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.

Systemic uptake of inhaled arsenic in rabbits.

Human occupational exposure to sufficiently high levels of arsenic in air has been associated with lung cancer, but generally not other types of cancer. Thus, a better understanding of the relationship between airborne arsenic exposures and systemic uptake is essential. In this study, rabbits were exposed to one of four levels of arsenic trioxide in air for 8 h/day, 7 days/week, for 8 weeks (0.05, 0.1, 0.22, or 1.1 mg/m3). Plasma levels of inorganic arsenic, monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) were measured following the last exposure. Although there was a dose-related increase in plasma levels of methylated arsenic metabolites, statistically significant increases in mean inorganic arsenic levels in plasma were observed only in male rabbits exposed to 0.22 mg/m3, and in both males and females exposed to 1.1 mg/m3. Mean inorganic arsenic levels in plasma in males and females exposed to 0.05 and 0.1 mg/m3, and females exposed to 0.22 mg/m3, were not significantly elevated compared to controls. These results suggest that arsenic inhalation has a negligible impact on body burden of inorganic arsenic until air levels are significantly elevated. Based on plasma measurements of inorganic arsenic, the two lowest exposure levels in this study (0.05 and 0.1 mg/m3) are indistinguishable from background.

Potential teratogenicity of di-n-butyltin dichloride and other dibutyltin compounds.

The developmental toxicity of di-n-butyltin dichloride (DBT-dC) was evaluated in Wistar rats following oral administration. No maternal toxicity, embryotoxicity, or malformations were observed at 1, 2.5, or 5 mg DBT-dC/kg body weight. Signs of maternal toxicity, including decreased food consumption, body weight gain, and thymus weight, were observed at 10 mg/kg body weight DBT-dC. At this dose, no evidence of embryotoxicity, including such measures as total resorptions, viable fetuses, or fetal weights, was noted in any litter data. There was a slightly increased frequency of total malformations at the 10 mg/kg dose level of 4/262 treated vs. 1/269 control fetuses. All defects occurred singly with no clustering nor organ system pattern of occurrence, which would be indicative of a teratogenic effect. The no-observed-adverse-effect-level (NOAEL) for prenatal as well as maternal toxicity was considered to be 5 mg DBT-dC/kg body weight. The interpretation and utility of previously published studies on the developmental toxicity of dibutyltin compounds are confounded by dose regimen and data reporting deficiencies. These studies suggest that, after oral administration during days 6-17 of pregnancy, the NOAEL for malformations in rats of different strains ranges from 1.7 to 5 mg/kg body weight. In these studies, the maternal LD50 was reported to be about 8 mg/kg body weight in one study but at greater than 15 mg/kg in others. Thus, the NOAEL for teratogenicity may be roughly estimated to be from one-tenth to one-third of the maternal LD50. When evaluated, thymus involution, a typical but reversible effect of di- and tri-butyltin compounds, was also observed at 5-10 mg/kg body weight. The most susceptible time for inducing teratogenic effects is reported to be days 7-9 of pregnancy, but malformations have also been found with dosing over longer duration at lower doses. It is doubtful that the findings of malformations at highly toxic doses in animals has any health hazard significance, especially when human exposure to dibutyltins typically occurs at several orders of magnitude lower than the doses used in these studies. Further comparative pharmacokinetic studies would be necessary in order to refine the hazard characterization.

Proposed occupational exposure limits for select ethylene glycol ethers using PBPK models and Monte Carlo simulations.

