Adaptation of the WHO guideline for residual DNA in parenteral vaccines produced on continuous cell lines to a limit for oral vaccines.

Although there is a WHO guidance for a limit on residual DNA for parenterally administered vaccines produced on continuous cell lines, there is no corresponding guidance for oral vaccines. To help determine an oral limit, we performed a study of Vero cell DNA uptake in rats, in which the relative uptake and persistence of Vero cell DNA administered orally was compared to its uptake when delivered intramuscularly (IM). The results of this study allowed the generation of an empirically derived IM versus oral factor (10(6)) representing the relative inefficiency of DNA uptake by oral administration. This factor was then applied to the WHO recommended parenteral limit of 10 ng/dose to determine a corresponding upper limit on the level of residual Vero cell DNA for an oral vaccine of 10 mg. As a conservative approach, this empirically determined limit was reduced 100-fold to 100 microg. Thus, the results of this animal study, together with additional evidence in the literature, support a residual DNA safety limit of 100 microg per dose for an oral vaccine produced on a continuous cell line.

P53+/- hemizygous knockout mouse: overview of available data.

The performance of the p53-/- transgenic (knockout) mouse model was evaluated through review of the data from 31 short-term carcinogenicity studies with 21 compounds tested as part of the International Life Sciences Institute's (ILSI) Alternatives to Carcinogenicity Testing (ACT) project, together with data from other studies which used comparable protocols. As expected based on the hypothesis for the model, a significant number (12/16 or 75%) of the genotoxic human and/or rodent carcinogens tested were positive and the positive control, p-cresidine, gave reproducible responses across laboratories (18/19 studies positive in bladder). An immunosuppressive human carcinogen, cyclosporin A, was positive for lymphomas but produced a similar response in wild type mice. Two hormones that are human tumorigens, diethylstilbestrol and 17beta-estradiol, gave positive and equivocal results, respectively, in the pituitary with p53-deficient mice showing a greater incidence of proliferative lesions than wild type. None of the 22 nongenotoxic rodent carcinogens that have been tested produced a positive response but 2 compounds in this category, chloroform and diethylhexylphthalate, were judged equivocal based on effects in liver and kidney respectively. Four genotoxic noncarcinogens and 6 nongenotoxic, noncarcinogens were also negative. In total (excluding compounds with equivocal results), 42 of 48 compounds or 88% gave results that were concordant with expectations. The technical lessons learned from the ILSI ACT-sponsored testing in the p53+/- model are discussed.

CB6F1-rasH2 mouse: overview of available data.

This article presents data from short-term carcinogenicity studies of compounds tested in the CB6F1-rasH2 transgenic mouse as part of the International Life Sciences Institutes' (ILSI) Health and Environmental Sciences' (HESI) Alternative to Carcinogenicity Testing (ACT) project. Additionally, data from other studies that were not conducted as part of the ILSI program, but used comparable or slightly modified protocols, are included here. A significant number (3 of 4) of the genotoxic carcinogens tested were positive in the rasH2 mouse; the other compound was equivocally positive. The positive control, N-Methyl-N-nitrosurea (MNU), gave reproducible responses across all participating laboratories with tumors noted at multiple sites in the animal. The immunosuppressive human carcinogen. Cyclosporin A, was equivocal. Two hormones that are human tumorigens. Diethylstilbestrol and 17beta-Estradiol, gave positive and negative results, respectively. Of the twelve additional compounds tested that are classified as non-genotoxic rodent carcinogens and putative human non-carcinogens, only the two peroxisome proliferators (clofibrate and diethylhexylphthalate(DEHP)) produced a positive response (liver effects). The three non-genotoxic non-carcinogens that were tested also gave negative responses in the rasH2 model. This result provides confidence that the model is likely to have a low false-positive rate.

Antigen levels and antibody titers after DNA vaccination.

