Near-total absence of the cerebellum.

We report five cases of near-total absence of the cerebellum with accompanying pontine hypoplasia. The cerebellar remnant in each case comprised only antero-superior masses, the posterior fossa being otherwise fluid filled. Three of these patients, two teenagers and an infant, presented a fairly consistent clinical and neuroradiological phenotype, and a few similar cases are recorded in the literature. The cerebellar remnant was irregular and asymmetrical, and no ventral pontine prominence was discernible. In at least the older two, cerebellar motor functions were not greatly compromised, and intellectual handicap was of a mild degree. We propose that these cases represent a distinct entity of "near-total absence of the cerebellum with flat ventral pons, and relatively mild clinical affection". All cases have been sporadic, implying that the risk of recurrence within a family may be low. Quite different clinical pictures, of considerably greater severity, are demonstrated in the remaining two cases. One had pontocerebellar hypoplasia type 2, while the other had a complex cerebellar and cerebral malformation.

Reproductive endpoints in general toxicity studies: are they predictive?

The ability to use necropsy and in-life vaginal cytology data from 90-d general toxicity studies to predict the outcome of more definitive reproductive toxicity tests was evaluated using data from 18 compounds tested by the National Toxicology Program. Sperm motility and vaginal cytology evaluations (SMVCE) were performed at the end of 90-d toxicity studies. When these same compounds were tested in the definitive Reproductive Assessment by Continuous Breeding (RACB) design, 13 of the 18 compounds were classified the same way by both tests. The different conclusions for five compounds can be explained by differences in dose used or in endpoints evaluated. We conclude that reproductive-system necropsy data from general toxicity studies can provide a valuable preliminary indication of the likely reproductive toxicity of the compound under study.

The relationships among reproductive endpoints in Swiss mice, using the reproductive assessment by Continuous Breeding database.

The database of Continuous Breeding mouse studies was evaluated to determine the relationships between the functional indicators of reproduction (pup measures) and the various necropsy endpoints collected for males and females. Of 72 chemicals studied, both males and females were affected in 33 studies, while females and/or conceptuses were affected in 7. Two compounds affected only males, 17 studies were negative, and in 13 studies with effects it was not possible to clearly determine the affected gender(s). Greater F0 dam weight was correlated with increased pup mass per litter; this relationship was strongest for the first litter, and weakest for the fifth litter. For both generations of treated females (F0 and F1), longer estrous cycles correlated with reduced numbers of pups; the relationship was stronger in F0 than in F1 females and was not seen in controls. Sperm parameters had different distributions in treated mice than in control mice. Fertility (total live pups/number of pairs cohabited) was reduced if there were > approximately 15% sperm abnormalities or if sperm motility (moving/not moving) was < approximately 37%. Both of these relationships appeared to have thresholds. Epididymal sperm count in treated animals, however, was linearly related to fertility, even within the control range, suggesting strongly that other factors are important. Using both treated and control data together, combining sperm count with motility could explain much (r = 0.77) of the variation in fertility; adding morphology did not significantly improve the correlation. The model was almost as strong using count and morphology, in which case adding motility did not strengthen the model. This analysis of these studies shows that while some endpoints (e.g., random-estrous-cycle-point ovary weight) correlate poorly with fertility, other necropsy endpoints (epididymal sperm count and motility, estrous cycle length, and testis and epididymal weights) can be useful (though not complete) surrogates of overall reproductive function. Indeed, over many studies, epididymal sperm count in treated animals correlates with fertility so well that even small reductions (approximately 20%) in count result in reduced fertility, suggesting that mice may be better models of human fertility than was previously believed.

Reproductive assessment by continuous breeding: evolving study design and summaries of ninety studies.

The Reproductive Assessment by Continuous Breeding (RACB) design has been used by the National Toxicology Program for approximately 15 years. This article details the evolutions in the thinking behind the design and the end points used in the identification of hazards to reproduction. Means of nominating chemicals are provided, and both early and current designs are described as well as some proposed changes for the future. This introduction is followed by a text and tabular summary of each study performed to date. We hope that this will not only be an explicit presentation of the findings of this testing program to date, but will help stimulate thinking about new ways to detect and measure reproductive toxicity in rodents, and help identify new relationships among the end points that are measured in such studies.

