Review of reproductive and developmental toxicity studies with isopropanol

Published studies for reproductive and developmental toxicity conducted with isopropanol have been conducted by the inhalation and oral gavage routes of administration. Interpretation of the data from these studies has resulted in discussions regarding NOAELs and additional benchmark dose modeling publications. Unpublished reproductive and developmental toxicity studies administered in the drinking water were also conducted by BIBRA, and the results of those studies are presented here. In addition, all of the reproductive and developmental toxicity studies conducted with isopropanol are summarized and evaluated for concordance of effects and NOAELs. Endpoints of concern for regulatory agencies were decreases in male mating index and reductions in postnatal pup survival. Original study reports were evaluated and data collated to address these two endpoints, and the data summarized. Data are presented suggesting that there were technical problems in the study that implied a decrease in male mating index, and based on the results from the drinking water studies, the weight of evidence suggests that isopropanol does not affect male mating or fertility at dose levels of up to 1000 mg/kg/day. The weight of evidence suggests that isopropanol can cause decreases in postnatal pup survival following oral gavage administration of 1000-1200 mg/kg/day to the dams. The NOAEL for this endpoint with oral gavage administration was 700 mg/kg/day. Indications of maternal toxicity were also an important predictor for decreased postnatal survival. Decreased postnatal pup survival was also noted in the drinking water studies with isopropanol with a LOAEL of 2278 mg/kg/day and a NOAEL of 1947 mg/kg/day.     

Cre activity in fetal albCre mouse hepatocytes: Utility for developmental studies

The albCre transgene, having Cre recombinase driven by the serum albumin (alb) gene promoter, is commonly used to generate adult mice having reliable hepatocyte‐specific recombination of loxP‐flanked (“floxed”) alleles. Based on previous studies, it has been unclear whether albCre transgenes are also reliable in fetal and juvenile mice. Perinatal liver undergoes a dynamic transition from being predominantly hematopoietic to predominantly hepatic. We evaluated Cre activity during this transition in albCre mice using a sensitive two‐color fluorescent reporter system. From fetal through adult stages, in situ patterns of Cre‐dependent recombination of the reporter closely matched expression of endogenous Alb mRNA or protein, indicating most or all hepatocytes, including those in fetal and juvenile livers, had expressed Cre and recombined the reporter. Our results indicate the albCre transgene is effective in converting simple floxed alleles in fetal and neonatal mice and is an appropriate tool for studies on hepatocyte development. genesis 47:789–792, 2009. © 2009 Wiley‐Liss, Inc.     

Monomethylarsonic acid and dimethylarsinic acid: developmental toxicity studies with risk assessment

BACKGROUND: The toxicity of arsenic compounds is highly dependent on the valence and methylation state of the compound. Although there is extensive published literature on the potential developmental toxicity of inorganic arsenic compounds, little exists on organic arsenic compounds and, in particular, studies conducted in accordance with conventional regulatory guidelines appropriate for risk assessment are rare. The organic arsenic compounds, monomethylarsonic acid (MMAV) and dimethylarsinic acid (DMAV, also called cacodylic acid), are the active ingredients in pesticide products that are used mainly for weed control. MMAV and DMAV are also metabolites of inorganic arsenic formed intracellularly by most living organisms (animals, plants and bacteria). In mammals, this occurs predominantly in liver cells. METHODS: Conventional developmental toxicity studies of orally administered MMAV and DMAV in the Sprague-Dawley rat and New Zealand White rabbit were conducted in commercial contract laboratories in the late 1980 s for regulatory compliance. The results of these studies are summarized and presented to broaden the data available in the public domain. RESULTS: In both species, data shows an absence of dose-related effects at organic arsenic exposures that were not maternally toxic. MMAV doses of 0, 10, 100, and 500 mg/kg/day (rat) and 0, 1, 3, 7, and 12 mg/kg/day (rabbit) and DMAV doses of 0, 4, 12, and 36 mg/kg/day (rat) and 0, 3, 12, and 48 mg/kg/day (rabbit) were administered by oral gavage daily during organogenesis (Gestation Day [GD] 6-15, rat; GD 7-19, rabbit) and the litters examined at maternal sacrifice (GD 20, rat; GD 29, rabbit). After treatment with MMAV, maternal and fetal toxicity were observed at the highest doses of 500 mg/kg/day (rat) and 12 mg/kg/day (rabbit), but no treatment-related developmental toxicity at the lower doses, even in the presence of minimal maternal toxicity in the rat at 100 mg/kg/d. There was no evidence of teratogenicity associated with MMAV treatment. With DMAV, maternal and developmental toxicity were observed in the rat at 36 mg/kg/day, with a higher than spontaneous incidence of fetuses with diaphragmatic hernia. In the rabbit at 48 mg/kg/day, there was marked maternal toxicity, culminating for most females in abortion and with no surviving fetuses for evaluation. There was no treatment-related maternal or developmental toxicity in the rat or rabbit at 12 mg/kg/day. Based on pregnancy outcome, the developmental toxicity no observed adverse effect level (NOAEL) for orally administered MMAV were 100 and 7 mg/kg/day in the rat and rabbit, respectively, and for DMAV were 12 mg/kg/day in both species. CONCLUSIONS: Margins of exposure estimated based on conservative estimates of daily intakes of arsenic in all of its forms indicate that exposure to MMAV or DMAV at environmentally relevant exposure levels, by the oral route (the environmentally relevant route of exposure) is unlikely to pose a risk to pregnant women and their offspring.     

