Absence of developmental toxicity in a canine model after infusion of a hemoglobin-based oxygen carrier: Implications for risk assessment.

Bovine-derived hemoglobin-based oxygen carriers (HBOCs) have been investigated for use in humans (HBOC-201) and approved for veterinary medicine (HBOC-301). We infused pregnant beagles with HBOC-201 to test whether HBOC-induced developmental toxicity previously observed in rats would occur in a species devoid of an inverted visceral yolk sac (invVYS). Phase 1 assessed developmental toxicity of 6g/kg HBOC-201 on gestational day (GD) 21. Phase 2 investigated single infusions of 6g/kg HBOC-201 on one of GDs 21, 25, 29 or 33. Phase 3 studied multiple sequential infusions on GDs 21, 23,25,27,29, 31, and 33 at 0.52g/kg/day (3.6g/kg total dose). Mild to moderate maternal toxicity occurred in all phases. There was an unequivocal absence of developmental toxicity in all phases. Overall, our hypothesis that HBOC, which interferes with the function of the invVYS, would not affect the offspring in dogs was supported. The implications relative to human risk are discussed.

Developmental toxicity in rats of a hemoglobin-based oxygen carrier results from impeded function of the inverted visceral yolk sac.

HBOC-201 is a bovine-derived, cross-linked, and stabilized hemoglobin (250kDa) in physiological saline. Daily intravenous infusions of HBOC (1.95, 3.90, or 5.85g/kg/day) during gestational days (GDs) 6-18 in Sprague-Dawley rats caused fetal mortality, reduced birth weight, and malformations. Subsequent single-day infusions (5.85g/kg/day) showed that developmental toxicity was limited to GDs 7-9 when histiotrophic nutrition via the inverted visceral yolk sac (invVYS) is essential. Histiotrophic nutrition is receptor-mediated endocytosis of bulk maternal proteins and subsequent lysosomal degradation providing amino acids and other nutrients for embryonic growth. Controls for protein content, oncotic properties, and hemoglobin content indicated that toxicity was due to hemoglobin. Rat whole embryo cultures verified HBOC interference with invVYS transport capacity and resultant deficient embryonic nutrition. These mechanisms of action are not expected to impact human development based on differences in VYS morphology and function, although a complete understanding of early human embryonic nutrition is lacking.

The case for integrating low dose, beneficial responses into US EPA risk assessments.

When conducting risk assessments, the US Environmental Protection Agency (EPA) does not currently consider the beneficial effects from exposure to concentrations of agents below the no observed adverse effect level (NOAEL). If such benefits were observed, and if the beneficial and toxicological mechanisms of action were identical, this would probably be represented as a 'j-shaped' hormetic dose-response curve. If such data are available, they should be considered when assigning uncertainty factors for safe exposure calculations. However, when such data are not readily available, as is likely the case when the mechanism of action of the benefit differs from that of toxicity, current US EPA methods appear adequate.

Teratogen update: inorganic arsenic.

BACKGROUND: Inorganic arsenic has been used by many laboratories to study the pathogenesis of exencephaly in rodents. These studies, which used predominantly injection exposures, coupled with the paucity of epidemiology data, resulted in the erroneous inference that inorganic arsenic should be considered a human teratogen. METHODS: This study assembles and assesses literature analyses of older human and animal investigations together with the results of new experimental studies. These recent studies were performed according to modern regulatory guidelines, and relevant exposure routes (inhalation and ingestion) were used to evaluate the potential risk of developmental effects in humans. RESULTS: The existing epidemiological data are inadequate to support risk assessment because of the failure to confirm or measure arsenic exposure during early gestation and the deficiencies in accounting for potential confounding factors. The animal data revealed that inorganic arsenic caused malformations in offspring only when it was injected into the veins or peritoneal cavity of pregnant animals during early gestation. Exposure via inhalation or oral ingestion, even at concentrations that were nearly fatal to pregnant females, caused no arsenic-related malformations. CONCLUSIONS: Inorganic arsenic poses virtually no danger to developing offspring when maternal exposure occurs by relevant routes (oral and inhalation) at concentrations that are likely to be experienced in the environment or in the workplace.

Anatomical and physiological parameters affecting gastrointestinal absorption in humans and rats.

