Neil Chernoff

An in vivo teratology screen utilizing pregnant mice

Twenty-eight compounds of known teratogenic potential were assayed by an in vivo screening procedure. Postnatal growth and viability of prenatally exposed offspring was used as a measure of developmental toxicity. Gravid CD-1 mice were administered maximum tolerated doses of the compounds for up to 5 consecutive days during the period of major organogenesis. The dams were allowed to give birth, and litter size and weight on postpartum d 1 and 3 were recorded and compared with concurrent controls. All 15 compounds that were teratogenic by standard teratology test criteria exhibited some form of developmental toxicity. Four chemicals known to produce only fetal toxicity (reduced weight or supernumerary ribs) were tested and the screen successfully identified those that reduced weight. Finally, of the 9 compounds that show no effect in standard tests, 6 were also negative in the screen and 3 demonstrated either reduced viability or weight.

A review of the literature on potential reproductive and developmental toxicity of electric and magnetic fields

The potential of electric and magnetic fields to adversely affect the health of the human population is an issue which continues to receive a great deal of attention in both public and scientific forums. One of the critical issues is the possibility that such fields may adversely affect the reproductive process. Numerous studies investigating the potential of electric and/or magnetic fields to alter reproduction in vertebrates have been conducted. These studies have, in many instances, yielded seemingly contradictory results. A number of epidemiological studies have been conducted as well. This review of the literature examines relevant studies and attempts to draw biologically rational conclusions from them. The studies are ordered in broad categories based upon both classification of the species studied (i.e. submammalian, mammalian exclusive of man and human) and the agent used (i.e. extremely low frequency electric, very low frequency electric, and magnetic fields). From our review we conclude that laboratory experimental and epidemiological results to date have not yielded conclusive data to support the contention that such fields induce adverse reproductive effects under the test or environmental conditions studied. Additional studies may, however, be warranted to clarify some of the experimental results obtained.

An overview of maternal toxicity and prenatal development: considerations for developmental toxicity hazard assessments

The objective of testing xenobiotics for potential developmental toxicity is to extrapolate laboratory animal information to the human species, thereby deriving biologically rational regulatory policies. One of the problems that significantly contributes to the difficulty of this task is the possibility that general effects on the maternal organism could affect the developing conceptus. Published data have indicated that factors intrinsic to the maternal organism affect developmental outcome. This overview examines factors which may bear directly or indirectly upon developmental outcome, with emphasis on those of greatest relevance to the hazard assessment process. Standard teratology testing protocols often call for dose levels that induce overt maternal toxicity, and the developmental effects of this toxicity (both alone, and with concurrent embryo/fetal insult) continue to present regulators with considerable interpretive difficulties. In response to these problems there have been both research and literature review efforts dealing with the relationship of maternal and developmental toxicity. Relevant studies are reviewed here, and suggestions for avenues of future research are offered including the identification of any syndromes of developmental effects occurring at maternally toxic levels irrespective of the causative agent, and experimental approaches for the characterization of maternal toxicity.

Teratogenic effects of benomyl in the Wistar rat and CD-1 mouse, with emphasis on the route of administration.

Abstract Benomyl, a systemic fungicide whose molecular basis of action is inhibition of tubulin polymerization, was administered during organogenesis via the dietary and gavage routes to pregnant Wistar rats, and via the gavage route to pregnant CD-1 mice. Benomyl was fetotoxic and teratogenic in both species via the po route of administration, producing a broad spectrum of malformations at a dose of 62.5 mg/kg/day in the rat and 100 mg/kg/day in the mouse. Via the dietary route of administration, benomyl produced fetotoxicity, but no teratogenic effects. The fetotoxic potential of benomyl from dietary exposure was approximately an order of magnitude less effective than from gavage exposure. Benomyl did not affect postnatal growth, viability, or locomotor activity at subteratogenic doses. The most sensitive indicator of perinatal exposure to benomyl via the po route of administration was a permanent reduction in testes and accessory sex gland weight noted in male offspring of dams receiving 31.2 mg/kg/day benomyl during gestation and lactation. No effects on any parameters were evident in rats receiving 15.6 mg/kg/day by po gavage. The relevance of the two routes of administration for risk extrapolation is discussed.