Genetic toxicities of human teratogens.

Birth defects cause a myriad of societal problems and place tremendous anguish on the affected individual and his or her family. Current estimates categorize about 3% of all newborn infants as having some form of birth defect or congenital anomaly. As more precise means of detecting subtle anomalies become available this estimate, no doubt, will increase. Even though birth defects have been observed in newborns throughout history, our knowledge about the causes and mechanisms through which these defects are manifested is limited. For example, it has been estimated that around 20% of all birth defects are due to gene mutations, 5-10% to chromosomal abnormalities, and another 5-10% to exposure to a known teratogenic agent or maternal factor [D.A. Beckman, R.L. Brent, Mechanisms of teratogenesis. Ann. Rev. Pharmacol. Toxicol. 24 (1984) 483-500; K. Nelson, L.B. Holmes Malformations due to presumed spontaneous mutations in newborn infants, N. Engl. J. Med. 320 (1989) 19-23.]. Together, these percentages account for only 30-40%, leaving the etiology of more than half of all human birth defects unexplained. It has been speculated that environmental factors account for no more than one-tenth of all congenital anomalies [D.A. Beckman, R.L. Brent, Mechanisms of teratogenesis, Ann. Rev. Pharmacol. Toxicol. 24 (1984) 483-500]. Furthermore, since there is no evidence in humans that the exposure of an individual to any mutagen measurably increases the risk of congenital anomalies in his or her offspring' [J.F. Crow, C. Denniston, Mutation in human populations, Adv. Human Genet. 14 (1985) 59-121; J.M. Friedman, J.E. Polifka, Teratogenic Effects of Drugs: A Resource for Clinicians (TERIS). The John Hopkins University Press, Baltimore, 1994], the mutagenic activity of environmental agents and drugs as a factor in teratogenesis has been given very little attention. Epigenetic activity has also been given only limited consideration as a mechanism for teratogenesis. As new molecular methods are developed for assessing processes associated with teratogenesis, especially those with a genetic or an epigenetic basis, additional environmental factors may be identified. These are especially important because they are potentially preventable. This paper examines the relationships between chemicals identified as human teratogens (agents that cause birth defects) and their mutagenic activity as evaluated in one or more of the established short-term bioassays currently used to measure such damage. Those agents lacking mutagenic activity but with published evidence that they may otherwise alter the expressions or regulate interactions of the genetic material, i.e. exhibit epigenetic activity, have likewise been identified. The information used in making these comparisons comes from the published literature as well as from unpublished data of the U.S. National Toxicology Program (NTP).

PBPK modeling/Monte Carlo simulation of methylene chloride kinetic changes in mice in relation to age and acute, subchronic, and chronic inhalation exposure.

During a 2-year chronic inhalation study on methylene chloride (2000 or 0 ppm; 6 hr/day, 5 days/week), gas-uptake pharmacokinetic studies and tissue partition coefficient determinations were conducted on female B6C3F1, mice after 1 day, 1 month, 1 year, and 2 years of exposure. Using physiologically based pharmacokinetic (PBPK) modeling coupled with Monte Carlo simulation and bootstrap resampling for data analyses, a significant induction in the mixed function oxidase (MFO) rate constant (Vmaxc) was observed at the 1-day and 1-month exposure points when compared to concurrent control mice while decreases in glutathione S-transferase (GST) rate constant (Kfc) were observed in the 1-day and 1-month exposed mice. Within exposure groups, the apparent Vmaxc maintained significant increases in the 1-month and 2-year control groups. Although the same initial increase exists in the exposed group, the 2-year Vmaxc is significantly smaller than the 1-month group (p < 0.001). Within group differences in median Kfc values show a significant decrease in both 1-month and 2-year groups among control and exposed mice (p < 0.001). Although no changes in methylene chloride solubility as a result of prior exposure were observed in blood, muscle, liver, or lung, a marginal decrease in the fat:air partition coefficient was found in the exposed mice at p = 0.053. Age related solubility differences were found in muscle:air, liver:air, lung:air, and fat:air partition coefficients at p < 0.001, while the solubility of methylene chloride in blood was not affected by age (p = 0.461). As a result of this study, we conclude that age and prior exposure to methylene chloride can produce notable changes in disposition and metabolism and may represent important factors in the interpretation for toxicologic data and its application to risk assessment.