Anatomical and physiological parameters of the gastrointestinal (GI) tract dramatically affect the rate and extent of absorption of ingested compounds. These parameters must be considered by nutritionists, pharmacologists and toxicologists when describing or modeling absorption. Likewise, interspecies extrapolation (e.g. from rat to human) requires species-to-species comparison of these parameters. The present paper (1) describes the alimentary canal and the barrier to absorption; (2) relates the major sites of absorption; (3) compares the dimensions and surface areas of human and rat intestinal tracts; (4) discusses motility of the gut and transit times through regions of the alimentary canal; (5) explains how luminal contents are altered by physical, chemical and metabolic processes; and (6) describes the flow of blood and lymph from the GI tract to the systemic circulation, including the enterohepatic circulation. Despite strong morphological similarities between humans and rats at the microscopic level, gross anatomical differences in the relative absorptive surface areas provide a basis for concluding that the human GI tract is capable of absorbing materials faster and to a greater extent than that of the rat. Differences in the environment of the GI lumen of the two species make it possible to infer which substances are more likely to be present in a dissolved/non-ionized state for each species. Taken together, these differences may be of sufficient magnitude to alter the assessment of risks/benefits for a given compound when those risks/benefits are based on interspecies extrapolations.

Assessment of the carcinogenicity associated with oral exposures to hydrogen peroxide.

Concern regarding hydrogen peroxide (H(2)O(2)) carcinogenicity arises from its ability to act as a strong oxidizing agent. In short-term genotoxicity tests, H(2)O(2) has given predominantly positive results; however, these assays have been performed using either bacterial strains engineered to be exquisitely sensitive to oxidant damage, or mammalian cells deficient in antioxidant enzymes. Significantly, the addition of antioxidant protective measures (normally present in vivo) to these assay systems protects against H(2)O(2) genotoxicity. In most whole animal studies, H(2)O(2) exposure neither initiates nor promotes tumors. In mice, however, 0.4% H(2)O(2) in drinking water was reported to induce hyperplastic lesions of the duodenum and to erode areas in the glandular stomach epithelium. Owing to the chemistry of dilute H(2)O(2) solutions and the anatomy/physiology of the gastrointestinal tract, it is unlikely that orally ingested H(2)O(2) reaches the duodenum. Instead, greatly decreased water consumption and the resultant abrasion of the luminal lining on ingestion of pelleted dry rodent chow is the most likely cause of the observed gastric and duodenal lesions following H(2)O(2) administration in drinking water. Significantly, when hamsters received high doses of H(2)O(2) by gastric intubation (and water intake was not affected), the gastric and duodenal epithelia appeared normal. In-depth analysis of the available data supports the conclusion that oral ingestion of H(2)O(2) should not be considered a carcinogenic threat.

Hydroxylamine moiety of developmental toxicants is associated with early cell death: a structure-activity analysis.

BACKGROUND: Cellular debris, an indicator of cell death, appears in limb buds of gestational day 12 rabbit embryos 4 hr after either a subcutaneous injection of hydroxyurea to pregnant rabbits or an injection of hydroxyurea into the exocoelomic cavities of the embryos. This episode of early cell death appears to be central to the teratogenic action of hydroxyurea. Several chemicals that are structurally related to hydroxyurea, and that possess a terminal hydroxylamine moiety (-NHOH), also produce limb abnormalities. METHODS: To investigate whether the hydroxylamine moiety is responsible for early cell death and, therefore, is likely to be associated with teratogenesis, five structurally related hydroxylamine-bearing chemicals (hydroxylamine hydrochloride, N-methylhydroxylamine hydrochloride, hydroxyurea, acetohydroxamic acid, and hydroxyurethane) were administered at equimolar doses to rabbits either by subcutaneous (8.55 mmol/kg) or intracoelomic (2.66 micromol/embryo) injection on gestational day 12. Five additional chemicals, structurally similar to the hydroxylamine-bearing compounds, but possessing a terminal amino group (-NH(2)) (ammonium hydroxide, methylamine, urea, acetamide, and urethane), were tested at equimolar or higher doses by an identical protocol. In a subsequent experiment, the antioxidant propyl gallate (3.0 mmol/kg or 1.30 micromol/embryo) was co-administered with the hydroxylamine-bearing compounds to determine its effect on early cell death. Embryos were harvested 4 or 8 hr after treatment and analyzed by light microscopy. RESULTS: Cellular debris was obvious in forelimb buds from embryos treated with the hydroxylamine-bearing compounds; however, none of the amino compounds produced an early episode of embryonic cell death. In all cases, the antioxidant propyl gallate prevented or delayed the early episode of cell death observed after treatment with the hydroxylamine-bearing compounds. CONCLUSIONS: These results are consistent with the concept that the rapidly occurring embryonic cytotoxicity induced by hydroxylamine-bearing compounds involves a free radical mechanism that requires the presence of a terminal hydroxylamine group for initiation.

