A critical look at adjusted fetal weights in rats.

BACKGROUND: A new derived (i.e., calculated) endpoint of developmental toxicology has appeared in a very few studies since 1990. This endpoint is adjusted mean live fetal weight per litter or adjusted fetal weight. Given our lack of familiarity with the endpoint, we evaluated the basis, prevalence, methods, and usefulness in embryo-fetal developmental toxicity (EFDT) studies in rats. METHODS: Literature searches were performed with key terms using PubMed and Google Scholar. Major textbooks were consulted but lack of any mention of the endpoint. Unpublished EFDT data, which are readily available online, were utilized to test adjustment methods. RESULTS: Pertinent information on factors that influence fetal weight goes back a century. Four papers utilizing rats were found in which fetal weights were adjusted using either statistical or formula-based methods to adjust fetal weights. Only one study showed a clear benefit to the endpoint when there was a marked decrease in live litter size; this pointed to situations in which the new endpoint might be useful. The lone formula-based adjustment method was found to be lacking adequate testing and justifications. A new experimental alternative formula-based adjustment is shown to produce results very similar to statistical methods. CONCLUSIONS: From this assessment, we recommend that adjusted fetal weight should not be a routine endpoint at this time. However, there are likely cases where this derived endpoint could aid interpretation. We encourage other investigators to examine previous EFDT study data to establish guidance on the use of adjusted mean live fetal weights.

Revisiting the testicular toxicity of cyanide: Assessment of the new and existing literature.

BACKGROUND: Based on new testing, we re-assess U.S. EPA and California OEHHA conclusions regarding male reproductive toxicity associated with cyanide exposure. METHODS: Literature identified by ATSDR, ECETOC and EPA was complemented by studies conducted after 2006. Relevant studies were scored for quality using ToxRTool. RESULTS: Eleven pertinent animal investigations were identified; five with quality scores of 1 were evaluated in-depth. The NTP 13-week drinking water study of NaCN in rats reported significantly decreased water intakes and reduced cauda epididymal weights; altered sperm parameters occurred in high-dose rats. When compared to contemporaneous historical control data (HCD), the mean cauda epididymal weights of cyanide-treated rats in the NTP study were within HCD, whereas control weights exceeded HCD. A new 13-week drinking water study used the same design with additional features (individually caged rats, "paired water" controls, thyroid hormone determinations, post-treatment recovery) and found a smaller decrease in water consumption (11% versus 18% at 300 ppm) and no treatment-related changes in male reproductive measures. Although thyroid/parathyroid weights were increased at 300 ppm, histopathology and thyroid hormone levels were unaffected. The remaining high-quality cyanide studies reported no adverse findings in male reproductive organs. Unconfounded sperm measures were not adversely affected in any quality 1 studies. CONCLUSIONS: Changes in the male reproductive system reported after cyanide exposure in the NTP study were not reproducible, unlikely to be treatment-related, and should not be used as the sole basis for human health assessments.

Comparative anatomy, pre- and postnatal changes during the development and maturation of the small intestine: Life-stage influences on exposure.

The gastrointestinal (GI) system absorbs nutrients and xenobiotics, excretes waste, and performs immunologic and endocrine functions. The subdivisions of the mature gut and the complexity of their corrugated, absorptive luminal surfaces differ greatly among mammals. Regardless, the embryonic gut tube in all mammalian species arises when cephalocaudal folding incorporates the roof of the yolk sac into the embryo. The gut tube quickly lengthens and bulges into the umbilical cord. Upon reentry into the abdominal cavity, the gut tube begins to differentiate-a process that continues until well into the lactation period. Differentiation of the small intestine involves (1) increasing the absorptive surface area of the lumen; (2) establishing mechanisms to control the pH of luminal contents; (3) forming a hierarchical vascular system for distribution of absorbed nutrients; (4) developing a complex enteric nervous system to control motility; (5) providing a system for replenishment of cells; and (6) contributing to the immunity of the organism. Because the length of gestation varies among species typically used in safety tests and is much shorter than human gestation, the state of GI maturation at the time of parturition differs significantly. Differences in GI maturation can contribute to species differences in the rate and extent of absorption; these differences must be considered when designing and interpreting pharmacological/toxicological studies and extrapolating safety test results to humans.

Systematic assessment of quaternary ammonium compounds for the potential to elicit developmental and reproductive effects.