A shared response model for clustered binary data in developmental toxicity studies

Existing distributions for modeling fetal response data in developmental toxicology such as the beta-binomial distribution have a tendency of inflating the probability of no malformed fetuses, and hence understating the risk of having at least one malformed fetus within a litter. As opposed to a shared probability extra-binomial model, we advocate a shared response model that allows a random number of fetuses within the same litter to share a common response. An explicit formula is given for the probability function and graphical plots suggest that it does not suffer from the problem of assigning too much probability to the event of no malformed fetuses. The EM algorithm can be used to estimate the model parameters. Results of a simulation study show that the EM estimates are nearly unbiased and the associated confidence intervals based on the usual standard error estimates have coverage close to the nominal level. Simulation results also suggest that the shared response model estimates of the marginal malformation probabilities are robust to misspecification of the distributional form, but not so for the estimates of intralitter correlation and the litter-level probability of having at least one malformed fetus. The proposed model is fitted to a set of data from the U.S. National Toxicology Program. For the same dose-response relationship, the fit based on the shared response distribution is superior to that based on the beta-binomial, and comparable to that based on the recently proposed q-power distribution (Kuk, 2004, Applied Statistics53, 369-386). An advantage of the shared response model over the q-power distribution is that it is more interpretable and can be extended more easily to the multivariate case. To illustrate this, a bivariate shared response model is fitted to fetal response data involving visceral and skeletal malformation.     

Reduced water intake: Implications for rodent developmental and reproductive toxicity studies

In developmental and reproductive toxicity studies, drinking water is a common means of delivering the test agent. Reduced consumption of toxicant-containing water raises questions about indirect effects of reduced maternal fluid consumption resulting from unpalatability, versus direct effects of the test compound. Issues to consider include: objective assessment of dehydration and thirst, the relative contributions of innate and learned behaviors to drinking behavior and flavor preference, and the objective assessment of physiologic stress. Not only do lab animals under ad lib conditions consume more water than the minimum required to maintain fluid balance, animals faced with water restriction have substantial physiologic capacity for protection of metabolic processes. Measures of blood biochemistry can provide quantifiable, objective indications of fluid balance, but changes in these parameters could result from other causes such as effects of a test toxicant. Consummatory behaviors in response to perceived need are highly influenced by learning. Hence, the drinking behavior, water intake, and flavor acceptance/preference of animals used in toxicology experiments could be subject to learning experiences with the test compound. Physiological symptoms of stress produced by water deprivation may be distinguishable from the symptoms associated with other generalized stressors, such as food deprivation, but doing so may be beyond the scope of most developmental or reproductive toxicity studies. Use of concurrent controls, paired to test groups for water consumption, could help distinguish between the direct effects of a test toxicant as opposed to effects of reduced water consumption alone. Birth Defects Res (Part B), 86:157–175, 2009. ©2009 Wiley-Liss, Inc.     