Appropriate use of animal models in the assessment of risk during prenatal development: an illustration using inorganic arsenic.

BACKGROUND: Assessing risks to human development from chemical exposure typically requires integrating findings from laboratory animal and human studies. METHODS: Using a case study approach, we present a program designed to assess the risk of the occurrence of malformations from inorganic arsenic exposure. We discuss how epidemiological data should be evaluated for quality and criteria for determining whether an association is causal. In this case study, adequate epidemiological data were not available for evaluating the potential effect of arsenic on development. Consequently, results from appropriately designed, conducted, and interpreted developmental toxicity studies, which have been shown to be predictive of human risk under numerous scenarios, were used. In our case study, the existing animal data were not designed appropriately to assess risk from environmental exposures, although such studies may be useful for hazard identification. Because the human and animal databases were deficient, a research program comprising modern guideline toxicological studies was designed and conducted. RESULTS: The results of those studies in rats, mice, and rabbits indicate that oral and inhalational exposures to inorganic arsenic do not cause structural malformations, and inhalational exposures produced no developmental effects at all. The new study results are discussed in conjunction with considerations of metabolism, toxicokinetics, and maternal toxicity. CONCLUSIONS: Based on the available experimental data, and absent contrary findings from adequately conducted epidemiological studies, we conclude that exposure to inorganic arsenic by environmentally relevant routes poses no risk of the occurrence of malformations and little risk of other prenatal developmental toxicity in developing humans without concomitant and near-lethal toxicological effects in mothers.

Workshop to identify critical windows of exposure for children's health: cardiovascular and endocrine work group summary.

The work group on cardiovascular and endocrine effects was asked to review the current state of knowledge about children's windows of vulnerability to developmental toxicants and to recommend how that information may be used to improve risk assessment and public health. We considered differences between structural defects, where periods of vulnerability are rather well defined, and functional defects, where periods of vulnerability are quite elusive.

Apparent lability of neural tube closure in laboratory animals and humans.

Neural tube defects (NTDs), a set of structural abnormalities affecting the brain, spinal cord, and the skeletal and connective tissues that protect them, are common malformations among humans and laboratory animals. The embryogenesis of the neural tube is presented to convey the complexity of the phenomenon, the multiplicity of requisite cellular and subcellular processes, and the precise timing of events that must occur for successful neural tube development. Interruption, even transitory, of any of these intricate processes or disruption of an embryo's developmental schedule can lead to an NTD. The population distribution of human NTDs demonstrates that genetic predisposition functions in susceptibility to NTDs. Data from animal studies support these concepts. NTDs are common outcomes in developmental toxicity safety assessments, occurring among control and treated groups. Numerous agents have caused increased levels of NTDs in laboratory animals, and species with shorter gestational periods appear more prone to toxicant-induced NTDs than those with longer gestations. Data from post-implantation whole embryo culture, although not predictive of human risk, are useful in studying neurulation mechanisms and in demonstrating the importance of maintaining embryonic schedules of development. We conclude that the concept that NTDs are produced by only a few toxicants that selectively target the developing nervous system is untenable. Rather, the combination of the time in gestation that an agent is applied, its dose, and its ability to disrupt critical processes in neurulation leads to NTDs. We further conclude that, because of both the relatively high prevalence and the multifactorial nature of NTDs, the mere occurrence of an NTD is insufficient for inferring that the defect was caused by an exogenous agent.

An assessment of the developmental toxicity of inorganic arsenic.

