The following abstract presents results of a study conducted by a contract laboratory for the National Toxicology Program. The findings were not evaluated in accordance with the levels of evidence for reproductive or developmental criteria established by NTP in March 2009. The findings and conclusions for this study should not be construed to represent the views of NTP or the U.S. Government.
The potential effects of lead acetate trihydrate on the Sprague-Dawley rat reproductive system was evaluated. This study was a collaborative study among Drs. Robert Chapin and G. Jean Harry of NIEHS, Dr. Rebecca Sokol of USC, Dr. Steve Schrader of NIOSH, and Dr. Joel Pounds of Wayne State University. Phase 1, the Pilot Study, was conducted to confirm similarities of response across laboratories. Phase 2, the Main Study, was conducted to determine if rats acclimatize to some of the effects of lead over a 6-month time frame.
For Phase 1, twenty-six male and sixteen female Sprague-Dawley rats/group were administered 0 or 0.3% lead acetate trihydrate in the drinking water for at least 28 days. Clinical observations, body weights, feed and water consumption data were collected. At necropsy blood was collected and samples sent to Dr. Sokol for lead, DNA, Androgen Binding Protein (ABP), and hormone analyses. A gross necropsy was performed, sperm analysis conducted, organ weights obtained, and tissues preserved. The left kidneys were examined microscopically. Tissue specimens were sent to Dr. Sokol for immuno-histochemistry, Northern analysis of DNA, and Western analysis of proteins. Sperm suspensions were sent to Dr. Schrader for analysis of chromatin structure.
During Phase 1, no treatment-related effects were observed in body weight, food consumption, mortality, clinical observation, or gross observation data. Mean water consumption values were decreased in the lead-treated group compared to controls. During the study, mean water consumption values were decreased by 22-28% in the lead-treated males compared to the controls. During Study Week 1, the mean water consumption value for the lead-treated females was decreased by 17% compared to controls; however, water consumption values were comparable between controls and lead-treated females for the remainder of the study. No treatment-related differences were observed in the organ weight data with the exception of kidney weights. The absolute and relative (to body weight) kidney weights were increased by 9-10% in the 0.3% females compared to controls. No treatment-related microscopic lesions were observed in the left kidneys. Epididymal sperm density and percent abnormal sperm were comparable between control and lead-treated groups. The number of homogenization-resistant spermatids per testis and per mg testis were increased by 18-20% in the 0.3% males compared to controls. The testes were not evaluated microscopically.
For Phase 2, four hundred male and four hundred female Sprague-Dawley rats were administered 0, 0.025, 0.05, 0.1, and 0.3% lead acetate trihydrate in the drinking water for up to six months. Body weights, food consumption, and water consumption were measured during Study Weeks 1, 4, 8, 12, 16, 20, and 24. Sixteen rats/sex/group were scheduled to be necropsied after 1, 4, 8, 16, and 24 weeks of treatment. At necropsy, blood was collected and the samples were sent to Dr. Sokol for DNA, ABP, and hormone analyses and to Dr. Flessel for lead analysis. A gross necropsy was performed, sperm analysis conducted, organ weights obtained, and tissues preserved. The left kidneys were examined microscopically for evidence of lead-related lesions. Tissue specimens were sent to Dr. Sokol for immuno-histochemistry, Northern analysis of DNA, and Western analysis of proteins. Sperm suspensions and unstained sperm smears were sent to Dr. Schrader for analysis of chromatin structure and morphometry. Sciatic nerve specimens were forwarded to Dr. Harry for analysis of nerve structure and mRNA levels. Bone samples were sent to NIEHS/RTI to determine skeletal lead levels. Other bone/tissue specimens (left tibia, calvaria, and 1/2" duodenum) were sent to Dr. Pounds to examine lead's effects on the vitamin D-dependent expression of osteocalcin.
Phase 2 male and female body weights were unaffected by lead acetate trihydrate administration, except during Study Week 4 when the 0.3% male body weights were decreased by 3%. No treatment-related differences were observed in food consumption values, clinical observations, or mortality. Mean water consumption values decreased with increasing concentration although the magnitude of the effect decreased with longer exposure.
No treatment-related changes were observed in gross observation or organ weight data except for kidney weights. Treatment-related increases in the kidney weights were observed in both sexes but the effect was more prominent in the females. Microscopic evaluation of the left kidneys revealed treatment-related lead inclusions seen in the nuclei of tubular epithelial cells and tubular epithelial cell megalocytosis. The incidence of these lesions increased with the duration of exposure: more than half the females in the 0.1% group showed lead inclusions at 16 and 24 weeks, while almost all the males and females in the 0.3% group were affected at 16 and 24 weeks. No treatment-related differences were observed in sperm motion parameters or in testicular spermatid head counts at any of the time points evaluated. After one week of treatment, the mean percent abnormal sperm values were increased by 83% and 72% in the 0.1 and 0.3% males, respectively, when compared to controls but were comparable among dose groups after 4, 8, 16, and 24 weeks of treatment. After one week of treatment, mean epididymal sperm density was decreased by 25% in the 0.3% group when compared to controls. Mean epididymal sperm density values were comparable among the dose groups after 4, 8, and 16 weeks of treatment, with an exception of an increased (23%) epididymal sperm density count in 0.05% males at Week 16. After 24 weeks of treatment, the mean epididymal sperm density values were decreased by 23 and 18% in the 0.1 and 0.3% males, respectively, when compared to controls. The testes were not evaluated microscopically.
Based on the data presented in this report (body weight, food and water consumption, clinical and gross observation, organ weight, and sperm analysis data), lead acetate trihydrate is not a selective male reproductive toxicant, because the minor effects on sperm density occurred concomitant with, or at doses greater than, those doses that produced microscopic changes in the left kidneys. The only female reproductive data presented in this report are the uterine and ovarian weights which were comparable among control and treated groups. The no-observable-adverse-effect level (NOAEL) was not established in this study as the 0.025% animals displayed decreases in water consumption. A maximum tolerated dose (MTD) was reached for the 0.3% dose group, based on decreased water consumption, increased kidney weights, and increased incidence of renal intranuclear lead inclusions and renal tubular megalocytosis. The remainder of parameters (immuno-histochemistry, Northern analysis of DNA, Western analysis of proteins, blood analyses, sperm chromatin structure, nerve structure and myelin mRNA levels, skeletal lead levels, and osteocalcin levels), analyzed by the collaborators, will be reported separately.
NTIS # PB97-125371