DNA vaccination generates strong cellular and humoral immunity in animal models. The mechanisms by which plasmid DNA uptake and expression after intramuscular injection lead to immune responses are not well understood. In particular, the importance of antigen expression levels on subsequent antibody immune responses has not been established. We found that a chemiluminescent assay for alkaline phosphatase allows measurement of antigen levels of secreted alkaline phosphatase (SEAP) in vivo after intramuscular injection of a wide range of plasmid doses. The mice produced antibodies to the alkaline phosphatase reporter gene and both antigen levels and antibody titers were measured over time. We found that the correlation between initial antigen level and antibody response was high (r = 0.74, p < 0.001) and remained high even after accounting for the dose of plasmid injected (r = 0.61, p < 0.001). The correlation between DNA dose and antibody titer was statistically significant (r = 0.53, p < 0.001) but was reduced to almost zero after we accounted for initial antigen levels.

Chronic nephropathy in ad libitum overfed Sprague-Dawley rats and its early attenuation by increasing degrees of dietary (caloric) restriction to control growth.

The early development and progression of chronic nephropathy and its amelioration by moderate and marked dietary restriction (DR) was determined in Sprague-Dawley (SD) rats at 20, 33, 60, and 113 weeks of age. Both sexes of SD rats were overfed ad libitum (AL) or DR-fed at 72-79%, 68-72%, or 47-48% of the adult AL intake. The AL-fed rats rapidly developed increased body and kidney size, increased glomerular area (GA) and urinary protein loss, followed by declining creatinine clearance. Early increased kidney growth and glomerular hypertrophy by 20 weeks preceded increases in glomerular sclerotic index (GSI), 7-day BrdU tubular labeling index (TLI), and the lesions associated with chronic nephropathy. The glomerular number (GN) or the number of nephrons did not differ between the groups over the course of the study. Moderate DR (68-79% of AL) prevented the increased kidney size and GA at 20 weeks and delayed increases in GSI and TLI until 60 weeks of age. Marked DR (47-48% of AL) prevented increases in kidney size, GA and TLI at 20 weeks, and GSI at 60 weeks of age. In AL-fed rats, the early increase in GA predicted the early onset of proteinuria and the later decrease in creatinine clearance, and increased GSI, TLI, and mortality from severe nephropathy. The temporal and dose-related effects of increasing degrees of DR demonstrated that while nephron numbers were unchanged with age, the early development of glomerular hypertrophy was the critical morphological biomarker predicting the progression and severity of chronic nephropathy. Caloric restriction by DR prevented or delayed the development of glomerulosclerosis, tubulointerstitial damage, functional changes, morbidity, and mortality associated with chronic nephropathy in AL-overfed SD rats by controlling initial body and kidney growth, glomerular size, and nephron hypertrophy. These results indicate that control of body and renal growth by DR may be essential to prevent the development and progression of glomerulosclerosis in spontaneous nephropathy of laboratory rats.

The relative protective effects of moderate dietary restriction versus dietary modification on spontaneous cardiomyopathy in male Sprague-Dawley rats.

The relative protective effects of modifying dietary protein, fat, fiber, and energy content vs moderate food or dietary restriction (DR) on spontaneous cardiomyopathy of Charles River male Sprague-Dawley (SD) rats was evaluated at 1 and 2 years. For 2 years, SD rats were fed Purina Rodent Chow 5002 (21.4% protein, 5.7% fat, 4.1% fiber, 3.1 kcal/g) or a modified rodent chow 5002-9 (13.6% protein, 4.6% fat, 15.7% crude fiber, 2.4 kcal/g) ad libitum (AL) or by moderate DR at approximately 65% of the caloric intake of the AL group fed the 5002 diet. Serum lipids, carcass composition, and organ weights were evaluated and hearts were qualitatively and quantitatively examined microscopically for male SD rats at 1 and 2 years. Cardiomyopathy was characterized by the colocalization of myocardial degeneration, the development of subepicardial, perivascular, subendocardial, and interstitial fibrosis, and mononuclear inflammatory cell infiltration that increased by incidence and severity in an age-dependent manner from 1 to 2 years. SD rats fed the 5002 diet AL had the greatest heart weights and the most severe cardiomyopathy, with the highest myocardial fibrotic index. These parameters were relatively decreased in the AL 5002-9 diet, the DR 5002 diet, and the DR 5002-9 diet rats at 1 and 2 years. Regardless of the type of diet fed, both AL groups had the most severe cardiomyopathy by 2 years. Moderate DR allowed isocaloric comparisons of the relative effects of modified diets on survival, obesity, and heart disease. Only slight improvements in the severity and progression of spontaneous cardiomyopathy were seen by modification of the protein, fiber, fat, and energy content of the diet if fed AL. However, moderate DR with either diet was more effective than changing the diet composition in preventing and controlling the progression of cardiomyopathy in male SD rats.

