Three-generation reproduction study of rats receiving acrylonitrile in drinking water.

Acrylonitrile, a high volume organic chemical, was tested for reproductive effects in a three generation drinking water study with two matings per generation. Sprague-Dawley rats were exposed to acrylonitrile in drinking water at 0, 100, or 500 ppm. This corresponds to 0, 11+/-5 and 37+/-10 mg/kg, respectively, for males and 0, 20+/-3 and 40+/-8 mg/kg per day for the females, respectively. Water consumption was reduced in F0 rats in the 100 and 500 ppm groups. At 500 ppm, acrylonitrile reduced body weight gain and food intake of the first generation parental rats (F0). These parameters were not investigated at subsequent generations. The pup survival (both viability and lactation indices) was reduced at the 500 ppm treatment level in both matings of all three generations. Fostering the 500 ppm pups onto untreated mothers following the second mating lessened mortality, suggesting a maternal effect consistent with decreased water consumption. There was no remarkable change in the reproductive capacity in any of matings in rats at the 100 ppm concentration. In contrast, in all three generations, the body weights of the pups of the 500 ppm treatment level were reduced on Day 21 at both matings. No adverse findings were observed in the tissues of a limited number of third generation weanlings (F3b) upon gross and microscopic evaluation. No effect on the sciatic nerve was evident among the adult female rats held for 20 weeks after weaning of the second litter. There was a dose-related effect of acrylonitrile on gross masses in female rats at each parental generation held 20 weeks after the weaning of the second litter. Histopathological evaluation of these dams showed an increase in astrocytomas and zymbal gland tumors.

Disposition of inhaled mercury vapor in pregnant rats: maternal toxicity and effects on developmental outcome.

The disposition and toxicity of inhaled elemental mercury (Hg0) vapor for pregnant Long-Evans rats, and potential adverse effects on reproductive outcome were investigated. Rats were exposed to 0, 1, 2, 4, or 8 mg Hg0/m(3) for 2 h/day from gestation day (GD) 6 through GD 15. Maternal toxicity occurred primarily in rats exposed to 4 and 8 mg/m(3) and was manifested as a concentration-related decrease in body weight gain and mild nephrotoxicity. Control rats gained about 13% of their initial body weight during the 10-day exposure. Rats exposed to 4 mg/m(3) Hg0 gained about 7% less than controls, and rats exposed to 8 mg/m(3) Hg0 lost about 17% of their initial body weight during the 10-day exposure period. Maternal kidney weights were significantly increased in the 4 and 8 mg/m(3) concentration groups, and urinalysis revealed increased levels of protein and alkaline phosphatase activity in urine of all Hg0-exposed rats. Dams exposed to 8 mg/m(3) were euthanized in moribund condition on postnatal day (PND) 1. There was no histopathological evidence of toxicity in maternal lung, liver, or kidney of exposed rats at GD 6, GD 15, or PND 1. The incidence of resorptions was significantly increased, litter size and PND 1 neonatal body weights were significantly decreased only in the 8-mg/m(3) group. Total Hg concentrations in maternal tissues increased with increasing number of exposure days and concentration. In general, approximately 70% of Hg was eliminated from maternal tissues during the week following the last exposure (GD 15 to PND 1). Elimination of Hg from maternal brain and kidney was slower than in other tissues, possibly due to higher levels of metallothionein. Total Hg concentrations in fetal tissues increased with increasing number of exposure days and concentration, demonstrating that a significant amount of Hg crossed the placenta. One week after the last exposure, significant amounts of Hg were still present in brain, liver, and kidney of PND 1 neonates. Metallothionein levels in neonatal tissues were not significantly increased by exposure to 4 mg/m(3) Hg0. The total amount of Hg in neonatal brain (ng/brain) continued to increase after termination of inhalation exposure, suggesting a redistribution of Hg from the dam to neonatal brain. These data demonstrate that inhaled Hg0 vapor is distributed to all maternal and fetal tissues in a dose-dependent manner. Adverse effects of Hg on developmental outcome occurred only at a concentration that caused maternal toxicity.

Valproate-induced limb malformations in mice associated with reduction of intracellular pH.