Comparison of the penetration of 14 pesticides through the skin of young and adult rats

In vivo percutaneous absorption of 14 pesticides was studied in young (33-d-old) and adult (82-d-old) female Fischer 344 rats, at three different dose levels. Carbon-14-labeled pesticides in acetone were applied to previously clipped middorsal skin. The treatment area was 2-3% of the body surface area. Penetration of the pesticides during a 72-h period ranged from approximately 1%-90%, depending on compound, dose, and age of animal. No clear age-related pattern of dermal absorption among compounds was found. Only chlordecone, folpet, and permethrin did not show significant age-dependent differences in skin penetration. Atrazine, carbaryl, chlorpyrifos, and hexachloro-biphenyl had greater absorption in the young, while carbofuran, captan, dinoseb, DSMA, MSMA nicotine, and parathion displayed greater absorption in the adult. The majority of the compounds showed dose-dependent penetration. The dose-response curves for penetration were not parallel for 8 of the 14 compounds studied.

Significance of Supernumerary Ribs in Rodent Developmental Toxicity Studies: Postnatal Persistence in Rats and Mice

Pregnant Sprague-Dawley rats and Swiss-Webster mice were gavaged with bromoxynil at 15 and 96.4 mg/kg/day, respectively, on Days 6-15 of gestation. The frequency of supernumerary ribs (SNR), which are here defined as any degree of ossification lateral to the first lumbar vertebrae, was determined in fetuses at term and offspring on Postnatal Days 6, 20, and 40. Bromoxynil induced significant increases in the incidence of SNR in fetuses of both species. In rats, SNR occurred in 62% of treated fetuses as compared to 14% in controls; in mice these values were 45% and 11%, respectively. The postnatal incidence and persistence of SNR was species dependent. In the rat, postnatal SNR incidence in treated animals did not differ significantly from controls. In contrast, in mice the bromoxynil-induced elevated incidence of SNR persisted through Day 40 (42.3% in treated vs 0% in controls). Analysis of SNR was also done on the basis of their length (greater or less than 1/2 the length of the 13th rib). In the mouse, the incidence of smaller SNR was much lower on Day 40 as compared to Day 20; in contrast the incidence of larger SNR persisted through Day 40. In the rat, the incidence of larger SNR was too small to draw conclusions as to the postnatal fate of these structures. As in the mouse, however, the incidence of smaller SNR was significantly lower by Day 40. The significance of SNR in developmental toxicity remains problematic. The impact of this anomaly on animals is difficult to assess.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship of maternal and fetal toxicity in developmental toxicology bioassays with notes on the biological significance of the “no observed adverse effect level”

Standard developmental toxicology bioassays are designed to identify agents with the potential to induce adverse effects and include dose levels that induce maternal toxicity. The work reported here was undertaken to evaluate the relationship of maternal and fetal toxicity. It constitutes an analysis of 125 developmental toxicity bioassays in the mouse, rat, and rabbit conducted by the National Toxicology Program. Although varying by species, general findings include: (1) most lowest observable adverse effect levels (LOAELs) were determined by reduced maternal gestational weight gain or fetal weight at term. (2) Maternal weight reductions are associated with reduced food intake for a variety of dissimilar test agents. (3) Lower fetal weights were associated with reduced maternal weight gains late in gestation. (4) The degree of fetal weight reduction is correlated with the extent of the maternal weight loss. In a substantial number of the studies, reduced fetal weights at term may, therefore, be due to maternal undernutrition caused by general toxicity rather than direct developmental insult. Consequently, such test agents may be erroneously classified as primary developmental toxicants. Experimental approaches to test the hypothesis that maternal undernutrition in standard developmental toxicology bioassays may be responsible for significant term fetal weight decrements are discussed.

Prenatal or postnatal exposure to bis(tri-n-butyltin)oxide in the rat: Postnatal evaluation of teratology and behavior