Diet, caloric restriction, and the rodent bioassay.

The diet can significantly alter the results of toxicity and carcinogenicity studies. Ad libitum (AL) overfeeding of excessive calories to sedentary adult rodents is one of the most poorly controlled variables affecting the current rodent bioassay. AL-overfed rodents develop an early onset of adverse metabolic events, endocrine-disruptive degenerative diseases, and tumors that result in early morbidity and mortality. AL food consumption is extremely variable, but has a strong correlation with adult body weight, obesity, and survival. AL feeding of diets with modified protein, fiber, and energy content are not as effective as simple, moderate dietary (caloric) restriction (DR) in controlling these study variables. Moderate DR (70-75% of adult AL) is operationally simple and controls adult body weights, prevents obesity, and improves health and survival by reducing or delaying diet-related endocrine, renal, and cardiac diseases. Moderate DR provides a uniform rodent model, increases treatment exposure time, and increases the statistical sensitivity of these chronic bioassays to detect true treatment effects. Feeding a balanced diet by a moderate DR regimen of 70-75% of the maximum, unrestricted adult AL food intake is recommended for conducting well-controlled toxicity and carcinogenicity studies.

Weighted multiplicity adjustments for animal carcinogenicity tests.

Sparse data is a difficulty in the analysis of animal carcinogenicity data: it is difficult to detect effects when the background tumor rates are low. The widely used "Haseman rule" and its variants provide more power to tests with low background rates, while maintaining a degree of control over the global false positive rate. In this article we explore the use of these rules, finding global error rates that are unacceptably high for many animal carcinogenicity studies. We provide alternative weighting methods that correct the deficiencies of the Haseman rule, and apply them to carcinogenicity data from a pharmaceutical company.

The effects of diet, overfeeding and moderate dietary restriction on Sprague-Dawley rat survival, disease and toxicology.

Overfeeding by ad libitum (AL) food consumption is the most significant, uncontrolled variable affecting the outcome of the current rodent bioassay. The correlation of food consumption, the resultant adult body weight and the 2-y survival in Sprague-Dawley rats is highly significant. Feeding natural ingredient diets that varied in protein, fiber and metabolizable energy content did not improve low 2-y survival if Sprague-Dawley rats were allowed AL food consumption. Moderate dietary restriction (DR) of all diets tested significantly improved survival and delayed the onset of spontaneous degenerative disease (i.e., nephropathy and cardiomyopathy) and diet-related tumors. By 2 y, moderate DR resulted in an incidence of spontaneous tumors similar to that seen with AL consumption; however, the tumors were more likely to be incidental and did not result in early mortality. There was a decreased age-adjusted incidence in pituitary and mammary gland tumors, but tumor volume and growth time were similar in the AL and DR groups, indicating a similar tumor progression with a delay in tumor onset. Moderate DR did not significantly alter drug-metabolizing enzyme activities or the toxicologic response to five pharmaceuticals tested at maximum tolerated doses (MTD). However, moderate DR did require higher doses of compounds to be given before classical MTD were produced with four pharmaceutical drug candidates. Toxicokinetic studies of two of these compounds demonstrated steady-state systemic exposures that were equal or higher in moderate DR-fed rats. These and other data indicate that moderate DR is the most appropriate method of dietary control for rodent bioassays used to assess human safety of candidate pharmaceuticals.