Valproic acid (VPA) is a commonly used antiepileptic agent that recently has been found useful in the treatment of affective disorders and prophylaxis of migraine. VPA induces congenital malformations, especially spina bifida, in the offspring of women treated with this agent during early pregnancy. The mechanism by which VPA induces abnormal development remains unknown despite many studies in experimental animals in which VPA causes malformations similar to those seen in human infants. Because of its chemical structure as a weak organic acid and its capability to induce postaxial forelimb ectrodactyly in C57BL/6 mice, we postulated that VPA acts to perturb limb morphogenesis by reducing embryonic intracellular pH (pHi). We administered VPA, 200 to 400 mg/kg, to C57BL/6 mice on day 9 of gestation. A dose-dependent incidence of postaxial forelimb ectrodactyly was observed. Forelimb bud pHi was estimated by computer-assisted image analysis from the transplacental distribution of 14C-DMO. At the highest doses, 300 and 400 mg/kg, a decrease of pHi of 0.2 to 0.3 pH units was observed uniformly throughout the limb bud 1 h after VPA treatment. None of these changes were seen after treatment with 2-en VPA, a nonteratogenic analog of VPA. Furthermore, the capability of VPA to induce postaxial forelimb ectrodactyly was greatly enhanced by coadministration of agents that inhibit pHi regulatory processes. These data support the hypothesis that VPA-induced postaxial ectrodactyly in murine fetuses can be attributed to reduction in limb bud pHi.

A physiologically based pharmacokinetic model for fluoride uptake by bone.

A sex-specific, physiologically based pharmacokinetic (pbpk) model has been developed to describe the absorption, distribution, and elimination of fluorides in rats and humans. Growth curves generated by plotting mean body weights (kg) against age (weeks or years) are included in the simulation model to allow the integration of chronic fluoride exposure from birth to old age. The model incorporates age and body weight dependence of the physiological processes that control the uptake of fluoride by bone and the elimination of fluoride by the kidneys. Six compartments make up the model. These are lung, liver, kidney, bone, and slowly and rapidly perfused compartments. The model also includes two bone subcompartments: a small, flow-limited, rapidly exchangeable surface bone compartment and a bulk virtually nonexchangeable inner bone compartment. The inner bone compartment contains nearly all of the whole body content of fluoride, which, in the longer time frame, may be mobilized through the process of bone modeling and remodeling. The model has been validated by comparing the model predictions with experimental data gathered in rats and humans after drinking water and dietary ingestion of fluoride. This physiological model description of absorption, distribution, and elimination of fluoride from the body permits the analysis of the combined effect of ingesting and inhaling fluorides on the target organ, bone. Estimates of fluoride concentrations in bone are calculated and related to chronic fluoride toxicity. The model is thus useful for predicting some of the long-term metabolic features and tissue concentrations of fluoride that may be of value in understanding positive or negative effects of fluoride on human health. In addition, the pbpk model provides a basis for across-species extrapolation of the effective fluoride dose at the target tissue, bone, in the assessment of risk from different exposure conditions.

Physiologically based pharmacokinetics of methoxyacetic acid: dose-effect considerations in C57BL/6 mice.

Methoxyacetic acid (MAA), a weak acid with a pKa of 3.57, was used to test the broad hypothesis that distribution of weak acids in maternal and fetal tissues is determined principally by the pKa of the acid and the pH values of tissue and fluid compartments and to examine tissue dose-teratogenesis relationships, as well as administered dose-teratogenesis relationships. Five related experimental studies were conducted in pregnant C57BL/6CrIBR mice: a conventional dose-response study of developmental toxicity and transplacental pharmacokinetics in mice, a second dose-response study in which reproductive outcomes in litters from individual dams were related to individual pharmacokinetic behavior, a protein-binding experiment, an embryo tissue localization study, and determination of pH in maternal and embryonic compartments after exposure to MAA. MAA was administered intraperitoneally at 9:00 a.m. on day 10 of gestation, at doses ranging from 88 to 164 mg/kg. Localization within the forelimb bud of the embryo, an MAA target site, was determined by computerized image analysis of the distribution of radiolabeled MAA. The kinetic predictions of a physiologically based model incorporating tissue pH values and MAA pKa agreed well with observed concentrations at the lowest dose. However, at intermediate and higher doses, concentrations in both maternal and embryonic tissues were consistently underestimated. MAA was bound neither to maternal plasma proteins nor to embryonic proteins. Intermediate and higher doses of MAA caused dose-dependent transient depressions in tissue pH, but these were not of sufficient duration to bring predicted tissue concentrations into congruence with the concentrations observed. Distribution of MAA within the forelimb bud was broadly consistent with the pH hypothesis, but MAA concentration was not increased in the distal postaxial sector that is the site of the precursor cells of the missing digits. Internal exposure to MAA, defined as the area under the maternal plasma or embryo concentration curve (AUC), was not proportional to administered dose, but AUC-response relationships generated by the group and individual dose-response studies were comparable. While AUC may be a useful measure of effective MAA dose, it cannot be accurately predicted at teratogenic doses of this agent by the model as it is presently structured.