A critical analysis of the literature base regarding the reproductive and developmental toxicity of arsenic compounds, with emphasis on inorganic arsenicals, was conducted. The analysis was stimulated by the great number of papers that have purported to have shown an association between exposure of pregnant laboratory animals to arsenic compounds and the occurrence of offspring with cranial neural tube defects, particularly exencephaly. For the most part, the literature reports of arsenic developmental toxicity in experimental animals are inadequate for human risk assessment purposes. Despite the shortcomings of the experimental database, several conclusions are readily apparent when the animal studies are viewed collectively. First, cranial neural tube defects are induced in rodents only when arsenic exposure has occurred early in gestation (on Days 7 [hamster, mouse], 8 [mouse], or 9 [rat]). Second, arsenic exposures that cause cranial neural tube defects are single doses that are so high as to be lethal (or nearly so) to the pregnant animal. Third, the effective routes of exposure are by injection directly into the venous system or the peritoneal cavity; even massive oral exposures do not cause increases in the incidence of total gross malformations. Fourth, repetition of similar study designs employing exaggerated parenteral doses is the source of the large number of papers reporting neural tube defects associated with prenatal arsenic exposure. Fifth, in five repeated dose studies carried out following EPA Guidelines for assessing developmental toxicity, arsenic was not teratogenic in rats (AsIII, 101 micromol/kg/d, oral gavage; 101 micromol/m3, inhalation), mice (AsV, 338 micromol/kg/d, oral gavage; est. 402 micromol/kg/d, diet), or rabbits (AsV, 21 micromol/kg/d, oral gavage). Data regarding arsenic exposure and adverse outcomes of pregnancy in humans are limited to several ecologic epidemiology studies of drinking water, airborne dusts, and smelter environs. These studies failed to (1) obtain accurate measurements of maternal exposure during the critical period of organogenesis and (2) control for recognized confounders. The lone study that examined maternal arsenic exposure during pregnancy and the presence of neural tube defects in progeny failed to confirm a relationship between the two. It is concluded that under environmentally relevant exposure scenarios (e.g., 100 ppm in soil), inorganic arsenic is unlikely to pose a risk to pregnant women and their offspring.

Protective effect of liposome encapsulation on paclitaxel developmental toxicity in the rat.

Paclitaxel is an anticancer drug that has demonstrated severe embryotoxicity in chicks. This embryotoxicity is reduced by liposome encapsulation of the drug. The current study was designed to evaluate the potential of liposome encapsulation for reducing paclitaxel embryotoxicity in rats. Wistar rats were treated with paclitaxel on day 8 of pregnancy (plug = day 0) at doses of 0.67, 2.0, or 10.0 mg/kg intravenously. The same doses of paclitaxel encapsulated in liposomes were administered intravenously to other groups of animals. Control animals were given blank liposomes. Free paclitaxel produced maternal and embryotoxicity at 10.0 mg/kg with three of seven dams dying and resorption of all embryos in surviving dams. Liposome encapsulation at 10.0 mg/kg was not associated with maternal death and there were live fetuses on evaluation at term, although litter size was reduced and malformations occurred in surviving fetuses. At 2.0 mg/kg free paclitaxel, fetal weight was decreased and resorptions increased. Liposome encapsulation at 2.0 mg/kg produced litter results similar to those obtained in control animals given empty liposomes. Malformations were prominent at 2.0 mg/kg free paclitaxel and at 10.0 mg/kg paclitaxel in liposomes and included exencephaly/anencephaly, ventral wall defects, facial clefts, anophthalmia, diaphragmatic hernia, and defects of the kidney, cardiovascular system, and tail. Liposome encapsulation appeared to shift the developmental response to paclitaxel such that 10 mg/kg encapsulated drug produced effects similar to 2.0 mg/kg free drug. These results may have implications for drug delivery of therapeutic agents used during human pregnancy.

Embryotoxicity of free and liposome-encapsulated taxol in the chick.