The results of a series of screening tests to determine the potential teratogenicity and neurotoxicity of developmental exposure to TBTO in rats are presented in this paper. For prenatal exposure, pregnant Long Evans rats were intubated with 0-16 mg/kg/day bis(tri-n-butyltin)oxide TBTO from Days 6 to 20 of gestation (GD 6-20). For postnatal exposure, rat pups were intubated with 0-60 mg/kg TBTO on Postnatal Day 5 (PND 5). Following prenatal exposure, dams were allowed to litter and pups were evaluated using a postnatal teratology screen. Postnatal evaluation for both exposures included motor activity (PND 13-64), the acoustic startle response (PND 22-78), growth, and brain weight. The maximally tolerated dose (MTD) in pregnant rats was 5 mg/kg/day, which is one-third the MTD in nonpregnant rats. There were decreased numbers of live births, and decreased growth and viability at dosages greater than or equal to 10 mg/kg/day. Cleft palate was found in 3% of the 12 mg/kg/day group. There was mortality following postnatal exposure to 60 mg/kg and all prenatal dosages greater than or equal to 10 mg/kg/day. Preweaning body weight was significantly decreased for all postnatal dosages, and all prenatal dosages greater than 2.5 mg/kg/day. Body weight reductions persisted to the postweaning period only in the high dose groups (10 mg/kg/day and 60 mg/kg). Behavioral evaluation demonstrated transient alterations in motor activity development (prenatal exposure only) and the acoustic startle response (postnatal exposure only). Persistent behavioral effects were observed only at dosages that produced overt maternal toxicity and/or postnatal mortality. The demonstration of the teratogenic and neurotoxic potential of TBTO in rats is confounded by associated maternal toxicity and/or pup mortality.

SUPERNUMERARY RIBS IN DEVELOPMENTAL TOXICITY BIOASSAYS AND IN HUMAN POPULATIONS: INCIDENCE AND BIOLOGICAL SIGNIFICANCE

Supernumerary or accessory ribs (SNR), either lumbar (LR) or cervical (CR), are a common finding in standard developmental toxicology bioassays. The biological significance of these anomalies within the regulatory arena has been problematic and the subject of some debate. In rodents, the spontaneous incidence of SNR is species and strain related and ranges from <1% to >30%. Compound-induced LR are induced by a wide variety of chemical and physical agents when pregnant animals are exposed during specific gestational periods. A significant portion of the agent-induced LR may be due to maternal factors, as it has been shown that stress alone will induce LR in rodents. SNR are not isolated phenomena and signify basic alterations in the architecture of the axial skeleton. LR are associated with longer ribs, increased numbers of vertebrosternal ribs, and the presence of extra presacral vertebrae (“anteriorization”). CR are associated with reduced numbers of vertebrosternal ribs (“posteriorization”). It is evident that SNR are not a single anomaly, but consist of two unrelated structures: an extra rib that has a cartilaginous segment at the distal end, and an ossification site that lacks cartilage. These have a bimodal size distribution, with the population of extra ribs being significantly longer than the ossification sites, and 0.6 mm can be used as an approximate length for distinguishing the two populations in mice. Extra ribs are permanent structures in contrast to ossification sites that disappear postnatally, probably becoming part of the lateral transverse vertebral processes. SNR are also found in humans although, in contrast to laboratory species, CR are more commonly noted. SNR are associated with adverse heath effects, and CR with inducing thoracic outlet disease characterized by diminished blood flow and altered position of the ganglia and nerve roots in the area of the C7–T1 vertebrae. LR are associated with lower back pain and L4–5 degeneration. The incidence of CR is greatly reduced in adult humans as compared to fetuses, and it has been hypothesized that fetal “SNR” may be largely composed of ossification sites that disappear postnatally. The mechanisms involved in the formation of extra ribs are not understood at this time, although the fact that the early sensitive periods for their initiation during embryogenesis is coupled with the associated changes in the axial skeleton argues for their induction being due to fundamental changes in gene expression. The sum of the experimental evidence supports the idea of SNR being composed of two different structures: extra ribs that are permanent dysmorphological structures that may be induced by xenobiotics and/or maternal stress, and ossification sites that may be transient variations in the formation of the lateral processes of the vertebrae.

Developmental effects of maternal stress in the CD-1 mouse induced by restraint on single days during the period of major organogenesis

Maternal stress during gestation can produce significant fetal and/or postnatal effects, and can enhance the teratogenicity of other agents. We have previously shown that restraint stress on gestational day 8 in CD-1 mice produces significant increases in encephaloceles and supernumerary and fused ribs. In the present study we have examined the effects of stress induced by restraint on individual days during the period of major organogenesis (days 6-14). Weight loss and stress-induced analgesia as assessed by the tail-flick method were used to determine the degree of stress induced by a 12-h restraint period. Restrained animals lost significantly more weight and had longer tail-flick latencies than the concurrent food and water deprived controls on all gestational days. Significant increases in embryo/fetal mortality were also observed in the offspring of restrained animals. An increased incidence of supernumerary ribs was found in mice restrained on days 7 and 8. Since maternal toxicity induced by chemical teratogens may be accompanied by a general increase in maternal stress, our data suggest that such stress may be an etiological factor in teratology bioassays in which dose levels are sufficiently high to induce overt maternal toxicity.