A physiologically based kinetic model of rat and mouse gestation: disposition of a weak acid.

A physiologically based toxicokinetic model of gestation in the rat and mouse has been developed. The model is superimposed on the normal growth curve for nonpregnant females. It describes the entire gestation period including the period of organogenesis. The model consists of uterus, mammary tissue, maternal fat, kidney, liver, other well-perfused maternal tissues, and other poorly perfused maternal tissues, embryo/fetal tissues, and yolk sac and chorioallantoic placentas. It takes into account the growth of maternal tissues such as uterus, mammary glands, fat, and liver during pregnancy, as well as growth of the conceptus. The gestation model is based on published values of organ volumes and blood flows for the rat throughout pregnancy. It is scaled to the mouse using conventional scaling procedures. Its descriptive utility has been examined with the test chemical 5,5'-dimethyloxazolidine-2,4-dione (DMO), a weak acid that is not bound measurably in plasma or tissues and is eliminated by excretion in the urine. Concentrations of DMO were monitored in maternal rat and embryo plasma and in homogenates of maternal rat muscle and whole embryo after ip administration at 9:00 AM on Day 13 of gestation. On the basis that distribution of DMO is determined solely by its pK and the pH's of body fluids, pH and excretion rate values were estimated by visual optimization of model predictions to the concentration profile. Successful prediction of concentrations of DMO in the same tissues of pregnant mice after its ip administration at 9:00 AM on Day 10 or 11 of gestation required only adjustment for pH's of mouse body fluids.

Consideration of the target organ toxicity of trichloroethylene in terms of metabolite toxicity and pharmacokinetics.

Trichloroethylene (TRI) is readily absorbed into the body through the lungs and gastrointestinal mucosa. Exposure to TRI can occur from contamination of air, water, and food; and this contamination may be sufficient to produce adverse effects in the exposed populations. Elimination of TRI involves two major processes: pulmonary excretion of unchanged TRI and relatively rapid hepatic biotransformation to urinary metabolites. The principal site of metabolism of TRI is the liver, but the lung and possibly other tissues also metabolize TRI, and dichlorovinyl-cysteine (DCVC) is formed in the kidney. Humans appear to metabolize TRI extensively. Both rats and mice also have a considerable capacity to metabolize TRI, and the maximal capacities of the rat versus the mouse appear to be more closely related to relative body surface areas than to body weights. Metabolism is almost linearly related to dose at lower doses, becoming dose dependent at higher doses, and is probably best described overall by Michaelis-Menten kinetics. Major end metabolites are trichloroethanol (TCE), trichloroethanol-glucuronide, and trichloroacetic acid (TCA). Metabolism also produces several possibly reactive intermediate metabolites, including chloral, TRI-epoxide, dichlorovinyl-cysteine (DCVC), dichloroacetyl chloride, dichloroacetic acid (DCA), and chloroform, which is further metabolized to phosgene that may covalently bind extensively to cellular lipids and proteins, and, to a much lesser degree, to DNA. The toxicities associated with TRI exposure are considered to reside in its reactive metabolites. The mutagenic and carcinogenic potential of TRI is also generally thought to be due to reactive intermediate biotransformation products rather than the parent molecule itself, although the biological mechanisms by which specific TRI metabolites exert their toxic activity observed in experimental animals and, in some cases, humans are not known. The binding intensity of TRI metabolites is greater in the liver than in the kidney. Comparative studies of biotransformation of TRI in rats and mice failed to detect any major species or strain differences in metabolism. Quantitative differences in metabolism across species probably result from differences in metabolic rate and enterohepatic recirculation of metabolites. Aging rats have less capacity for microsomal metabolism, as reflected by covalent binding of TRI, than either adult or young rats. This is likely to be the same in other species, including humans. The experimental evidence is consistent with the metabolic pathways for TRI being qualitatively similar in mice, rats, and humans. The formation of the major metabolites--TCE, TCE-glucuronide, and TCA--may be explained by the production of chloral as an intermediate after the initial oxidation of TRI to TRI-epoxide.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacokinetically based risk assessment of workplace exposure to benzene.