Relationship between bovine oocyte morphology and in vitro developmental potential

We investigated the relationship between the morphology of oocytes collected from small antral follicles and their developmental capacity. Immature oocytes were classified into seven groups and cultured in vitro for maturation (IVM), fertilization (IVF) and development to blastocysts (IVC). After IVF, sperm penetration and normal fertilization rates were higher in the oocytes whose cytoplasm appeared brown. The rate of polyspermy was highest in the oocytes whose cytoplasm was black. After IVC, the rates of cleavage and of development to the blastocyst stage were also higher in the brown oocytes. Although the oocytes with dark clusters in a pale cytoplasm showed lower cleavage rates, cleaved zygotes had high developmental rates the same as the oocytes with a brown cytoplasm. Transmission electron microscopy showed that the oocytes with a pale or black cytoplasm had organelles arranged differently from other oocytes before IVM. Most of the oocytes with a brown and homogeneous cytoplasm or small diameter had the characteristics of immature cytoplasm (large clusters of cortical granules) even after IVM. On the other hand, the brown oocytes with a dark zone at the periphery or with dark clusters showed the same arrangement of organelles as in vivo matured oocytes. The oocytes with a pale or black cytoplasm appeared to be degenerating and/or ageing. In conclusion, a dark ooplasm indicates an accumulation of lipids and good developmental potential, while a light-coloured ooplasm indicates a low density of organelles and poor developmental potential. A black ooplasm indicates ageing and low developmental potential.     

Debendox in early pregnancy and fetal malformation.

During the mid-1960s, 22 977 pregnant women in Scotland and England were followed up prospectively for the incidence of malformations in their infants evident at birth or within six weeks. During the first 13 weeks of gestation 620 of these women had been prescribed Debendox (dicyclomine-doxylamine-pyridoxine) and 743 other women agents other than Debendox containing pyridoxine. Of the 620 women given Debendox, 589 (95%) had a normal outcome of pregnancy, 8 (13%) delivered a malformed infant, and 23 (3.7%) had other outcomes. Of the 22 357 women who were given Debendox, 445 (2.0%) produced infants with malformation; and the rates for all abnormal outcomes among women given Debendox and those not given the drug were 5.0% and 5.4% respectively. These results support the hypothesis that Debendox is not teratogenic.     

Relationship between transferrin C2 and birth weight

In previous investigations transferrin C2 has been found to be associated with spontaneous abortion and premature birth. In a study of newborn infants from northern Sweden, no significant correlation was found between birth weight and transferrin C2 of the infants. Thus transferrin C2 appears to be associated with reproductive disturbances and with premature birth, but not with birth weight in mature infants.     

Relationship between polyamines and paraquat toxicity in sunflower leaf discs

Polyamines have been reported as efficient antioxidantcompounds in plants. Sunflower leaf discs, treatedwith 100 µM paraquat (PQ), a well known oxidativestress inducer, showed decreased levels of putrescine(Put), spermidine (Spd) and spermine (Spm) (between33% and 80% with respect to the controls). Argininedecarboxylase (ADC) and ornithine decarboxylase (ODC)activities decreased 42% and 33% respectively. Amongthe markers of oxidative stress measured after PQtreatment, chlorophyll and glutathione content werereduced (30% and 49% respectively) andthiobarbituric acid reactive substances (TBARS)content increased (60%). Superoxide dismutase (SOD)activity declined 60% with respect to the control andlipoxygenase (LOX) increased 25% when leaf-discs weretreated with the herbicide. Pretreatment withexogenous polyamines (1 mM) reversed paraquat toxicityto different degrees according to the polyamine and/orthe tested parameter. Spermidine was able to inhibitchlorophyll loss, while Spm reverted the effect of PQon the level of TBARS almost completely and alsorestored SOD activity close to control values.Putrescine was the least effective as an oxidantprotectant. These results provide support for theargument that polyamines are effective antioxidantsthrough their ability to act as radical scavengers.     