Methoxyethanol (ethylene glycol monomethyl ether, EGME), ethoxyethanol (ethylene glycol monoethyl ether, EGEE), and ethoxyethyl acetate (ethylene glycol monoethyl ether acetate, EGEEA) are all developmental toxicants in laboratory animals. Due to the imprecise nature of the exposure data in epidemiology studies of these chemicals, we relied on human and animal pharmacokinetic data, as well as animal toxicity data, to derive 3 occupational exposure limits (OELs). Physiologically based pharmacokinetic (PBPK) models for EGME, EGEE, and EGEEA in pregnant rats and humans have been developed (M. L. Gargas et al., 2000, Toxicol. Appl. Pharmacol. 165, 53-62; M. L. Gargas et al., 2000, Toxicol. Appl. Pharmacol. 165, 63-73). These models were used to calculate estimated human-equivalent no adverse effect levels (NAELs), based upon internal concentrations in rats exposed to no observed effect levels (NOELs) for developmental toxicity. Estimated NAEL values of 25 ppm for EGEEA and EGEE and 12 ppm for EGME were derived using average values for physiological, thermodynamic, and metabolic parameters in the PBPK model. The uncertainties in the point estimates for the NOELs and NAELs were estimated from the distribution of internal dose estimates obtained by varying key parameter values over expected ranges and probability distributions. Key parameters were identified through sensitivity analysis. Distributions of the values of these parameters were sampled using Monte Carlo techniques and appropriate dose metrics calculated for 1600 parameter sets. The 95th percentile values were used to calculate interindividual pharmacokinetic uncertainty factors (UFs) to account for variability among humans (UF(h,pk)). These values of 1.8 for EGEEA/EGEE and 1.7 for EGME are less than the default value of 3 for this area of uncertainty. The estimated human equivalent NAELs were divided by UF(h,pk) and the default UFs for pharmacodynamic variability among animals and among humans to calculate the proposed OELs. This methodology indicates that OELs (8-h time-weighted average) that should protect workers from the most sensitive adverse effects of these chemicals are 2 ppm EGEEA and EGEE (11 mg/m(3) EGEEA, 7 mg/m(3) EGEE) and 0.9 ppm (3 mg/m(3)) EGME. These recommendations assume that dermal exposure will be minimal or nonexistent.

Safety assessment of DHA-rich microalgae from Schizochytrium sp.

Schizochytrium sp. (DRM) contains oil rich in highly unsaturated fatty acids (PUFAs). Docosahexaenoic acid (DHA) is the most abundant PUFA component of the oil (approx. 35% w/w). DHA-rich extracted oil from Schizochytrium sp. is intended for use as a nutritional ingredient in foods. As part of a comprehensive safety assessment program, the developmental toxicity of DRM was assessed in Sprague-Dawley derived rats [25/group, provided DRM in the diet at 0.6, 6, and 30% on gestation days (GD) 6-15] and in New Zealand White (NZW) rabbits (22/group, dosed with DRM at levels of 180, 600, and 1800 mg/kg/day by oral gavage on GD 6-19). Fish oil was used as a negative control at dose levels to provide an equivalent amount of fat to that received by the high-dose DRM rabbits. Maternal food consumption, body weights, and clinical signs were recorded at regular intervals throughout these studies. Animals were sacrificed on GD 20 (rats) and GD 29 (rabbits) and examined for implant status, fetal weight, sex, and morphologic development. No clinical signs of toxicity were observed. Maternal exposure to DRM during organogenesis did not adversely affect the frequency of postimplantation loss, mean fetal body weight/litter, or external, visceral, or skeletal malformations in either the rat or the rabbit. In the rats, neither maternal nor developmental toxicity was observed at any dietary concentration of DRM. Thus, 22 g/ kg/day(1) of DRM administered in the feed to pregnant rats during organogenesis was the NOEL (no-observed-effect level) for both maternal and developmental toxicity. In rabbits, no maternal toxicity was expressed at DRM dose levels of 180 and 600 mg/kg/day. As a possible consequence of the high-fat content of the fish oil and DRM, reductions in food consumption and body weight gain and a slight increase in abortions occurred in the fish oil control and 1800 mg/kg/day DRM groups. Developmental toxicity was not observed at any DRM dose level. Based on the results of this study, the NOEL for maternal toxicity of DRM was 600 mg/kg/day, and the NOEL for developmental toxicity was 1800 mg/kg/day in NZW rabbits.

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.

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 10mg/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 10mg/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 5mg/kg/day group caused the maternal no-observed-adverse-effect level (NOAEL) to be determined as 2. 5mg/kg/day. Intrauterine parameters were unaffected by arsenic trioxide. No treatment-related foetal malformations were noted in any dose group. Increased skeletal variations at 10mg/kg/day were attributed to reduced foetal weight at that dose level. The developmental NOAEL was thus 5mg/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.

Interpreting the toxicologic significance of alterations in anogenital distance: potential for confounding effects of progeny body weights.