Cancer risk from exposure to benzene for a working lifetime was estimated from data obtained in studies with rodents. Cancers of the Zymbal gland and the blood-forming system were selected as endpoints for the assessment because of their consistent occurrence. The combined metabolites were judged from toxicological data to be the best representative of the reactive agent. Because of similarity in the percentages of lifetime exposed in the rodent studies and in the occupational setting, the amount metabolized/day as a result of exposures 5 days a week for a lifetime was judged to be an appropriate dose paradigm for this assessment. Derived Michaelis-Menton constants were used to convert the doses of combined metabolites from the pharmacokinetic studies to the doses used in the bioassays. Scaling across species was based on allometric relationships. Experimental data were used to scale doses across species with body weight ratios raised to the exponents of 0.74 for the inhalation route and 1.0 for the oral route. The occupational lifetime cancer risk estimated from rodent data was 6 to 14 cases/1000 workers, which is consistent with the 9.5 to 174 leukemia cases/1000 estimated by others from epidemiological data. Implications of these estimates and uncertainties associated with making them are discussed.

Dose paradigms for inhaled vapors of primary carcinogens and their impact on risk assessment.

In the assessment of risk, several factors affect predictions: selection of reactive agent, selection of tumor incidence data, modeling of dose, scaling across species, adjustment for differences in duration and frequency of exposure, and selection of the most suitable risk extrapolation model. If the endpoints, exposure regimen, and the model for risk extrapolation are constant, then the review of dose paradigms will illustrate the effect of dose modeling on risk, since by definition the reactive agent is the primary carcinogen. The response incidence in lifetime inhalation bioassays of two primary carcinogens, ethylene oxide and formaldehyde, was used with different dose paradigms to estimate risk from maximum lifetime occupational exposures. The dose paradigms that will be considered include: concentration, concentration time product, retained dose, integrated blood concentration, and tissue exposure. The basis for across-species scaling and the assumptions underlying each dose paradigm were discussed.

Risk assessment and oncodynamics of ethylene oxide as related to occupational exposure.

Two rat inhalation bioassays have been integrated into the risk assessment on the carcinogenicity of ethylene oxide (EO). The carcinogenic findings as well as relevant metabolism and pharmacokinetic data are reviewed. Brain tumors were selected as the endpoint for the assessment of risk because of the indication that adverse effects on the nervous system, related to EO exposure, were consistent across species. Two methods, time-exposure concentration product and area under the plasma concentration-time curve (AUC) are used as a basis for calculating effective dose. Scaling of the dose to man from both rat and dog is explored based on pharmacokinetic studies. Two different mathematical risk extrapolation models, the probit and the multi-stage, are used to estimate the cancer risk for daily exposures to EO of 1.8 microgram/liter over a working lifetime. The use of AUC as a basis for dose from a daily exposure of 1.8 microgram/liter over a working lifetime gives the higher risk rates (90-142/10,000 workers). The implication of the simulated dose using plasma concentrations versus the time-concentration product approach is discussed in relation to threshold effects.

Biological basis for extrapolation across mammalian species.

The rationale for extrapolation or "scaling" across species is founded in the commonality of anatomic characteristics and the universality of physiologic functions and biochemical reactions. The development of the allometric equation, Y = aWn, relating species body size (W) with various morphological, physiological, biochemical, pharmacological, and toxicological characteristics, as the fundamental basis for extrapolation of biological data from laboratory animals to man is outlined. The familiar methods of extrapolation on the basis of "milligrams per kilogram body weight" and "body surface area" are simply examples, W1.0 and W0.67, respectively, of this equation. The experimental observations used to support these two, and other extrapolation bases, are reviewed. Criteria for the selection of an appropriate base for transfer of specific biologic data from laboratory animals to man, and the expected reliability of the extrapolation, are discussed with the enunciation of four guiding principles. The application of these principles to the extrapolation to man of dose-tumor incidence data from carcinogenicity bioassays of laboratory animals is discussed. The components are identified, and illustrative examples are given.

Chronic toxicity and three-generation reproduction study of styrene monomer in the drinking water of rats.

Chronic toxicity and reproductive performance were evaluated in groups of rats receiving styrene monomer in their drinking water at nominal concentrations of 0, 125, or 250 ppm. Fifty male and 70 female rats in each test group and 76 males and 104 females in the control group were placed on a 2-year study and followed for observations of general health which included measurement of body weight, food and water consumption, hemograms, clinical chemistries, urinalysis, and histopathological examination. Ten males and 20 females from each group in the study were mated to produce F1 pups. These pups were subsequently mated to produce three generations of offspring, all maintained on styrene-treated drinking water. For each generation, the following were evaluated: fertility, litter size, pup viability, pup survival, sex ratio, pup body weight, weanling liver and kidney weight, and marrow cytogenetics. Except for a statistically significant reduction in water consumption for styrene-treated rats, no treatment-related changes, including mortality patterns, were reported for animals in the chronic study. The data evaluated for reproductive performance also showed no evidence of styrene-related changes. It was concluded that the administration of styrene in the drinking water of rats for 2 years produced no deleterious dose-related effects or decrements in reproductive performance.

