Prenatal Developmental Toxicity Research Articles

Prenatal developmental toxicity evaluation of 2′,3′‐dideoxyinosine (ddI) and 2′,3′‐didehydro‐3′‐deoxythymidine (d4T) co‐administered to Swiss Albino (CD‐1) mice

In pregnant women, antiretroviral drugs improve maternal health and reduce vertical transmission of human immunodeficiency virus to the infant. However, few nonclinical studies have examined the potential for adverse drug interactions. On gestational days (GD) 6-16, mice were dosed with vehicle, ddI (360, 1440, or 2,880 mg/kg/day, p.o.), d4T (60, 240, or 480), or ddI/d4T combinations (360/60, 1,440/240, or 2,880/480). Daily doses were divided into two equal parts that were administered >or=6-hr apart. Body weight, clinical signs, and feed consumption were monitored. Pregnancies (22-24/group) were confirmed at necropsy. Maternal liver and gravid uterine weights (GUW), uterine implants (resorption, live or dead fetus), fetal body weight, gender, and morphologic anomalies (external, visceral, skeletal) were recorded. Maternal body weight, clinical signs, and GUW were unaffected. Maternal weight change corrected for GUW was greater than controls at 60 and 480 d4T. Relative feed consumption during treatment was increased relative to controls at 1,440 and 2,880 ddI and 2,880/480 ddI/d4T. Relative maternal liver weight was elevated above controls at 240 and 480 d4T and 2,880/480 ddI/d4T, and above the constituent dose of ddI at 1,440/240 and 2,880/480 ddI/d4T. Liver weight was not affected by ddI and there was no significant drug interaction. Prenatal mortality and morphologic anomalies were not increased. Fetal body weight showed only a decreasing trend for ddI/d4T, no effect for ddI or d4T, and no statistically significant drug interaction. In pregnant mice, ddI/d4T combinations were not associated with well-defined developmental toxicity or adverse drug interactions.

Prenatal developmental toxicity study of the basic rubber accelerator, 1,3-di- o-tolylguanidine, in rats

Pregnant rats were given 1,3-di- o-tolylguanidine (DTG) by gavage at 0, 10, 20 or 40 mg/kg bw/day on days 6–19 of pregnancy and the pregnancy outcome was determined on day 20 of pregnancy. At 40 mg/kg bw/day, deaths were observed in four out of 24 females. The incidences of females showing mydriasis at 20 and 40 mg/kg bw/day and showing decreased locomotor activity at 40 mg/kg bw/day were significantly increased. Alopecia, bradypnea, prone position and tremor were also observed at 40 mg/kg bw/day. The maternal body weight gain at 20 and 40 mg/kg bw/day and food consumption at 40 mg/kg bw/day were significantly reduced. A significantly decreased weight of the gravid uterus, increased incidence of postimplantation loss, decreased number of live fetuses, and lowered weights of fetuses and placentae were found at 40 mg/kg bw/day. The incidences of the total number of fetuses with external malformations at 40 mg/kg bw/day and with skeletal malformations at 20 and 40 mg/kg bw/day were significantly increased. Significantly higher incidences of fetuses with brachydactyly and short tail and defects of caudal vertebrae, phalanges and metacarpals were observed at 40 mg/kg bw/day. Delayed ossification was also noted at 40 mg/kg bw/day. The data indicate that DTG is teratogenic at maternal toxic doses and the NOAELs of DTG for maternal and developmental toxicity are 10 mg/kg bw/day in rats.

Prenatal developmental toxicity study with 7-hydroxymatairesinol potassium acetate (HMRlignan) in rats

Plant lignan 7-hydromatairesinol, a novel precursor of the mammalian lignan enterolactone was evaluated in a prenatal developmental toxicity study conducted in the Wistar rat. Mated female rats were fed diets containing 0, 0.25, 1, and 4% (w/w) of 7-hydroxymatairesinol in the form of potassium acetate complex (HMRlignan; potassium acetate level approximately 20% w/w within the preparation) for days 0–21 of gestation. Test substance intake was calculated to be 0.14–0.18, 0.46–0.74, and 1.19–2.93 g/kg body weight/day for the low, mid, and high-dose groups, respectively. The rats were sacrificed on day 21 of the gestation period and examined for standard parameters of reproductive performance (fecundity index, gestation index, number of corpora lutea, number of implantations, pre- and post-implantation loss, number of early- and late resorptions, number of live- and dead fetuses, sex-ration and the weight of the reproductive organs). The fetuses were examined for external, visceral, and skeletal alterations. The results from this study showed no effects on reproductive performance or any treatment related findings following external, visceral, and skeletal examination of the fetuses. However, approximately half of the mated dams of the high-dose failed to thrive due to an unexpected large decrease in their food intake, and were sacrificed early. Body weights of the remaining animals of the high-dose group were decreased. Food consumption was decreased in all treatment groups during the first three days of the gestation period as a result of decreased palatability of the feed. In conclusion, the no-observed-effect level (NOEL) for maternal effects was 1%, whereas the NOEL for fetal development following daily oral HMRlignan administration throughout the gestation was equivalent to 4% in the diet.

