduced anti-sheep red blood cell (SRBC) titres for immunoglobulins (Ig) M and G; increased IgG titres to pneumococcal antigens, but not to the tetanus toxoid antigen; reduced T-helper/inducer mean lymphocyte numbers and the mean T-helper/inducer:T-suppressor/cytotoxic cell ratio and reduced respiratory burst activity in peripheral blood monocytes and granulocytes, albeit no changes on the phagocytic activity of these cells were detected. The above noted effects although not statistically significant (P0.05) suggest that chronic exposure to low levels of toxaphene may be immunosuppressive in cynomolgus monkeys and may pose a hazard to human health. To advance our understanding of the degree of hazard that toxaphene may pose to human health, we have undertaken additional chronic studies with a larger number of animals. Particular attention is focused on determining the potential immunotoxic effects of toxaphene in offspring following in utero exposure.

The elimination and estimated half-lives of specific polychlorinated biphenyl congeners from the blood of female monkeys after discontinuation of daily dosing with Aroclor® 1254

The levels of thirty polychlorinated biphenyl congeners in the blood of female rhesus monkeys, previously dosed with Aroclor 1254 for over six years, were monitored every two weeks during the first year and monthly during the subsequent two years after dosing was discontinued. Both blood lipid and polychlorinated biphenyl congener levels generally declined during this post dosing period. The percent distribution of the PCB congeners during the post dosing period remained relatively constant with more than half of all polychlorinated biphenyls consisting of the mono-orthochlorine substituted biphenyls. The contribution of the mono-orthochlorine substituted biphenyls was significantly different for one out of three monkeys in two of the three dose groups, during the post dosing period. Half-life, estimations for nine of the congeners ranged from 0.3-7.6 years.

Postmortem tissue levels of polychlorinated biphenyls in female rhesus monkeys after more than six years of daily dosing with Aroclor 1254 and in their non-dosed offspring.

Polychlorinated biphenyl (PCBs) analyses were made on prenecropsy blood samples and postmortem adipose, liver, kidney, and brain tissues from female rhesus monkeys fed a daily dose of 0, 5, 20, 40, or 80 μg Aroclor® 1254/kg body weight for approximately 6 years. During this time, the females were bred with non-dosed males. All resulting offspring were nursed for 22 weeks and fed no additional PCBs until they were necropsied at approximately 120 weeks after birth. PCBs were also measured in necropsied infant tissues to determine PCB levels due to intake of PCB-contaminated milk from the dosed dams, in addition to in utero exposure.Polychlorinated biphenyl levels in all tissues of the adult monkeys increased with their dosage. The highest PCB levels were found in adipose tissue and the lowest levels were found in the brain. Polychlorinated biphenyl residues in the cortex of the kidney were lower than in the medulla, while in the brain no appreciable differences were observed between the occipital and frontal lobes. Necropsy tissues of infants from dosed dams contained more PCBs than those nursed by controls, but less than tissues from stillborn infants. Although no differences were observed between PCB tissue levels from monkeys having offspring and those having no offspring, those having a stillborn infant had higher PCB levels in their tissues than those with a viable infant. Similarly, monkeys that were euthanized because of poor health had higher PCB levels in their tissues than those necropsied at the conclusion of the study and showed a dramatic shift from tetra- and hexachlorobiphenyls to penta-and heptachlorobiphenyls in their tissues. The PCB distribution pattern in tissues from a dosed mother/infant pair differed considerably. A larger percentage of heptachlorobiphenyls was found in the infant than in its dam. The adipose/blood PCB ratio increased with dosage, while the brain/blood PCB ratio in the adult monkeys remained remarkably constant.

Capillary GC-ECD and ECNI GCMS characterization of toxaphene residues in primate tissues during a feeding study

Toxaphene is a pesticide whose use was banned in North America because of concerns regarding its toxicity. To obtain better data on the metabolism and toxicity of toxaphene in primates, a one year feeding study was carried out in cynomologous monkeys at a dose of 1 mg/kg/day for one year. Levels of toxaphene residues in blood and adipose tissue during the dosing period were measured by GC-ECD and ECNI GCMS. The dosing toxaphene mixture was found to be extensively metabolized. Four chlorinated bornane congeners were the predominate residues found in the tissue samples. Blood levels of toxaphene residues plateaued at 40 ppb, adipose levels at approximately 4000 ppb. Kidney, liver, feces and urine were analyzed for toxaphene residues after necropsy.

Effects elicited by toxaphene in the cynomolgus monkey (Macaca fascicularis) : a pilot study

Toxaphene, which was added to glycerol/corn oil, was administered at a level of 1 mg/kg body weight/day in gelatin capsules to four healthy young adult cynomolgus (Macaca fascicularis) monkeys for 52 weeks. Four control monkeys ingested capsules containing only glycerol/corn oil. Each group had two males and two females. On a daily basis, each monkeys feed and water consumption was determined, its health was monitored and the females were swabbed to evaluate menstrual status. On a weekly basis, each monkeys body weight was determined and a detailed clinical evaluation was performed. At 4-week intervals, blood samples were taken for serum biochemistry, haematology and toxaphene analysis. Also, a local anaesthetic was administered to the nuchal fat pad area of each monkey, and adipose samples were obtained for toxaphene analysis. 1 day prior to the biopsies, a 24-h urine and faecal collection was obtained for toxaphene analysis. After 34 weeks of treatment, the immune system of the monkeys was evaluated. After 52 weeks of dosing, all treated and two control animals were necropsied. Liver samples were obtained and microsomal fractions were prepared immediately. A portion of liver and kidney was taken for toxaphene analysis. All of the major internal organs were weighed and bone marrow evaluations were conducted. Organ and tissue samples were fixed in 10% formalin and processed for light microscopy. There was no effect of treatment on body weight gain, feed consumption, water consumption or haematological parameters. Two major clinical findings were inflammation and/or enlargement of the tarsal gland and impacted diverticulae in the upper and lower eye lids. At necropsy, the relative spleen and thymus weights were greater for the treated monkeys than the controls. Toxaphene administration produced an increase in metabolism of aminopyrene, methoxyresorufin and ethoxyresorufin, three substrates that are altered specifically by cytochrome P450-based hepatic monooxygenase enzymes. Histopathological examination of tissues was unremarkable by light microscopy. Tissue analysis for toxaphene and immunology findings have been published elsewhere.