Endogenous formation of N-nitrosomorpholine in mice from 15NO2 by inhalation and morpholine by gavage.

Male CD-1 mice were exposed to an nominal concentration of 20 p.p.m. of 15N-nitrogen dioxide (15NO2) for 6 h/day for 4 days and for 2 h on the day 5, and to 1 g morpholine/kg body wt by gavage daily for five consecutive days. N-Nitrosomorpholine (NMOR) was found in whole mice, stomachs, skins with hair, and remains. The sum of individual tissue concentrations measured separately was 3421 ng/tissue, where the average skin weighed 4.3 g, the average stomach weighed 1.0 g and the average remains weighed 22.2 g. The average whole mouse weighed 27.7 g and contained a total of 3903 ng of NMOR. The concentration of NMOR was highest in the skin, next highest in the stomach, and lowest in the remains. However, the total quantity of NMOR per tissue, while highest in the skin (83%), was next highest in the remains (14.8%) and lowest in the stomach (2.2%). GC-MS analysis served to distinguish between the NMOR of 15NO2 origin and that of other origin. All of the NMOR in the whole mouse homogenates was identified as 15NMOR. In the stomach 73% was identified as 14NMOR, representing 1.6% of the total NMOR in the mouse, and 27% as 15NMOR, representing 0.6% of the total NMOR in the mouse. N-Nitrosamine formation in vivo is discussed as a possibly ongoing mammalian process.

Comparative toxicity of arsine gas in B6C3F1 mice, Fischer 344 rats, and Syrian golden hamsters: system organ studies and comparison of clinical indices of exposure.

In order to examine possible species differences in response to arsine exposure, multiple inhalation studies consisting of acute (1-day), subacute (14- and 28-day), and subchronic (90-day) exposures to this agent were conducted using three different species of rodents. Evaluations of hematopoietic organs and alterations in the heme biosynthetic pathway were the focus of these studies. Species used were B6C3F1 mice (exposed 1, 14, or 90 days), Fischer 344 rats (exposed 14, 28, or 90 days), and Syrian Golden hamsters (exposed 28 days). All arsine exposures were at concentrations of 0.5, 2.5, or 5.0 ppm except for 90-day studies, in which concentrations were lowered to 0.025, 0.5, or 2.5 ppm. No changes in body weight gain were observed in either sex of mice or hamsters. The only decrease in body weight gain occurred in male rats exposed to 5.0 ppm arsine for 28 days. Significant exposure-related increases in relative spleen weights occurred in both sexes of mice and rats in the 0.5 (except 14-day female rats), 2.5, and 5.0 ppm exposure groups from all studies and in hamsters in the 2.5 and 5.0 ppm exposure groups. Generally, increases in relative liver weight occurred in fewer exposure groups and were of a lesser magnitude than increases in spleen weight. Other parameters affected included decreased packed cell volumes (mice, rats, and hamsters), hematology profiles (rats), and an increase in delta-aminolevulinic acid dehydratase activity in all species. Arsenic content was measured in livers of rats after 90 days of exposure. Concentrations increased in relation to atmospheric concentrations of arsine. Histopathologic changes included increased hemosiderosis and extramedullary hematopoiesis in spleen and intracanalicular bile stasis (mice only) in liver. Additionally, bone marrow hyperplasia was observed in rats. Effects on other organs were not observed, suggesting that the hematopoietic system is the primary target for arsine. In conclusion, we have determined that the effects of arsine exposure upon mice, rats, and hamsters are similar. Most importantly, even though no effects on the hematopoietic system were observed following a single exposure to 0.5 ppm arsine which is 10 times the Threshold Limit Value (TLV) set by the American Conference of Governmental Industrial Hygienists, repeated exposure to 0.025 ppm (one-half the TLV) caused a significant anemia in rats.

Hepatic cholesterol metabolism following exposure to carbon disulfide in phenobarbital-treated rats.