Prenatal developmental toxicity study with 7-hydroxymatairesinol potassium acetate (HMRlignan) in rats

Plant lignan 7-hydromatairesinol, a novel precursor of the mammalian lignan enterolactone was evaluated in a prenatal developmental toxicity study conducted in the Wistar rat . Mated female rats were fed diets containing 0, 0.25, 1, and 4% (w/w) of 7-hydroxymatairesinol in the form of potassium acetate complex (HMRlignan; potassium acetate level approximately 20% w/w within the preparation) for days 0–21 of gestation. Test substance intake was calculated to be 0.14–0.18, 0.46–0.74, and 1.19–2.93 g/kg body weight/day for the low, mid, and high-dose groups, respectively. The rats were sacrificed on day 21 of the gestation period and examined for standard parameters of reproductive performance (fecundity index, gestation index, number of corpora lutea, number of implantations, pre- and post-implantation loss, number of early- and late resorptions , number of live- and dead fetuses , sex-ration and the weight of the reproductive organs). The fetuses were examined for external, visceral, and skeletal alterations. The results from this study showed no effects on reproductive performance or any treatment related findings following external, visceral, and skeletal examination of the fetuses. However, approximately half of the mated dams of the high-dose failed to thrive due to an unexpected large decrease in their food intake, and were sacrificed early. Body weights of the remaining animals of the high-dose group were decreased. Food consumption was decreased in all treatment groups during the first three days of the gestation period as a result of decreased palatability of the feed. In conclusion, the no-observed-effect level (NOEL) for maternal effects was 1%, whereas the NOEL for fetal development following daily oral HMRlignan administration throughout the gestation was equivalent to 4% in the diet.

Rentech in another GTL licensing move

Prenatal developmental toxicity (PDT) as observed with some petroleum substances (PS) has been associated with the presence of 3–7 ring polycyclic aromatic hydrocarbons (PAHs). In the present study, the applicability of ES-D3 cell differentiation assay of the EST to evaluate in vitro embryotoxicity potencies of PS and gas-to-liquid (GTL) products as compared to their in vivo potencies was investigated. DMSO-extracts of a range of PS, containing different amounts of PAHs, and GTL-products, which are devoid of PAHs, were tested in the ES-D3 cell proliferation and differentiation assays of the EST. The results show that PS inhibited the differentiation of ES-D3 cells into cardiomyocytes in a concentration-dependent manner at non-cytotoxic concentrations, and that their potency was proportional to their PAH content. In contrast, as expected, GTL-products did not inhibit ES-D3 cell viability or differentiation at all. The in vitro PDT potencies were compared to published in vivo PDT studies, and a good correlation was found between in vitro and in vivo results (R2 = 0.97). To conclude, our results support the hypothesis that PAHs are the primary inducers of the PDT in PS.

In Vivo Assessment of Prenatal Developmental Toxicity in Rodents

This unit presents two basic protocols for testing prenatal developmental toxicity in rodents. The standard protocol for assessing prenatal developmental toxicity, often referred to as a Segment II study, describes the methods used for performing cesarean sections and examining cesarean-delivered fetuses for external, skeletal, and visceral alterations. The protocol for the Chernoff/Kavlock assay, a developmental toxicity screen, describes methods for examining parturition, and postnatal growth and viability of prenatally exposed litters.