Effect of early body weight and moderate dietary restriction on the survival of the Sprague-Dawley rat.

The effects of ad libitum (AL) feeding, moderate dietary restriction (DR), and initial (6-week) and one-year body weights on the two-year survival of the Sprague-Dawley (SD) rat were evaluated. DR-fed rats were given approximately 75 percent of the adult AL food intake. At two years, body weights of DR-fed males and females were approximately 69 and 58 percent of the AL-fed male and female body weights, respectively. The 2-year survival rate was 80 and 74 percent in DR-fed males and females, respectively, and 28 and 38 percent in AL-fed males and females, respectively. This increase in longevity indicates that DR-fed males and females in carcinogenicity studies would have 14.8 and 9.1 additional weeks of exposure in a 2-year period to test compounds, respectively, compared to AL-fed animals. There was no correlation between initial body weight and 2-year survival in DR or AL-fed rats. There was no association between 1-year body weight and 2-year survival among DR-fed rats. However, AL-fed rats with the greatest 1-year body weight had a lower 2-year average survival compared with the lightest AL-fed rats; this trend was statistically significant only in males. Body weights between the first and second years were statistically significantly correlated for both genders and feeding regimens but no correlation was observed between pretest and 2-year body weights. These findings demonstrate that initial body weight is not the determining factor of 2-year survival, but that the total adult food (caloric) intake is important. In conclusion, moderate dietary restriction prevented excessive body weight gain and greatly increased the 2-year survival of the SD rat. Initial body weights did not correlate to 2-year body weight gain and were not a predictive biomarker of 2-year SD rat survival.

The effects of diet, ad libitum overfeeding, and moderate dietary restriction on the rodent bioassay: the uncontrolled variable in safety assessment.

Ad libitum (AL) overfeeding is the most significant, uncontrolled variable affecting the outcome of the current rodent bioassay. There is a highly significant correlation between AL food consumption, the resultant obesity and body weight, and low 2-yr survival in rodents. AL feeding of diets with lowered protein, metabolizable energy (ME), and increased fiber does not improve survival. Only dietary restriction (DR) of all diets tested significantly improves survival and delays the onset of spontaneous degenerative disease (i.e., nephropathy and cardiomyopathy) and diet-related tumors. Moderate DR results in an incidence of spontaneous tumors similar to AL-fed rats, but the tumors are found incidentally and do not cause early mortality. There is a decreased age-adjusted incidence of pituitary and mammary gland tumors in moderate DR-fed rats, but tumor growth time is similar between AL and DR rats with only a delay in tumor onset time seen in DR-fed groups. Moderate DR does not significantly alter drug-metabolizing enzyme activities nor the toxicologic response to 5 pharmaceuticals tested at maximum tolerated doses (MTDs). However, moderate DR-fed rats did require much higher doses of 4 additional pharmaceutical compounds before classical MTDs were produced. Toxicokinetic studies of 2 of these compounds demonstrated equal or higher steady-state systemic exposures to parent drug and metabolites in moderate DR-fed rats. Markers of oxidative stress (lipid peroxidation, protein oxidation) are decreased and cytoprotective anti-oxidant markers are preserved in moderate DR-fed rats. But moderate DR does not delay reproductive senescence in female rats. Only marked DR delays reproductive senescence compared to AL and moderate DR-fed female rats. These and other data indicate that moderate DR is the most appropriate method of dietary control for the rodent bioassay when used to assess pharmaceuticals for human safety and compounds for risk assessment.

The effects of overfeeding and moderate dietary restriction on Sprague-Dawley rat survival, pathology, carcinogenicity, and the toxicity of pharmaceutical agents.