Teratology and reproduction studies with triclopyr in the rat and rabbit.

Triclopyr (3,5,6-trichloro-2-pyridyloxyacetic acid), being developed as a new herbicide for use on brush and weeds, was evaluated for its potential effects on reproduction, and embryonal and fetal development. Pregnant Sprague-Dawley rats were given doses of 0, 50, 100, or 200 mg/kg/day by gavage on Days 6 through 15 of gestation. Dose-related signs of maternal toxicity were observed during the treatment period. No teratogenic effects were observed at any dose level, though slight fetotoxicity, possibly secondary to maternal toxicity, occurred at the high dose level (200 mg/kg/day). Pregnant New Zealand White rabbits were given doses of 0, 10, or 25 mg/kg/day by gavage on Days 6 through 18 of gestation which produced transient, dose-related decreases in maternal body weight gain. However, there were no indications of any treatment-related effects on fetal growth and development among rabbits. Male and female Sprague-Dawley rats maintained on diets supplying 0, 3, 10, or 30 mg/kg/day over three generations exhibited no consistent treatment-related effects on reproductive performance, pregnancy, parturition, or neonatal survival. These data indicated that triclopyr had little or no potential for teratogenic or reproductive toxicity even when the level of exposure approached that which elicited maternal toxicity.

Testing of selected workplace chemicals for teratogenic potential.

The reproductive toxicity and teratogenic potential of 19 industrial chemicals have been investigated during the past 3 a. Preliminary studies utilizing intraperitoneal treatments of rats on days 1-15 of gestation have been conducted on the following ten chemicals: allyl chloride, bisphenol A, copper naphthenate, ethylene dibromide, hexachlorobutadiene, 2-mercaptobenzothiazole, methyl styrene, naphthalene, 2-nitropropane, and 1,2,3-trichloropropane. Studies utilizing inhalation exposure of rats and rabbits on days 1-19 and 1-24, respectively, of gestation have been conducted on the following nine chemicals: butylene oxide, carbon disulfide, 2-ethoxyethanol, ethyl benzene, methyl bromide, nitrous oxide, styrene oxide, tetrachloroethylene, and trichloroethylene. In the preliminary studies, evidence of teratogenic potential was seen with allyl chloride and bisphenol A, and fetal toxicity was found in the absence of maternal toxicity with methyl styrene and 2-nitropropane. In the inhalation studies, 2-ethoxyethanol was strongly embryotoxic at the higher exposure levels employed and was teratogenic at the lower concentration.

Teratology study in mice subjected to inhalation of diethylhydroxylamine, nitroethane, and diethylamine hydrogen sulfite.

Pregnant female mice were exposed from Days 6 to 17 of pregnancy to 8.9 +/- 2.0 ppm of diethylhydroxylamine and 14.3 +/- 2.0 ppm of nitroethane. Exposures were for 8.25 +/- 2.25 hours/day including weekends. The mice were also exposed continuously for 24 hours a day to the vapor of diethylamine hydrogen sulfite. The exposure produced no effect on the dams compared to the control group. There was no evidence of compound-induced terata, variation in sex ratio, embryotoxicity, or inhibition of fetal growth and development.

The effect of massive transplacental iron loading.

Intravenous injection of three doses of 50 mg Fe/kg (total dose 150 mg Fe/kg) as iron dextran into rabbits late in pregnancy (days 26, 28, and 30 of gestation) reduced the weight gain of the dams and increased fetal mortality. Three doses of 20 mg Fe/kg also increased fetal mortality, while three doses of 5 mg Fe/kg were without effect. Liver and kidney iron concentrations of the dams and offspring were markedly increased at the time of parturition by treatment with a total dose of 150 mg Fe/kg. At 6 weeks after birth the liver and kidney iron concentrations of offspring from treated dams were comparable to those from control dams. The liver and kidney iron concentrations of the treated dams were significantly reduced from the levels found immediately post-partum. In the rat, four i.v. doses of 200 mg Fe/kg as iron dextran on days 17, 18, 19, and 20 of gestation (total dose 800 mg Fe/kg) produced tremors, reduced body weight gain, and reduced food consumption in the dams. The growth and survival of the offspring were adversely influenced by these effects on the dam. The liver iron concentration in offspring of rats treated with 800 and 400 mg Fe/kg was increased at parturition, but had returned to normal at 4 weeks of age. No iron-induced pathology was evident in the offspring of either rabbits or rats after 14 and 18 weeks, respectively.