Developmental toxicity of oral n-butylamine hydrochloride and inhaled n-butylamine in rats

Pregnant Wistar rats were administered 0, 100, 400 or 1000 mg mono- n-butylamine hydrochloride/kg body weight/day by gavage on days 6 through 15 post coitum (sperm-positive=day 0), or inhaled mono- n-butylamine 0, 17, 50 or 152 ppm (whole-body exposure), 6 h/day on days 6 through 19 post coitum. Oral n-butylamine HCl 1000 mg/kg reduced maternal feed consumption, increased early post-implantation losses (embryonic resorptions), reduced fetal and placental weight, and retarded skeletal development (incomplete skull and sternebral ossification), and produced malformations (filiform/kinked tail, enlarged cardiac ventricular chamber(s), malpositioned heart, aortic arch atresia, diaphragmatic hernia); 100 mg/kg was the no-observed-adverse effect level (NOAEL) for prenatal developmental toxicity; 400 mg/kg, the maternal no-effect level, produced only malformations (aortic arch atresia, malpositioned heart, diaphragmatic hernia). Inhaled n-butylamine produced concentration-dependent nasal epithelial hyperplasia and squamous metaplasia, inflammation and necrosis; the maternal NOAEL was less than 17 ppm. There were no treatment-related signs of embryo/fetotoxicity, particularly, no effects on fetal morphology. The developmental NOAEL was 152 ppm. The neutralization of n-butylamine by hydrochloride converts it from a strong alkali causing tissue burns into a weak acid/base which is fetotoxic. Possible mechanisms of fetotoxicity are free radical production, metabolic acidosis, and lysosomotrophy.

Developmental toxicity but no immunotoxicity in the rat after prenatal exposure to diethylstilbestrol

Studies with dioxins and PCB's have shown that the developing immune system may be especially vulnerable to xenobiotics during the perinatal period. However, current guidelines for reproductive toxicity testing do not include immune parameters. In the present study, we have explored the usefulness of including immune parameters within the prenatal developmental toxicity study in rats, using the treatment protocol as described in the OECD 414 developmental toxicity test guideline. In addition, the experimental protocol was enhanced by including ten dose groups to facilitate dose–response analysis. Diethylstilbestrol (DES) was used as the model compound, as it is known to be toxic both for embryofetal development and for the immune system. The results show developmental toxicity in terms of decreased fetal survival and decreased pup body weight in the presence of reduced maternal food consumption and reduced body weight gain. However, immune parameters, including histopathology, hematology, and antibody responses to sheep red blood cells (SRBC) in pups at 4 weeks of age were uncompromised. It is speculated that rather than the prenatal exposure protocol used here, the generation study design with both pre- and postnatal exposure may be preferable as a general screen to detect developmental immunotoxic injury after xenobiotic exposure.

Prenatal Oral (Gavage) Developmental Toxicity Study of Decabromodiphenyl Oxide in Rats

Decabromodiphenyl oxide (DBDPO) is a highly effective flame retardant that is primarily used in electrical and electronic equipment with a secondary, but important, application in upholstery textiles. DBDPO, the second largest volume brominated flame retardant in use today, has undergone a wide range of toxicology tests in mammalian species with the results indicating a no-adverse-effect level of approximately 1000 mg/kg/day in oral repeated-dose studies. An oral prenatal developmental toxicity study of the commercial DBDPO product (97% purity) was performed under current EPA OPPTS and OECD guidelines. Female Sprague-Dawley rats (25 mated females/group) received 0,100, 300 or 1000 mg DBPDO/kg/ day via gavage in corn oil during gestation days 0 through 19. All females survived until scheduled sacrifice. No clinical signs of toxicity were observed. Pregnancy rates in the control and treated groups ranged from 96% to 100% and provided 23 or more litters in each group for evaluation on gestation day 20. No effect of treatment was seen in maternal gestational parameters (body weight, body weight gain, and food consumption), uterine implantation data, liver weight, or necropsy findings. Likewise, no effect of treatment was seen in fetal body weights, fetal sex distribution, or during the fetal external, visceral, or skeletal examinations. The NOEL (no-observable-effect level) for maternal and developmental toxicity was 1000 mg DBPDO/kg/day, the highest dose level administered on gestation days 0 to 19.