Ad libitum (AL) overfeeding is the most significant uncontrolled variable effecting the rodent bioassay. There is a highly significant correlation between food consumption, the resultant body weight, and two-year survival in laboratory rats. We have studied the effects of AL overfeeding, moderate dietary restriction (DR) and several modified diets on Sprague-Dawley (SD) rat longevity, spontaneous disease, carcinogenesis and the toxicity of pharmaceuticals. AL feeding of diets varying in protein, fiber and metabolizable energy content did not significantly alter two-year rat survival. Moderate DR (within the range of reported AL food intake) of all diets tested significantly improved survival and delayed the onset of spontaneous degenerative disease and diet-related tumors compared to AL-fed rats. Moderate DR resulted in a similar incidence of spontaneous tumors by 2 years, however, the tumors were more likely to be incidental and not result in early mortality. There was a decreased, age-adjusted incidence of pituitary and mammary gland tumors, but tumor volume and growth time was similar between AL and DR groups indicating similar tumor progression with a delay in tumor onset. Moderate DR did not change Phase I and Phase II drug metabolizing enzyme levels and did not significantly alter the toxicological response to 5 pharmaceuticals tested at maximum tolerated doses (MTDs). Additional studies with 4 pharmaceutical candidates did demonstrate that moderate DR allowed higher doses of compounds to be given before classical MTDs were observed. However, toxicokinetic studies of two of these compounds demonstrated steady state systemic exposures that were either equal of higher in the moderate DR fed rats. These and other data indicate that the moderate DR fed SD rat is a more appropriately controlled rodent model for toxicity and carcinogenicity studies to assess human safety of candidate pharmaceuticals.

Extent and persistence of streptozotocin-induced DNA damage and cell proliferation in rat kidney as determined by in vivo alkaline elution and BrdUrd labeling assays.

The extent of DNA damage and cellular proliferation induced in rat kidneys by single doses of the diabetogenic alkylating agent streptozotocin (STZ) and the time course of repair of that damage were evaluated using an in vivo alkaline elution assay for DNA strand breaks and a bromodeoxyuridine (BrdUrd) labeling assay for cell replication. Male Sprague-Dawley rats were given iv injections of 0.25 to 60 mg/kg STZ and kidneys were harvested 3 hr later for alkaline elution. A dose of 2.5 mg/kg STZ was the lowest dose to induce detectable DNA strand breaks and extensive damage was produced by the commonly used diabetogenic dose of 60 mg/kg. To characterize the repair of the drug-induced DNA damage, kidneys were harvested from a 60 mg/kg group of animals 3 hr to 27 days after dosing. BrdUrd-labeled kidney sections were also evaluated to assess any cellular proliferative response associated with STZ administration. Significant DNA damage was detected up to 14 days after dosing with return to near background levels by 20 days. Similarly, treatment with 60 mg/kg STZ was associated with increases in BrdUrd labeling indices 4 and 9 days after treatment with resolution by 27 days. These results indicate that the cellular and molecular repair responses to a single diabetogenic dose of STZ are prolonged, requiring up to 3 weeks to complete. Thus, to avoid potential additive or synergistic effects on STZ-induced nephrotoxicity and/or genotoxicity, a delay in the start of experimental therapies in this model (other than insulin) should be considered.

Induction of minisatellite DNA rearrangements by genotoxic carcinogens in mouse liver tumors.

We investigated whether somatic rearrangements in minisatellite DNA are more frequent in chemically induced mouse liver tumors than they are in spontaneous tumors. CD-1 mouse liver tumors were induced by either a single dose or 15 consecutive daily doses of 7,12-dimethylbenz[alpha]anthracene, 4-aminoazobenzene, N-hydroxy-2-acetyl-aminofluorene or diethylnitrosoamine (DEN). Using DNA fingerprinting analysis, we found that the single- and multiple-dose carcinogen treatments caused a 2- to 5-fold higher frequency of minisatellite DNA rearrangements compared with that found in spontaneous tumors--with the exception of single-dose DEN tumors, which showed no increase in rearrangements. Our results suggest that DNA fingerprinting may be a valuable assay for differentiating certain chemically induced tumors from spontaneous tumors.