Male F344 rats were exposed either to 1.87 mg/L (600 ppm) carbon disulfide (CS2) for 6 hr/day by inhalation for 1, 2, or 3 consecutive days, or to 0.1% phenobarbital (PB) in the drinking water starting 5 days before exposure to CS2, or to both. Combined treatments (CS2 + PB) resulted in a decrease in hepatic cholesterol synthesis, increases in hepatic cholesterol concentration and relative liver weight, and histopathologic damage. Maximal inhibition of cholesterol synthesis was observed following 1 day of combined treatments, while the increases in hepatic cholesterol concentration were similar following 1, 2, or 3 days of combined treatments. Exposure to CS2 only produced a pattern of inhibition of cholesterol synthesis that was similar to, though less extensive than, that seen following combined treatments. All reported alterations caused by combined treatments of CS2 + PB were reversible; recovery was, in all cases, essentially complete by day 11 after a single exposure to 1.87 mg/L CS2. With the exception of cholesterol concentration where time to recovery was decreased by continuation of PB, the time required for recovery from the effects of combined treatments of CS2 + PB was not affected by whether or not PB was continued after CS2 exposure. The reported observations support the theory that metabolism of CS2 is involved in the expression of CS2-mediated alterations of hepatic cholesterol metabolism.

Hepatic cholesterol metabolism as a function of carbon disulfide concentration and treatment with phenobarbital.

Male F344 rats were exposed to carbon disulfide (CS2) at 0, 30, 75, 150, 300, or 600 ppm for 6 hr by inhalation in the presence or absence of 0.1% phenobarbital (PB) in the drinking water starting 5 days before exposure to CS2. Exposure to 600 ppm CS2 only resulted in a decrease in hepatic cholesterol synthesis and an increase in the liver-to-body-weight ratio (relative liver weight); however, it caused no histopathological damage and had little or no consistent effect on the concentration of hepatic cholesterol or on hepatic water content. Treatment with PB alone resulted in increases in the concentration of hepatic cholesterol and relative liver weight. Exposure to 300 ppm CS2 + PB or to 600 ppm CS2 + PB resulted in a decrease in hepatic cholesterol synthesis and increases in the concentration of hepatic cholesterol, relative liver weight, hepatic water content, and histopathological damage. A concentration-response relationship was demonstrated between exposure to CS2 only and decreased hepatic cholesterol synthesis. A concentration-response relationship also was demonstrated between exposure to CS2 in rats that had been treated with PB and decreased hepatic cholesterol synthesis, increased hepatic cholesterol concentration, increased relative liver weight, increased hepatic water content, and histopathological damage. Treatment with PB lowered the concentration of CS2 required to alter hepatic cholesterol metabolism. The reported observations are consistent with the theory that oxidative metabolism is involved in the expression of CS2-mediated alterations of hepatic cholesterol metabolism.

Failure of carbon disulfide and levothyroxine to modify the cardiovascular response of rabbits to a high-cholesterol diet.

Exposure of rabbits for 12 weeks to 300 ppm carbon disulfide (CS2) for 6 h/day, 5 days/week, or to 25 mg/day of thiourea or 2% cholesterol in the diet, or to any combination thereof caused a significant reduction in the concentration of serum thyroxine (T4). The reduction of the concentration of serum T4 in rabbits by the treatments was completely offset by the inclusion of 0.1 mg/day of sodium levothyroxine in the diet. Ingestion of feed containing 2% cholesterol significantly increased the degree of atherosclerosis present in the aortic arch and significantly increased the oil red O positive lipid present in the heart and the aorta, with the aortic arch being the most severely affected. The response of the aorta and the heart to the 2% cholesterol diet was not significantly modified by concurrent exposure to CS2 by inhalation or by treatment with thiourea, a metabolite of CS2. We found no evidence that the development of cardiovascular lesions induced by a 2% cholesterol diet in rabbits was mediated by a mechanism involving a component of hypothyroidism.

In vivo formation of N-nitrosomorpholine in CD-1 mice exposed by inhalation to nitrogen dioxide and by gavage to morpholine.