Recent developments in regulatory requirements for developmental toxicology

A number of legislative and regulatory changes have occurred over the past 5 years to prompt the re-evaluation of the regulatory requirements for developmental toxicity testing and use of the data for risk assessment. In particular, passage of the 1996 Food Quality Protection Act (FQPA) in the United States required the USEPA to evaluate children's health risks in a more rigorous fashion, and to apply an additional 10-fold safety factor if data were inadequate or children appeared to be more sensitive than adults. A review of the testing protocols required by USEPA led to extension of the dosing period to term in the prenatal developmental toxicity study and the addition of endpoints to the 2-generation reproduction study protocol as indicators of possible neurologic, reproductive, or immune alterations. Revised testing guidelines for pesticides and toxic substances were published by USEPA in 1998, including a developmental neurotoxicity testing protocol. Further review for FQPA implementation resulted in the proposal for a core set of required toxicology studies, including routine developmental neurotoxicity, adult neurotoxicity, and adult immunotoxicity studies. In addition, development of new testing guidelines in several areas was recommended, these guidelines to be used in conjunction with or as follow-up when indicated from standard testing: developmental immunotoxicity, carcinogenesis, specialized neurotoxicity studies, endocrine disruptor studies, pharmacokinetics, and direct dosing of neonates. The impact of these efforts on the policies for toxicity testing of pesticides are discussed, and these issues are currently being reviewed on a broader scale, in particular, by evaluating the adequacy of the methods used for reference values (e.g. chronic RfD, RfC). Three major areas of focus for this review include life stages evaluated, endpoints assessed, and the duration of exposure used in various studies. A major focus of these efforts is to ensure that children's health risks are being adequately addressed in the risk assessment process.

Evaluation of the prenatal developmental toxicity of orally administered arsenic trioxide in rats

A thorough review of the literature revealed no published repeated-dose oral developmental toxicity studies of inorganic arsenic in rats. In the present study, which was conducted according to modern regulatory guidelines, arsenic trioxide was administered orally beginning 14 days prior to mating and continuing through mating and gestation until gestational day 19. Exposures began prior to mating in an attempt to achieve a steady state of arsenic in the bloodstream of dams prior to embryo-foetal development. Groups of 25 Crl:CD ®(SD)BR female rats received doses of 0, 1, 2.5, 5 or 10 mg/kg/day by gavage. The selection of these dose levels was based on a preliminary range-finding study, in which excessive post-implantation loss and markedly decreased foetal weight occurred at doses of 15 mg/kg/day and maternal deaths occurred at higher doses. Maternal toxicity in the 10 mg/kg/day group was evidenced by decreased food consumption and decreased net body weight gain during gestation, increased liver and kidney weights, and stomach abnormalities (adhesions and eroded areas). Transient decreases in food consumption in the 5 mg/kg/day group caused the maternal no-observed-adverse-effect level (NOAEL) to be determined as 2.5 mg/kg/day. Intrauterine parameters were unaffected by arsenic trioxide. No treatment-related foetal malformations were noted in any dose group. Increased skeletal variations at 10 mg/kg/day were attributed to reduced foetal weight at that dose level. The developmental NOAEL was thus 5 mg/kg/day. Based on this study, orally administered arsenic trioxide cannot be considered to be a selective developmental toxicant (i.e. it is not more toxic to the conceptus than to the maternal organism), nor does it exhibit any propensity to cause neural tube defects, even at maternally toxic dose levels.

Large-scale double-staining of rat fetal skeletons using Alizarin Red S and Alcian Blue

A critical component in the conduct of a prenatal developmental toxicity study is the evaluation of fetal skeletal development. As the developing rodent fetus is typically evaluated at gestation day 20, at a time when ossification of the skeleton is incomplete, a thorough assessment of skeletal development would include both ossified and cartilaginous structures. Current methods to double-stain the fetal skeleton using Alizarin Red S and Alcian Blue are typically described for small sample sizes or using time allotments for each processing step that are unsuitable for industry. In an industrial setting, there is a need for an effective means to double-stain fetal skeletons on a large scale (i.e., hundreds of fetuses simultaneously). This article describes a method used in our laboratory to stain both fetal bone and cartilage using solutions and procedures on an industrial scale. Teratology 61:273–276, 2000. © 2000 Wiley-Liss, Inc.

Large-scale double-staining of rat fetal skeletons using Alizarin Red S and alcian blue.

A critical component in the conduct of a prenatal developmental toxicity study is the evaluation of fetal skeletal development. As the developing rodent fetus is typically evaluated at gestation day 20, at a time when ossification of the skeleton is incomplete, a thorough assessment of skeletal development would include both ossified and cartilaginous structures. Current methods to double-stain the fetal skeleton using Alizarin Red S and Alcian Blue are typically described for small sample sizes or using time allotments for each processing step that are unsuitable for industry. In an industrial setting, there is a need for an effective means to double-stain fetal skeletons on a large scale (i.e., hundreds of fetuses simultaneously). This article describes a method used in our laboratory to stain both fetal bone and cartilage using solutions and procedures on an industrial scale.