Diet, overfeeding, and moderate dietary restriction in control Sprague-Dawley rats: I. Effects on spontaneous neoplasms.

This study was designed to compare the effects of ad libitum (AL) overfeeding and moderate dietary restriction (DR) of two different diets on Sprague-Dawley (SD) rat 2-yr survival and the development of spontaneous neoplasms. SD rats were fed Purina Rodent Chow 5002 or a modified Rodent Chow 5002-9 containing lower protein, fat, metabolizable energy and increased fiber by AL or by DR at 65% of the AL amount by measurement or time (6.5 hr). At 106 wk, rats fed the 5002-9 diet AL did not have significantly improved survival over rats fed the 5002 diet AL. The 5002 diet fed DR by time (6.5 hr) improved survival for males but not females. Only DR by measurement of both diets resulted in lower mortality for both sexes. The most common cause of death in rats of both sexes fed either diet AL was pituitary tumors followed by mammary gland tumors in females and renal and cardiovascular disease in males. The overall tumor incidence by 106 wk was remarkably similar between AL and DR groups. However, compared to the 5002 AL group, a decrease in the age-adjusted (Peto analysis) incidence of pituitary adenoma was observed in all other male groups. This effect was noted in the female DR by measurement groups only. For males, compared to the 5002 AL group, a decrease in the age-adjusted incidence of pancreatic islet carcinoma was observed in the DR by measurement groups only. In females, compared to the 5002 AL group, the only other difference in tumor incidence was the mammary gland tumors, which showed a significant decrease in the age-adjusted tumor incidence or multiplicity in the 5002-9 AL, 5002-9 DR, and 5002 DR groups. Additional analyses of mammary gland tumors showed growth time (time from initial palpation until death), tumor doubling time, and tumor volume were generally not statistically significantly different between AL and DR groups, although AL females could sustain larger tumor volumes. Compared to the 5002 AL group, there were no other significant differences in the age-adjusted incidence of any other tumor site in animals fed a modified diet or subjected to moderate DR of either diet. The conclusion from this study is that moderate DR delays death due to fatal cardiovascular or renal degenerative disease and spontaneous tumors, particularly those of the pituitary and mammary gland.(ABSTRACT TRUNCATED AT 400 WORDS)

Diet, overfeeding, and moderate dietary restriction in control Sprague-Dawley rats: II. Effects on age-related proliferative and degenerative lesions.

This study compared the effects of ad libitum (AL) overfeeding and moderate dietary restriction (DR) of 2 different diets on Sprague-Dawley (SD) rat survival and spontaneous, age-related proliferative and degenerative lesions. SD rats were fed Purina Rodent Chow 5002 or a modified Rodent Chow 5002-9 containing lower protein, fat, metabolizable energy, and increased fiber by AL or by DR at 65% of the AL amount by measurement or time (6.5 hr). At 106 wk, rats fed the 5002-9 diet AL did not have significantly improved survival over rats fed the 5002 diet AL. The 5002 diet fed DR by time (6.5 hr) improved survival for males but not females. Only DR by measurement of both diets resulted in lower mortality for both sexes. By 106 wk rats fed either diet by AL had the same brain weights as DR fed rats, but AL fed rats had greater body weight, body fat content, and increased heart, lung, kidney, liver, adrenal, thyroid, and pituitary weights that correlated with an increased incidence and severity of degenerative and/or proliferative lesions in these organs. Moderate DR delayed the progression of chronic nephropathy by delaying the early development of glomerular hypertrophy that initiates the development of glomerular sclerosis and nephron loss in AL overfed rats. Moderate DR lowered the incidence, severity, and progression of cardiomyopathy and other degenerative, age-related lesions and appeared to delay the development of reproductive senescence in SD females. The conclusion from this study is that moderate DR delayed onset and progression of degenerative lesions, and death due to cardiovascular or renal disease, and thus potentially improves the bioassay to detect compound-specific chronic toxicity.