Male CD-1 mice were exposed to approximately 20 ppm nitrogen dioxide (NO2) for 5-6 hours, to 1 g morpholine/kg body weight by gavage, or to both. Treatments were repeated daily for 5 consecutive days. N-nitrosomorpholine (NMOR) was found in whole carcasses (16-146 ng NMOR/mouse) in all animals that had been exposed to both NO2 and to morpholine, but NMOR was not found in tissues from animals that had been exposed to either chemical alone. Approximately one-third of the NMOR was found in the gastrointestinal tract, mainly in the stomach. The coadministration of 2 g sodium ascorbate/kg body weight or 1 g alpha-tocopheryl acetate/kg body weight had no effect on the amount of NMOR that was found in any tissue. Another possible product of the interaction of NO2 and morpholine, N-nitromorpholine, was not detected in any tissue. We concluded that the repeated, concurrent exposures of mice to NO2 by inhalation and to morpholine by gavage resulted in the in vivo formation of significant quantities of NMOR. The biological significance of the observation remains unknown.

Time-varying concentration profile as a determinant of the inhalation toxicity of carbon tetrachloride.

Certain limitations on the flexibility of small-animal inhalation exposure systems are overcome by the machine control and monitoring of the concentration of the gas or vapor of interest. Computer assistance of chamber operation allows the user to simulate time-varying concentration profiles accurately and repeatedly. We exposed rats to seven different profiles in which the maximum concentration of carbon tetrachloride (CCl4) was 1500 ppm and the product of concentration times time (C X T) was 4500 ppm X h. The purpose was to determine the effects of systematically varying the shape of the concentration profile on the expression of hepatotoxicity of a chemical about which much was already known. All of the exposures were conducted within a span of 6 h. Examination of the severity of vacuolation and pattern of necrosis could be used to distinguish some of the exposure profiles from others. For example, vacuolation was less severe when two equal pulses were presented with an interval of 60 min, rather than 180-240 min. The indexes of necrosis varied in a more complex way, and the differences among the profiles that accounted for the differences in the patterns of the histopathological changes were not immediately apparent. We concluded that the characteristic of a time-related variation in concentration is one of the determinants of the inhalation hepatotoxicity of CCl4 and that the simple, time-weighted, average concentration may not always fairly represent the best model for the study of problems in inhalation toxicology.

Evaluation of a new antihelminthic for trichuriasis, hookworm, and stronglyloidiasis.

Mebendazole was tested in a double-blind trial for its efficacy in the treatment and control of enteric helminths. One hundred and twenty-two children from a community near the Gulf of Carpentaria, and from a community in Cape York Peninsula in northern Queensland were divided into two equal groups to receive a course of either mebendazole or placebo after the identification of one or more intestinal helminths in a single pretreatment specimen of faeces. Between the tenth and twentieth days after a four-day course of treatment, three specimens of faeces were collected from each child. Mebendazole cured trichuriasis in 75% of cases, and over-all egg reduction rate was 96%. The drug also cured hookworm in 13 out of 15 cases and Strongyloides sterocoralis infestation in 14 out of 21 cases. Mebendazole was ineffective against Hymenolepis nana. No cases of Ascaris lumbricoides infestation were present. No side effects or adverse reactions to the drug were noted and patient acceptance was excellent. Mebendazole appears to be a safe drug for use in the treatment of human parasitic intestinal nematode infestations, and should be especially useful in the treatment and control of trichuriasis.

PIP: Mebendazole was tested in a double-blind trial for its efficacy in the treatment and control of enteric helminths. 122 children from a community near the Gulf of Carpentaria, and from a community in Cape York Peninsula in northern Queensland were divided into 2 equal groups to receive a course of either mebendazole or placebo after the identification of 1 or more intestinal helminths in a single pretreatment specimen of feces. Between days 10-20 following a 4-day course of treatment, 3 specimens of feces were collected from each child. Mebendazole cured trichuriasis in 75% of the cases, and the overall egg reduction rate was 96%. The drug also cured hookworm in 13 of 15 cases and Strongyloides sterocoralis infestation in 14 of 21 cases. Mebendazole was ineffective against Hymenolepsis nana. No cases of Ascaris lumbricoides infestation were present. No side effects or adverse reactions to the drug were noted and patient acceptance was excellent. Mebendazole appears to be a safe drug for use in the treatment of human parasitic intestinal nematode infestations and should be especially useful in the treatment and control of trichuriasis.