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

Assessing risks to human development from chemical exposure typically requires integrating findings from laboratory animal and human studies. 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. 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. 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.

Absence of prenatal developmental toxicity from inhaled arsenic trioxide in rats.

A review of the literature revealed no published inhalational developmental toxicity studies of arsenic performed according to modern regulatory guidelines and with exposure throughout gestation. In the present study, inorganic arsenic, as arsenic trioxide (As(+3), As2O3), was administered via whole-body inhalational exposure to groups of twenty-five Crl:CD(SD)BR female rats for six h per day every day, beginning fourteen days prior to mating and continuing throughout mating and gestation. Exposures were begun prior to mating in order to achieve a biological steady state of As(+3) in the dams prior to embryonal-fetal development. In a preliminary exposure range-finding study, half of the females that had been exposed to arsenic trioxide at 25 mg/m3 died or were euthanized in extremis. In the definitive study, target exposure levels were 0.3, 3.0, and 10.0 mg/m3. Maternal toxicity, which was determined by the occurrence of rales, a decrease in net body weight gain, and a decrease in food intake during pre-mating and gestational exposure, was observed only at the 10 mg/m3 exposure level. Intrauterine parameters (mean numbers of corpora lutea, implantation sites, resorptions and viable fetuses, and mean fetal weights) were unaffected by treatment. No treatment-related malformations or developmental variations were noted at any exposure level. The no-observed-adverse-effect level (NOAEL) for maternal toxicity was 3.0 mg/m3; the NOAEL for developmental toxicity was greater than or equal to 10 mg/m3, 760 times both the time-weighted average threshold limit value (TLV) and the permissible exposure limit (PEL) for humans. Based on the results of this study, we conclude that arsenic trioxide, when administered via whole-body inhalation to pregnant rats, is not a developmental toxicant.

The application of benchmark dose methodology to data from prenatal developmental toxicity studies

The benchmark dose (BMD) concept was applied to 246 prenatal-developmental toxicity (DT) datasets from government, industry and commercial laboratories. Five modeling approaches were used, 2 generic and 3 specific to DT models. BMDs for both quantal and continuous data were compared with statistically derived no observed adverse effect levels (NOAELs) to determine similarities. Quantal (Q) endpoints included litter responses (e.g., one or more dead or malformed implants), and QBMDs were calculated using a Q Weibull (QW) model. Two types of continuous (C) data were modeled, the proportion of implants affected per litter, and the change in fetal weight (both mean and distribution); continuous power (CP) and DT models were used to calculate CBMDs. QBMDs for a 5% change in response (QBMD 05,) were 6-fold lower, on average, than the corresponding NOAEL. CBMD 05s on average were similar to the corresponding NOAELs, and CBMD 05s from different models were similar to each other. Including litter size but not threshold improved the fit of the DT models. For fetal weight data, specific cutoff values were used to calculate BMDs that were similar on average to the corresponding NOAELs: (1) changes from the control mean (5% of the mean, 25th percentile of the control distribution, or a decrease of 0.5 standard deviation), and (2) a 5 or 10% decrease in the proportion of fetuses below the 5th or 10th percentile, respectively, of the control distribution. These results support the use of BMDs as providing a more consistent basis for risk assessment than do NOAELs.

Studies on the Toxicity of Phthalates via Ingestion

Studies on the Toxicity of Phthalates via Ingestion

Studies on the toxicity of phthalates via ingestion.

Some recent publications have revealed the presence of phthalates in rather unexpected locations (1-4). Mayer (3) reported that di-n-butyl phthalate and di-2-ethylhexyl phthalate had measurable effects on the Daphnia, an accepted subject for studying the aquatic food chain in the environment. The ubiquity of phthalates has been well documented. Their presence in stored blood samples and in some randomly sampled humans is reason for concern, even though no directly related harmful effect has been clearly demonstrated to date. Numerous nontechnical writings displayed serious concern about the subject of phthalates in the environment. Some of these writings showed signs of emotional reaction, inappropriate generalizations and an unawareness of the content of technical studies in the literature concerning the subject. Nearly one billion pounds of phthalates are consumed in the U.S.A. per year. They are a critically necessary component of plastics, coatings and other industries that are an integral part of our life. In order to encourage mature perspective concerning the presence and effects of phthalates in our environment, one must retain perspective concerning the relative hazard or safety of various substances as a