Cancer incidence in a group of workers potentially exposed to ethylene oxide.

BACKGROUND: Cytogenetic changes associated with ethylene oxide (ETO) exposure at a worksite prompted a study of cancer incidence in that cohort. METHOD: Cancer incidence through 31 December 1987 was ascertained in a cohort of 1132 individuals employed at the worksite at any time from 1 July 1974 through 30 September 1980, the period of potential exposure to ETO at the plant. The number of observed cancers was compared with that expected based on age- and sex-specific incidence rates reported by the National Cancer Institute's Surveillance Epidemiology and End Results Program. Standardized morbidity ratios (SMR) were calculated separately for regular and temporary employees. RESULTS: Of the 28 cancers observed in the cohort, 12 were breast cancers. The SMR for breast cancer among regular female employees ranged from 2.55 (95% CI: 1.31-4.98, P = 0.02) to 1.70 (95% CI: 0.89-3.23, P = 0.09) depending on calendar year of follow-up, assumptions about completeness of follow-up, and the reference rates used. The excess of breast cancer over expected in regular female employees diminished over time. No statistically significant excess of breast cancer was noted for temporary female employees at any point during follow-up. No increase in cancer incidence was found over that expected for any cancer sites associated with ETO in previous studies--leukaemia, brain, pancreas and stomach. CONCLUSIONS: Factors such as appropriateness of latency periods, length of follow-up and lack of a common histopathological type need to be considered in evaluating the excesses in observed breast cancer incidence, which diminished over time.

Developmental neurotoxicity evaluation of acrylamide in Sprague-Dawley rats.

Based on the literature to-date, the potential of acrylamide (ACRL) to cause developmental neurotoxicity in laboratory animals has not been assessed. We examined this potential in Sprague-Dawley rats using a study design similar to that proposed by the USEPA. Dosages of 0 (deionized water), 5, 10, 15, or 20 mg/kg/day were administered at 5 ml/kg by oral gavage from gestational day 6 to lactational day 10 to groups of 12 mated females each. Females were allowed to deliver and the offspring were evaluated for survival, growth, development, behavior, and histological changes to brain, spinal cord, and peripheral nerve. Behavioral assessments consisted of open-field motor activity, auditory startle habituation, and passive avoidance tests during both the preweaning and adult periods (1 animal/sex/litter). All F0 and F1 animals in the 20 mg/kg/day group were euthanized early in the lactation period due to high pup mortality. Significantly increased pup mortality was also present in the 15 mg/kg/day group. There were dose-related decreases in average F0 maternal body weight gains during the dosing period in the 10, 15, and 20 mg/kg/day groups, and characteristic hindlimb splaying was observed in dams of the two highest dosage groups. Pup body weight proved to be the most sensitive indicator of developmental toxicity. Dose-related decrease in preweaning average weights were observed at all dose levels, although only transiently in the 5 mg/kg/day group. Average weight gain during the postweaning period was significantly decreased only in males of the 15 mg/kg/day group. Significant decreases in average horizontal motor activity and auditory startle response were observed only in weanlings of the 15 mg/kg/day group. The only behavioral effect in F1 adult animals was a decrease in auditory startle response in females of the 15 mg/kg/day group. There were no effects in the passive avoidance test or in the histological examination of the nervous system of preweaning pup or adult animals. Based on these results, the NOAEL (No Observed Adverse Effect Level) for developmental toxicity is less than 5 mg/kg/day, the NOAEL for maternal toxicity is 5 mg/kg/day, and that for developmental neurotoxicity is 10 mg/kg/day. Thus behavioral changes in the offspring were observed only at a dose which was also maternally toxic. These results suggest that acrylamide may be a selective developmental toxicant but not a selective developmental neurotoxicant, because a conventional measure of offspring toxicity (i.e., pup body weight) was affected at a dosage lower than that which produced maternal effects or offspring behavioral effects.

Short-term carcinogenesis bioassay of genotoxic procarcinogens in PIM transgenic mice.