Taxol, an inhibitor of microtubule disaggregation, is used in the therapy of breast, ovarian, and other human malignancies. The toxicity of taxol administration is due in part to the polyoxyethylated castor oil (Cremaphor) vehicle in which it is administered; taxol embryotoxicity appears also to be partially attributable to vehicle toxicity. Liposome encapsulation is a novel vehicle for drug administration. The administration of taxol encapsulated in liposomes was evaluated in the chick embryo. Albumen injections of taxol doses up to 30 micrograms/egg were used to characterize dose-response curves for free and liposome-encapsulated taxol, compared to liposome-only and saline-injected control eggs. Sixty percent embryotoxicity (death or malformation) occurred with taxol doses of 1.5 micrograms/egg. A 20-fold higher dose was necessary to produce the same degree of toxicity with liposome-encapsulated taxol. Curve-fitting equations were used to estimate median effective doses (ED50s) and slope functions of the dose response curves. The ED50 for taxol was more than an order of magnitude lower than that for liposome-encapsulated taxol. Estimated slope functions for the two dosage forms of taxol were the same, suggesting similar mechanisms of toxicity. The toxicity of liposomes alone was low.

D-mannitol, a specific hydroxyl free radical scavenger, reduces the developmental toxicity of hydroxyurea in rabbits.

Hydroxyurea (HU) is a potent mammalian teratogen. Within 2-4 hours after maternal injection, HU causes 1) a rapid episode of embryonic cell death and 2) profound inhibition of embryonic DNA synthesis. A variety of antioxidants delays the onset of embryonic cell death and reduces the incidence of birth defects. Antioxidants do not block the inhibition of DNA synthesis, indicating that early embryonic cell death is not caused by inhibited DNA synthesis. We have suggested that some HU molecules may react within the embryo to produce H2O2 and subsequent free radicals, including the very reactive hydroxyl free radical. The free radicals could cause the early cell death; antioxidants are believed to terminate the aberrant free radical reactions resulting in lessened developmental toxicity. To investigate whether hydroxyl free radicals cause the early episode of cell death, pregnant New Zealand white rabbits were injected subcutaneously on gestational day 12 with a teratogenic dose of HU (650 mg/kg) in the presence or absence of 550 mg/kg of D-mannitol (Man), a specific scavenger of hydroxyl free radicals. Osmotic control rabbits received HU plus 550 mg/kg of xylose (Xyl, a nonactive aldose). At term, the teratologic effects of HU were ameliorated by Man as evidenced by decreased incidences of the expected limb malformations. Xyl exerted no demonstrable effect on HU teratogenesis. Histological examination of limb buds at 3-8 hours after maternal injection, showed that Man delayed the onset of HU-induced cell death by as much as 4 hours. Xyl had no effect. That Man acts within the embryo was shown by performing intracoelomic injections on alternate implantation sites with Man, Xyl, or saline followed by subcutaneous injection of the pregnant doe with HU. Embryos were harvested 3-8 hours later. Limb buds from saline- and Xyl-injected embryos exhibited the typical pattern of widespread HU-induced cell death at 3-4 hours, whereas Man-injected embryos did not exhibit cell death until 5-8 hours. These results are consistent with those reported for antioxidant-mediated amelioration of HU-induced developmental toxicity and with the hypothesis that hydroxyl free radicals are the proximate reactive species in HU-induced early embryonic cell death.

Taxol and embryonic development in the chick.

Taxol, an inhibitor of microtubule disassembly, is currently under investigation in the therapy of several human cancers. The current investigation was undertaken to characterize potential taxol developmental toxicity in chicks. On one of days 1-4 of incubation, taxol was administered in dimethylsulfoxide (DMSO) or olive oil in a range of doses, the highest of which produced a high incidence of early embryo death. Production of gross structural malformations was sporadic and occurred in vehicle-treated as well as taxol-treated embryos. A more common manifestation of taxol toxicity was a syndrome of visceral abnormalities, including regression of the vitelline circulation, dilatation of the atria, and hemorrhage in the left side of the head and thorax, often with decreased eye pigmentation. Regardless of the day of treatment, this syndrome occurred at 4.5-5 days. To investigate the possibility that taxol induced its effect through disruption of angiogenesis in the vitelline circulation, filters soaked in taxol were applied to the margin of the germ disc. No inhibition of vessel development was demonstrated. We conclude that taxol decreases the viability of embryos and that this impairment of survival precludes the development of birth defects. Solvent toxicity is an important confounder in the investigation of taxol embryotoxicity.

Have animal data been used inappropriately to estimate risks to humans from environmental trichloroethylene?