Anogenital distance (AGD) is an endpoint that was recently added to the U.S. EPA testing guidelines for reproductive toxicity studies. This endpoint is sensitive to hormonal effects of test chemicals. It is possible that apparent alterations in AGD might occur after treatment with agents that affect overall pup body size. In such cases, hormonal activity might be associated incorrectly with the test treatment. The analyses in this report evaluated statistical correlations between pup body weight and AGD in control litters. AGDs were measured on postnatal day 1 in 1501 pups derived from 113 untreated female Sprague-Dawley rats in two independent two-generation reproductive toxicity studies. Significant correlations were detected between AGD and body weight and between AGD and the cube root of body weight. In males, AGD increased 0.26 mm for each 1 g increase in body weight. In females, AGD increased 0.13 mm per 1 g increase in body weight. Although there were essentially no differences between the regression models developed to predict AGD in either males or females using body weight as a covariate and those based on the cube root of body weight, such similarities in predictivity might not occur in larger animals with broader weight ranges than those encountered in this analysis. Normalization of AGD by dividing by body weight significantly overcompensated for differences in body size. Normalizing with the cube root of body weight resulted in an AGD/cube root of body weight ratio that was constant across the range of body weights observed in this study. In conclusion, as a preferred method to account for body size effects on AGD, analysis of covariance is recommended. If a normalization is done directly, the ratio of AGD to the cube root of body weight is the more appropriate metric.

Comparative effects of single intraperitoneal or oral doses of sodium arsenate or arsenic trioxide during in utero development.

Numerous studies have suggested that single-day intraperitoneal (IP) injection of inorganic arsenic results in failure of neural tube closure and other malformations in rats, hamsters, and mice. Most of these studies involved treatment of limited numbers of animals with maternally toxic doses of arsenic (generally As(V)), without defining a dose-response relationship. In the present Good Laboratory Practice-compliant study, sodium arsenate (As(V)) was administered IP and arsenic trioxide (As(III)) was administered either IP or orally (by gavage) on gestational day 9 to groups of 25 mated Crl:CD(R)(SD)BR rats. Only at dose levels that caused severe maternal toxicity, including lethality, did IP injection of arsenic trioxide produce neural tube and ocular defects; oral administration of higher doses of arsenic trioxide caused some maternal deaths but no treatment-related fetal malformations. In contrast, IP injection of similar amounts of sodium arsenate (based on the molar amount of arsenic) caused mild maternal toxicity but a large increase in malformations, including neural tube, eye, and jaw defects. In summary, neural tube and craniofacial defects were observed after IP injection of both As(V) and As(III); however, no increase in malformations was seen following oral administration of As(III), even at maternally lethal doses. These results demonstrate that the frequently cited association between prenatal exposure to inorganic arsenic and malformations in laboratory animals is dependent on a route of administration that is not appropriate for human risk assessment.

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.

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.

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.

A communicating intestinal duplication in a Sprague-Dawley rat.

Duplications of the gastrointestinal tract are exceedingly rare in laboratory animals. We report a case of a communicating intestinal duplication in a 17-wk-old Sprague-Dawley (SD) rat. The duplication was present in the mesenteric border of the ileum, and both proximal and distal ends were communicated with the lumen of ileum. Histologically, the duplicated portion had a thick muscle wall and a mucosa similar to that of the small intestine. This is the first reported case of intestinal duplication in an SD rat.

Developmental toxicity assessment of arsenic acid in mice and rabbits.

To evaluate potential effects of exposure to inorganic arsenic throughout major organogenesis, CD-1 mice and New Zealand White rabbits were gavaged with arsenic acid dosages of 0, 7.5, 24, or 48 mg/kg/d on gestation days (GD) 6 through 15 (mice) or 0, 0.19, 0.75, or 3.0 mg/kg/d on GD 6 through 18 (rabbits) and examined at sacrifice (GD 18, mice; GD 29, rabbits) for evidence of toxicity. Two high-dose mice died, and survivors at the high and intermediate doses had decreased weight gains. High-dose-group fetal weights were decreased; no significant decreases in fetal weight or increases in prenatal mortality were seen at other dosages. Similar incidences of malformations occurred in all groups of mice, including controls. At the high dose in rabbits, seven does died or became moribund, and prenatal mortality was increased; surviving does had signs of toxicity, including decreased body weight. Does given lower doses appeared unaffected. Fetal weights were unaffected by treatment, and there were no effects at other doses. These data revealed an absence of dose-related effects in both species at arsenic exposures that were not maternally toxic. In mice, 7.5 mg/kg/d was the maternal No-Observed-Adverse-Effect-Level (NOAEL); the developmental toxicity NOAEL, while less well defined, was judged to be 7.5 mg/kg/d. In rabbits, 0.75 mg/kg/d was the NOAEL for both maternal and developmental toxicity.

Subchronic, developmental, and genetic toxicology studies with the ethane sulfonate metabolite of alachlor.