E mu-pim-1 transgenic mice, which overexpress the pim-1 oncogene in lymphoid tissues, have shown increased susceptibility to induction of T cell lymphomas by N-ethyl-N-nitrosourea, a direct-acting chemical carcinogen (Nature, 340, 61-63, 1989). We sought to further evaluate E mu-pim-1 transgenic mice as a potential test animal for a short-term carcinogenesis bioassay. We chose to test four genotoxic procarcinogens; 2-acetylaminofluorene (2-AAF), N-nitro-sodiethylamine (NDEA), 1,2-dichloroethane (1,2-DCE) and benzene (BEN). These compounds require metabolic activation and, with the exception of benzene, are not mouse lymphomagens. Compounds were administered by gavage daily for 38 (NDEA and 2-AAF) or 40 (BEN and 1,2-DCE) weeks to groups of 25-29 male and female PIM mice at 1 and 3 mg/kg for NDEA, 50 and 100 mg/kg for BEN, 25-100 mg/kg for 2-AAF and 100-300 mg/kg for 1,2-DCE. Small but statistically significant increases in the incidence of malignant lymphoma were seen for three of the four carcinogens tested; in high dose males treated with 2-AAF, high and low dose females treated with NDEA and high dose females treated with 1,2-DCE. Results for BEN, the only mouse lymphomagen tested, did not show a statistically significant increase in the incidence of malignant lymphomas in transgenic mice within the 40 week duration of the study. NDEA also produced a high incidence (> 70%) of hepatic hemangiosarcomas in both sexes at the low and high dose levels. These results demonstrate that over-expression of the pim-1 oncogene in lymphoid tissue can confer susceptibility of this tissue to chemical carcinogenesis by genotoxic procarcinogens. However, whereas potent genotoxic carcinogens produced only small increases in the incidence of lymphoma and since BEN, a mouse lymphomagen, was negative, PIM transgenic mice may lack sufficient sensitivity to established carcinogens to justify their routine use in a short-term carcinogenesis screening assay.

Qualitative and quantitative evaluation of prostatic histomorphology in rats following chronic treatment with finasteride, a 5-alpha reductase inhibitor.

OBJECTIVE: To determine any potential direct and/or indirect effects of elevated intraprostatic T levels on the prostates of rats chronically (1-2 years) exposed to high doses (160 mg/kg/day) of finasteride, a selective inhibitor of 5-alpha reductase. METHODS: Sprague-Dawley male rats were administered daily finasteride by oral gavage. Prostates from all rats were weighed, fixed in 10% neutral buffered formalin, and processed for light microscopic examination. The volume fractions of the prostatic glandular and stromal compartments were quantitated by morphometric analysis. RESULTS: Administration of finasteride at doses of 20, 40, and 80 mg/kg/day for one year resulted in a significant (P < or = 0.05) decrease in prostatic weight; prostatic atrophy was evident by light microscopy. Morphometric analysis of the prostate showed that chronic finasteride administration resulted in a significant (P < or = 0.001) decrease in the absolute volume of both glandular (-65.2%) and stromal (-57.1%) compartments of the prostate. Furthermore, the total number of epithelial and stromal cells per gland were significantly (P < or = 0.002) decreased in finasteride-treated rats compared with vehicle controls; the magnitude of mean decrease was 69.8 percent and 50.6 percent of controls in epithelial and stromal cells, respectively. In addition, prostates from all two hundred fifty rats in a two-year study were qualitatively evaluated by light microscopy. Administration of finasteride at doses ranging from 2.5 mg/kg/day to 160 mg/kg/day for two years did not result in an increase over the background incidence of prostatic focal hyperplasia or adenoma. No malignant tumors of the prostate were seen in any of the groups. CONCLUSIONS: These studies have demonstrated that the expected pharmacologic effects of finasteride on the prostate are maintained following chronic treatment and that there was no evidence of a direct and/or an indirect effect of elevated intraprostatic T on prostatic morphology in rats.