Relationship between surplus energy and body weight in fish populations

This paper theoretically analyses the relationship between surplus energy, which is available for either somatic growth or reproduction, and body weight. From the data of metabolism and growth of the biwamasu, Oncorhynchus rhodurus, obtained by Miura et al., a Bernoulli's differential equation is induced to represent the relationship between body weight and the sum of surplus energy and active metabolic rate. Solving this equation gives the amount of surplus energy, f(W_x), as follows:f(W_x) = (αW_x^1−γ+β^1−γ)^1/(1−γ)−W_x, in which α, β and γ are constants and W_x is body weight at age x. The function is applied to ten fish populations and consequently it is found to be useful for a wider age range and a wider variety of fishes than the conventional function.     

BDE 49 and developmental toxicity in zebrafish

The polybrominated diphenyl ethers (PBDEs) are a group of brominated flame retardants. Human health concerns of these agents have largely centered upon their potential to elicit reproductive and developmental effects. Of the various congeners, BDE 49 (2,2',4,5'-tetrabromodiphenyl ether) has been poorly studied, despite the fact that it is often detected in the tissues of fish and wildlife species. Furthermore, we have previously shown that BDE 49 is a metabolic debromination product of BDE 99 hepatic metabolism in salmon, carp and trout, underscoring the need for a better understanding of biological effects. In the current study, we investigated the developmental toxicity of BDE 49 using the zebrafish (Danio rerio) embryo larval model. Embryo and larval zebrafish were exposed to BDE 49 at either 5 hours post fertilization (hpf) or 24 hpf and monitored for developmental and neurotoxicity. Exposure to BDE 49 at concentrations of 4iμ-32 μM caused a dose-dependent loss in survivorship at 6 days post fertilization (dpf). Morphological impairments were observed prior to the onset of mortality, the most striking of which included severe dorsal curvatures of the tail. The incidence of dorsal tail curvatures was dose and time dependent. Exposure to BDE 49 caused cardiac toxicity as evidenced by a significant reduction in zebrafish heart rates at 6 dpf but not earlier, suggesting that cardiac toxicity was non-specific and associated with physiological stress. Neurobehavioral injury from BDE 49 was evidenced by an impairment of touch-escape responses observed at 5 dpf. Our results indicate that BDE 49 is a developmental toxicant in larval zebrafish that can cause morphological abnormalities and adversely affect neurobehavior. The observed toxicities from BDE 49 were similar in scope to those previously reported for the more common tetrabrominated congener, BDE 47, and also for other lower brominated PBDEs, suggest that these compounds may share similarities in risk to aquatic species.     

Relationship between fetal adrenal morphology and anterior pituitary function

A comparative study of adrenal morphology between normal fetuses and those with anencephaly or congenital adrenal hyperplasia (CAH) was performed in order to examine the hypothesis that fetal adrenal mass and structure are adrenocorticotrophin (ACTH)-dependent throughout gestation. Combined adrenal weight in 102 normal fetuses was used to establish a reference range for the gestational ages of 15-27 weeks. During this period, mean adrenal weight showed a 6-fold linear increase. In 38 anencephalic fetuses of similar gestation age, adrenal weight was below the normal range and did not show a rise. Three fetuses with CAH (18, 22 and 30 weeks gestation) had adrenal weights considerably above the normal range. Adrenal cortical thickness was significantly increased in CAH fetuses, largely as a consequence of cell hypertrophy, whereas decreased cortical thickness in the anencephalic group represented cellular hypoplasia. Conspicuous secretory granules in the cytoplasm was the electron-micrographic feature of the adrenal gland in the 22-week fetus with CAH. These observations are consistent with close dependency of fetal adrenal growth and development upon fetal pituitary function from an early age, mediated primarily through ACTH.     