Trichloroethylene (TCE) is widely viewed as an environmental hazard. Its major metabolite, chloral hydrate, is a currently used medicine. Regulation of TCE is based on a linear extrapolation from effects of high doses in rodents to risks for humans at low doses. However, metabolic, toxicologic, and epidemiologic data on trichloroethylene and chloral hydrate as well as water chlorination studies call this approach into question. The mechanism of carcinogenesis of TCE and chloral hydrate in rodents is nonlinear: very high doses, sufficient to cause cellular necrosis, are necessary. Malignancy arises from repeated cycles of necrosis and regeneration with the ultimate emergence of hyperplasia and then neoplasia. Metabolites of TCE, trichloroacetic acid and dichloroacetic acid, mediate this toxic effect of TCE. These chloroacetic acids also induce similar lesions in rodents given high doses of the medicine, chloral hydrate. Human epidemiologic data show no increase in mortality or malignancy from substantial chronic exposure to trichloroethylene. Chlorination of drinking water produces much higher levels of chloroacetic acids than could be obtained from metabolizing TCE under current regulations. We conclude that the assumptions underlying current regulations are not applicable to TCE. Instead of a straight-line extrapolation model, a threshold model may be more appropriate. The data suggest that it is possible to increase substantially the allowable trichloroethylene in drinking water without increasing health hazards.

The relevance to humans of animal models for inhalation studies of cancer in the nose and upper airways.

While nasal cancer is relatively rare among the general population, workers in the nickel refining, leather manufacturing, and furniture building industries exhibit increased incidences of nasal cancer. To investigate the causes of nasal cancer and to design ameliorative strategies, an appropriate animal model for the human upper respiratory regions is required. The present report describes, compares, and assesses the anatomy and physiology of the nasal passages and upper airways of humans, rats, and monkeys for the purpose of determining a relevant animal model in which to investigate potential causes of nasal cancer. Based on the mode of breathing, overall geometry of the nasal passages, relative nasal surface areas, proportions of nasal surfaces lined by various epithelia, mucociliary clearance patterns, and inspiratory airflow routes, the rat, which is very different from humans, is a poor model. In contrast, the monkey exhibits many similarities to humans. Although the monkey does differ from humans in that it exhibits a more rapid respiratory rate, smaller minute and tidal volumes, larger medial turbinate, and a vestibular wing that creates an anterior vortex during inspiration, it offers a more appropriate model for studying the toxic effects of inhaled substances on the nasal passages and extrapolating the findings to humans.

Methotrexate-induced developmental toxicity in rabbits is ameliorated by 1-(p-tosyl)-3,4,4-trimethylimidazolidine, a functional analog for tetrahydrofolate-mediated one-carbon transfer.

Dihydrofolate reductase reduces folic acid to tetrahydrofolate as a prerequisite to one-carbon metabolism, which is required for normal embryonic de novo DNA synthesis. The developmental toxicity of methotrexate (MTX) has been attributed to MTX's ability to inhibit the activity of dihydrofolate reductase and thereby indirectly suppress one-carbon metabolism. The compound 1-(p-tosyl)-3,4,4-trimethylimidazolidine (TTI), which is structurally unrelated to folate, reestablishes one-carbon metabolism by the biomimetic transfer of single carbon units. Whether the developmental toxicity of MTX is indeed caused via suppressed one-carbon metabolism was tested in New Zealand white rabbits following concurrent maternal treatment with MTX and TTI. TTI reduced MTX developmental toxicity judged by increased mean fetal body weights, decreased percentage of malformed fetuses, and reduced incidences of major malformations. Two doses of TTI (90 mg/kg, each) at 1 hr prior to and 1 hr after MTX also reduced the developmental toxicity, but was no more effective than the single-injection regimen. Treatment with TTI alone caused no developmental toxicity. Histologically, MTX caused enlarged intercellular spaces in limb bud mesenchyme that began at 6-8 hr and increased in size until 16 hr. Mesenchymal nuclei appeared basophilic, with angular contours. Pretreatment with TTI delayed MTX-induced histological changes until 20-24 hr after MTX in 36-50% of embryos and completely protected the remainder. The sequence of MTX-induced changes was not altered among affected embryos, although the severity of the lesions did not appear as great. Saline-only or TTI-only treatments caused no alterations in limb buds. These data are consistent with the concept that impaired one-carbon metabolism is indeed the fundamental process underlying MTX developmental toxicity.