INTRODUCTION: Quaternary ammonium compounds (QUATs) are commonly found in cleaning products, disinfectants, hand sanitizers, and personal care products. They have been used for >50 years and are considered safe when used according to directions. Recent papers report reduced fertility and neural tube defects in rodents after low-level exposures. To determine if QUATs interfere with mammalian reproduction and development, we conducted a methodical assessment of all available data. METHODS: A systematic literature search identified 789 potential articles. Review of titles and abstracts found eight relevant studies, including two dissertation chapters; to these, 10 unpublished, guideline-compliant developmental and reproductive toxicity (DART) studies of QUATs (alkyldimethylbenzylammonium chloride [ADBAC] and dialkyldimethylammonium chloride [DDAC]) were added. ToxRTool was utilized to evaluate all 18 studies for data quality. RESULTS: Six studies were scored as "reliable without restriction"; four studies were considered "reliable with restriction" (mainly due to small rabbit group sizes). No test article-related, adverse DART endpoints were reported in these studies. ToxRTool scored the remaining eight studies as "not reliable." The unreliable studies failed to fully describe methods and/or endpoints, did not quantify (and in some cases, did not verify) exposures, utilized non-standard test methods, reported endpoints incorrectly, and assessed endpoints at inappropriate times. Some (not all) unreliable studies reported adverse effects after 7.5 mg QUATs/kg/day (mice), but these results were inconsistent. The reliable studies tested exposures ≥100 mg/kg/day (rats) with no effects. CONCLUSIONS: The available weight of evidence indicates no adverse DART effects after QUATs exposures at anticipated concentrations and normal use.

Teratogen update: Topical use and third-generation retinoids.

The first pharmaceutical retinoids approved by the U.S. Food and Drug Administration were given black-box warnings against their use in pregnancy due to potential teratogenic effects. These first- and second-generation retinoids were initially formulated for oral dosing and are structurally very similar to vitamin A, which has beneficial effects on skin as well as plays a vital role in driving healthy embryogenesis. Some of these early retinoids have since been reformulated for topical application, which has resulted in their diminished potential for systemic exposures. Additionally, rational drug design has been applied to create today's third-generation retinoids (adapalene, tazarotene, and bexarotene). These compounds include aromatic rings within their molecular cores to provide structural rigidity that contrasts with the flexible aliphatic backbone of vitamin A and the earlier generations of retinoids, and thus limits their off-target activity. As a result of these design features, the teratogenic potential in animals of the third-generation retinoids and those reformulated for topical use is generally less than seen with oral administration of earlier generations of retinoids. The available, but limited, epidemiologic data further show little-to-no teratogenic potential associated with real-life use of these compounds in humans. Given the paucity of epidemiologic data available at this time, however, it is recommended that the use of topical retinoids during pregnancy be avoided. However, in circumstances when inadvertent exposure in pregnancy may occur, the available data provide some reassurance that adverse pregnancy outcomes are unlikely.

Of embryos and tumors: Cyclopia and the relevance of mechanistic teratology.

Embryos and tumors share several characteristics, but embryos differ from tumors in their coordination of cellular- and tissue-level processes, including organized differentiation, remodeling of tissues through apoptosis, and disciplined migrations of cells. Embryonic cellular events are kept on track through orderly cell-cell communication via signal transduction pathways. If the pathways are disrupted, development is perturbed, and malformation may result. Despite profound differences between embryos and tumors, the study of one has benefited our understanding of the other. Using cyclopia as an example, the history of humans' beliefs concerning and reactions to this horrific malformation are explored. During the latter half of the 20th century, interest in cyclopic sheep from high pastures in western Idaho led to the discovery that cyclopia occurred after pregnant ewes foraged in fields containing corn lily (Veratrum californicum). Eventually, the proximate teratogen was identified as cyclopamine (a steroidal alkaloid). The teratogenic mechanism was identified as inhibition of the sonic hedgehog (Shh) signal transduction pathway. Alert cancer researchers noted that a prominent form of medulloblastoma (a devasting childhood brain tumor) overexpressed Shh. Cyclopamine effectively inhibited the tumor in mice and killed human medulloblastoma cells in vitro. Thus, over a 60-year period, a molecule causing hideous malformations and much emotional pain was discovered and then found capable of restraining a destructive tumor, potentially saving children's lives and sparing emotional devastation of their families. The success of identifying cyclopamine as a cause of cyclopia and a potential cure for medulloblastoma emerged from mechanistic research shared by multiple disciplines.

Trichloroethylene in drinking water throughout gestation did not produce congenital heart defects in Sprague Dawley rats.

BACKGROUND: Trichloroethylene (TCE) was negative for developmental toxicity after inhalation and oral gavage exposure of pregnant rats but fetal cardiac defects were reported following drinking water exposure throughout gestation. Because of the deficiencies in this latter study, we performed another drinking water study to evaluate whether TCE causes heart defects. METHODS: Groups of 25 mated Sprague Dawley rats consumed water containing 0, 0.25, 1.5, 500, or 1,000 ppm TCE from gestational day 1-21. TCE concentrations were measured at daily formulation, when placed into water bottles each day and when water bottles were removed from cages. Four additional mated rats per group were used for plasma measurements. At termination, fetal hearts were carefully dissected fresh and examined. RESULTS: All TCE concentrations were >90% of target when initially placed in water bottles and when bottles were placed on cages. All dams survived with no clinical signs. Rats in the two higher dose groups consumed less water/day than other groups but showed no changes in maternal or fetal weights. The only fetal cardiac observation was small (<1 mm) membranous ventricular septal defect occurring in all treated and water control groups; incidences were within the range of published findings for naive animals. TCE was not detected in maternal blood, but systemic exposure was confirmed by detecting its primary oxidative metabolite, trichloroacetic acid, although only at levels above the quantitation limit in the two higher dose groups. CONCLUSIONS: Ingesting TCE in drinking water ≤1,000 ppm throughout gestation does not cause cardiac defects in rat offspring.