The role of maternal toxicity in lovastatin-induced developmental toxicity

The role of maternal toxicity in lovastatin-induced developmental toxicity in rats was examined in a series of studies. The first study administered lovastatin at 100, 200, 400, or 800 mg/kg/day (mkd) orally to mated rats from Gestation Day (GD) 6 through 20. Maternal toxicity was observed as transient dose-related body weight losses at the initiation of dosing; there were also deaths and/or morbidity at 400 and 800 mkd. These toxicities occurred in conjunction with forestomach lesions. Mean fetal weights were decreased in all groups (-5 to -16%), and the incidence of skeletal malformations, variations, and incomplete ossifications was increased. The 2 highest doses produced the most severe maternal and developmental effects. Using the same dosages, the second study avoided gestational maternal weight losses and morbidity by starting treatment 14 days before mating with dosing continued to GD 20. There were transient dose-related body weight losses after the start of dosing and deaths in the 400- and 800-mkd groups; however, there was no evidence of maternal toxicity during gestation. Developmental toxicity was evident only as slight, but generally significant (p< or =0.05) decreases in mean fetal weights in groups given > or =200 mkd (-2 to -5%). Significantly, no skeletal abnormalities were observed. A third study administered the pharmacologically active metabolite of lovastatin subcutaneously at dose levels that matched oral maternal drug exposures. In the high-dose group, maternal weight gain and mean fetal weight were slightly decreased but there were no treatment-related skeletal abnormalities. Finally, a series of toxicokinetic studies assessed whether the 2 different developmental toxicity profiles were due to differences in drug exposure between the developmentally toxic and non-toxic dosing regimes. The data showed that groups with no skeletal abnormalities had maternal and embryonic/fetal drug concentrations similar to or even greater than the groups with fetal abnormalities. These results indicate that fetal skeletal abnormalities observed at lovastatin dose levels > or =100 mkd are not due to a direct teratogenic effect, but are the result of excessive maternal toxicity, which most likely involves a nutritional deficiency associated with forestomach lesions and reduced maternal food intake.     

Postnatal abstinence or acute toxicity can account for morbidity in developmental studies with opiates

The incidence of neonatal morbidity and mortality in rats exposed to opiates $\text{in utero}$ is generally high. To determine the extent to which neonatal opioid intoxication and/or withdrawal contribute to this effect, addicted pups from dams treated chronically with the long-acting opioid levo-alpha-acetylmethadol (LAAM) and appropriate controls were injected within 12 h of birth with saline, an opioid agonist (LAAM and metabolites) or an antagonist (naloxone). The incidence of neonatal mortality for pups born to dams maintained on a high dose of LAAM was 52%. A single injection of agonist on the first day of life reduced mortality in this group to 29% while a single injection of the antagonist increased mortality to 88%. In contrast, administration of the agonist to control pups and pups born to dams maintained on lower doses of LAAM resulted in increased mortality. Naloxone was apparently innocuous in non-dependent neonates. These data show that, despite LAAM's long duration of action in the mature rat, newborn rats experience withdrawal soon after drug exposure is terminated. These data also indicate that continued opioid exposure is a highly effective means of treating/preventing severe spontaneous withdrawal in the newborn.     

Relationship between cellular glutathione and hyperthermic toxicity in mammary carcinoma in mice

This investigation evaluates in an in vivo system the possible correlation between the intracellular content of GSH and cysteine and thermal sensitivity and thermotolerance. The studies were performed on C3H mammary carcinomas, located on the hind paw of CBA mice. Intracellular thiols were measured by the HPLC technique and the degree of thermotolerance induction was determined from tumour growth rate studies. It was found that the intracellular GSH levels did not change significantly during thermotolerance induction, and that subtoxic hyperthermia induced a pronounced transient decrease in GSH down to 30 per cent of the control level. When the intracellular GSH level was decreased to the same extent, by pretreatment with D,L-buthionine-S-R-sulphoximine (BSO), thermotolerance was still inducible. Thus, the induction of heat-induced thermal resistance did not seem to be dependent on the intracellular GSH level. When hyperthermia and BSO were combined, the GSH levels were further reduced. Treatment with BSO slightly increased the toxicity of both thermotolerance-inducing and subtoxic hyperthermia. The cysteine concentrations increased several fold after BSO and heat treatments and contributed, under these conditions, to more than 25 per cent of the intracellular free reduced thiols. In general, there was no direct correlation between GSH and cysteine levels. It is concluded that thermotolerance induction does not depend on or cause changes in intracellular GSH levels and that subtoxic heat treatments induce a pronounced transient decrease in GSH concentration.