Effects of toxaphene on the immune system of cynomolgus (Macaca fascicularis) monkeys.

Toxaphene, dissolved in glycerol/corn oil, was administered at 0.1, 0.4 or 0.8 mg/kg body weight/day in gelatin capsules to groups of 10 young adult female cynomolgus monkeys (Macaca fascicularis), while a group of five male monkeys (Macaca fascicularis) received 0.8 mg/kg body weight/day. Control male (a group of five) and female (a group of 10) monkeys ingested the glycerol/corn oil vehicle only. Treatment continued for 75 weeks. Testing for immune effects was initiated at 33 weeks of treatment. Immunization was initiated at 44 weeks of treatment. Pairwise comparisons between each of the treated female groups to the control indicated that the mean primary (post-immunization weeks 1-4) and secondary (post-immunization weeks 5-8) anti-SRBC IgM responses were significantly reduced at the 0.4 and 0.8 mg/kg body weight/day doses compared to the control (P< or =0.05). The mean primary (post-immunization weeks 1-4) anti-SRBC IgG response was significantly reduced compared to the control (P< or =0.05), while the secondary (post-immunization weeks 5-8) anti-SRBC IgG was not significantly affected by treatment (P>0.05). The mean anti-tetanus toxoid IgG response in the 0.8 mg/kg body weight/day dose group The mean primary anti-SRBC (IgM) response in the treated males was significantly different from the control (P<0.05), while the primary anti-SRBC IgG response was not affected by treatment. The mean absolute B-lymphocyte numbers in the female group administered 0.8 mg/kg of toxaphene was significantly reduced compared to the control (P< or =0.05). All other parameters including the natural killer cell activity, the delayed-type hypersensitivity response, the lymphoproliferative response of peripheral blood leukocytes to the mitogens Con A and PWM and the serum cortisol levels were not affected significantly by treatment (P>0.05). The no-observed-adverse-effect level (NOAEL) for the female monkeys based on the toxaphene effects on humoral immunity was 0.1 mg/kg body weight/day.

Consequences of Aroclor 1254 ingestion on the menstrual cycle of rhesus (Macaca mulatta) monkeys.

A group of 80 female rhesus (Macaca mulatta) monkeys were randomly distributed to four similar test rooms (20 monkeys/room) and then randomly allocated to one of five test groups (four females/test group/room). The objective of the study was to ascertain the toxicological and reproductive effects of Aroclor 1254 ingestion at dose levels of 0, 5, 20, 40 or 80 microg Aroclor 1254/kg body weight per day (Arnold et al., 1993a,b, 1995, 1996, 1997). It was deemed necessary to establish the menstrual patterns for all the monkeys both before and after the start of dosing so as to provide an appropriate baseline from which potential treatment effects could be ascertained. The data presented herein were obtained during the first 3 years after the start of dosing, or the studys pre-mating phase. At the end of the first 2 years of dosing, the monkeys attained a qualitative pharmacokinetic steady state regarding the levels of polychlorinated biphenyls in their adipose tissue. Upon termination of the study, a number of monkeys were found to have endometriosis, adenomyosis or uterine leiomyomas (Arnold et al., 1996, 1997). These monkeys were designated as having gynecological abnormalities which were considered to be a factor in the analysis of the menstrual data. The menstrual data (i.e. menses frequency, cycle length and menses duration) were subjected to a statistical assessment to see whether year, quarter, gynecological abnormalities or dose of Aroclor 1254 had any effect on menses frequency, menstrual cycle length (i.e. the first day of menses until the day prior to the start of the next menses) and/or menses duration (i.e. the number of days of haemorrhagic discharge). The only consistent statistically significant effect found was that gynecological abnormalities increased menses duration (P<0.05) in all 12 quarters of the premating observation period. This effect was significant during both the pre- (P=0.0004) and post- (P< or =0.0001) pharmacokinetic steady-state intervals. While there was some indication of seasonality regarding menstrual cycle length and menses duration when these data were compared on a quarterly basis during the first 2 years of the study (P=0.043; P< or =0.0001, respectively), this effect was not evident during the third year (P=0.21; P=0.31, respectively). In particular, the effect of quarter on menses cycle length was most evident during the first year, with the shortest cycles occurring during the first or spring quarter and the longest in the third or fall quarter. However, menses duration was shortest in the first quarter during the first 2 years and tended to peak in the second quarter of all 3 years, while generally diminishing in the third and fourth quarters. There was also an increase in menses duration with increasing time on test for all groups. In addition, Aroclor 1254 treatment appeared to have some effect on menses duration when menses duration was plotted against dose group, but the effect was not statistically significant (P>0. 05). It was concluded that the ingestion of Aroclor 1254 at dose levels up to 80 microg/kg body weight/day by rhesus monkeys did not have any appreciable biological effect on menstrual frequency, menstrual cycle length or menses duration. However, gynecological abnormalities significantly increased menses duration during the three-year observation period.