The arrogance of teratology: A brief chronology of attitudes throughout history.

While the discipline of Teratology has existed for about 60 years, there has been a deep interest in the causes of human malformations for millennia. Absent the scientific method and acting on fervent beliefs that made sense to ancient/medieval populations, "mechanisms" were described and prognostications of future events were assigned to terata resulting in tragic (and unwarranted) sequelae. This article examines the collective beliefs and thinking within various eras in the hope of providing lessons to inform future behavior. The eugenics movement is an informative, recent example. Science of the 19th century had unraveled some of the mysteries of development and the role of genetics in determining birth outcomes. There was, however, a deep misunderstanding about the enormous amount of information that had yet to be uncovered. Based on immature science and faulty assumptions, it was suggested that "unfit" individuals be euthanized and their parents sterilized. Such "solutions" would be considered deplorable today. Surprisingly, such a reprehensible program was supported (at least in part) by many intelligent and highly regarded individuals. Today, it is imperative that we enter into the era of molecular biology and gene editing cautiously and perspicaciously. The history of teratology has elucidated our inability to understand where our new technologies and actions might take us and how unintended consequences could disrupt even our most carefully thought-out plans.

Impact of chloroform exposures on reproductive and developmental outcomes: A systematic review of the scientific literature.

AIMS: We assessed the animal and epidemiological data to determine if chloroform exposure causes developmental and/or reproductive toxicity. RESULTS AND DISCUSSION: Initial scoping identified developmental toxicity as the primary area of concern. At levels producing maternal toxicity in rats and mice, chloroform caused decrements in fetal weights and associated delays in ossification. In a single mouse inhalation study, exposure to a high concentration of chloroform was associated with small fetuses and increased cleft palate. However, oral exposure of mice to chloroform at a dose 4 times higher was negative for cleft palate; multiple inhalation studies in rats were also negative. Epidemiologic data on low birth weight and small for gestational age were generally equivocal, preventing conclusions from being drawn for humans. The animal data also show evidence of very early (peri-implantation) total litter losses at very high exposure levels. This effect is likely maternally mediated rather than a direct effect on the offspring. Finally, the epidemiologic data indicate a possible association of higher chloroform exposure with lower risk of preterm birth (<37 weeks gestation). CONCLUSIONS: The available animal data suggest that exposures lower than those causing maternal toxicity should be without developmental effects in the offspring. Also, most studies in humans rely on group-level geographic exposure data, providing only weak epidemiologic evidence for an association with development outcomes and fail to establish a causal role for chloroform in the induction of adverse developmental outcomes at environmentally relevant concentrations.

Bone development in laboratory mammals used in developmental toxicity studies.

Evaluation of the skeleton in laboratory animals is a standard component of developmental toxicology testing. Standard methods of performing the evaluation have been established, and modification of the evaluation using imaging technologies is under development. The embryology of the rodent, rabbit, and primate skeleton has been characterized in detail and summarized herein. The rich literature on variations and malformations in skeletal development that can occur in the offspring of normal animals and animals exposed to test articles in toxicology studies is reviewed. These perturbations of skeletal development include ossification delays, alterations in number, shape, and size of ossification centers, and alterations in numbers of ribs and vertebrae. Because the skeleton is undergoing developmental changes at the time fetuses are evaluated in most study designs, transient delays in development can produce apparent findings of abnormal skeletal structure. The determination of whether a finding represents a permanent change in embryo development with adverse consequences for the organism is important in study interpretation. Knowledge of embryological processes and schedules can assist in interpretation of skeletal findings.

Vascular ontogeny within selected thoracoabdominal organs and the limbs.

The cardiovascular system is fundamental to life. Its vessels are the conduits for delivery of nutrients and oxygen to organs and the removal of wastes. During embryonic development, the vascular system is instrumental in the formation of organs. It contributes to the form and pattern of organs as diverse as the limbs and the gonads. Recent advances in molecular biology and genomics have afforded great insight to the control of vascular development at subcellular levels of organization. Nevertheless, there is little assembled information concerning the vascular development of the organ systems of the body. This paper begins by reviewing the modes of formation of embryonic blood vessels. This is followed by summaries of the ontogeny of the vasculature that supplies selected major thoracic and abdominal organs (heart, gut, liver, gonads, and kidney). The paper concludes with a description of the arterial development of the upper and lower extremities.