The ethane sulfonate (ESA) metabolite of the herbicide alachlor is formed in soil by microbial action. The present studies were conducted to assess the toxicity of ESA and provide a base set of data for risk assessment. ESA did not induce chromosomal effects in a mouse bone marrow micronucleus assay following acute administration. Administration of ESA to rats in drinking water at concentration of 200, 2000, and 10,000 ppm for 91 days elicited biologically significant indications of toxicity only at the high-dose level (1002 mg/kg/day). The observed responses included decreases in body weights and food consumption as well as effects on clinical chemistry values. Many of the changes appeared to be due to decreased palatability of the drinking water. There were no ESA-induced gross pathology findings, organ weight changes, or microscopic lesions. ESA did not produce any adverse effects in pregnant rats or their offspring even at 1000 mg/kg/day, the highest dose tested. These findings show that the subchronic and developmental toxicity of ESA are low. Furthermore, comparison of results from studies with alachlor and its metabolite shows that the toxicity of ESA is substantially lower. Margins of exposure for ESA range from 133,824 to 2,573,529 even using worst-case estimates of exposure, indicating that the metabolite poses little risk of producing adverse effects at the very low levels occasionally encountered. These results and accompanying analyses support the conclusion that ESA is not of toxicological concern.

Developmental toxicity of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). I. Multireplicated dose-response studies in four inbred strains and one outbred stock of mice.

A large-scaled multireplicated developmental toxicity study was conducted in various strains/stocks of mice with the herbicide, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), by gavage on Gestational Days 6 through 14. The most important attributes of the study design were replicated test groups, a minimum of four dose levels per replicate, use of multiple stocks/strains of animals to obtain an estimate of the range in sensitivities due to genotype, complete pathological evaluation of maternal animals, and histopathological as well as teratological evaluation of the fetuses. Developmental toxicity was observed at doses below those producing discernible or measurable maternal toxicity. Regression and/or probit analyses were conducted to determine whether a dose-response relationship existed. Reduced fetal weight and increased incidence of cleft palate and embryolethality were the most significant prenatal effects of 2,4,5-T exposure observed in this study. Each strain/stock exhibited a dose-related decrease in fetal weight with the CD-1 mice having the steepest slope and the A/J mice having the shallowest slope. There was a striking similarity among the slopes of the dose-response curves for the various strains/stocks. The mean incidence of embryolethality in the A/J strain was significantly greater than that of the other strains or stocks. There was substantial variation among replicates within strains. The use of the replicated study design was logistically necessary due to the magnitude of the study and it also served to increase the statistical power of the study.

Developmental toxicity of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). II. Multireplicated dose-response studies with technical and analytical grades of 2,4,5-T in four-way outcross mice.

A series of multireplicated developmental toxicity studies were conducted in four-way outcross mice and CD-1 outbred mice administered either analytical or technical grades of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by gavage on Gestational Days 6 through 14. The formulations of 2,4,5-T differed by a factor of 10-fold in 2,3,7,8-tetrachlorodibenzo-p-dioxin levels. Reduced fetal weight and increased incidences of cleft palate and embryolethality were the most significant prenatal effects of both formulations of 2,4,5-T observed in all strains/stocks of mice. Both the outcross and outbred mice exhibited a dose-response relationship with each of the above endpoints and the dose-response curves were parallel. There were no embryotoxic or fetotoxic differences between the technical and analytical grades of 2,4,5-T with regard to extent of fetal weight reduction, resorption rate, or cleft palate incidence. There was little difference in the results between the four-way outcross mouse and the CD-1 outbred mouse.

Blood flow during pregnancy in the rat: I. Flow patterns to maternal organs.

Normal blood flow patterns to several maternal organs were characterized in individual CD rats, nonpregnant (NP) or on day 6, 7, 8, 10, 11, 12, 13, 16, 18, or 20 of gestation using the radioactive microsphere technique. Weights and flow values were determined for several uterine tissue samples as well as maternal organs. No significant changes were found in blood flow to the stomach, spleen, and urinary bladder of these animals. There also were no remarkable changes as pregnancy progressed in blood flow to the lungs, suggesting that no marked arterial-venous shunting occurs in maternal placental tissues over gestation. Slight but consistent decreases in absolute (ml/min) and relative (ml/min/g tissue) blood flow to the brain were noted, and percent cardiac output (CO) was significantly decreased on all days of gestation except day 7. Complex changes were observed in blood flow to the kidneys, liver, adrenals, and heart. Absolute flow to the kidneys and liver reached maximum values on day 11, although percent CO delivered to both organs was consistently reduced throughout gestation. Absolute flow to the heart and adrenals peaked on day 13 and days 11-12, respectively. Absolute flow to the ovaries increased nearly 5-fold from the NP state (0.36 +/- 0.11) to day 20 of pregnancy (1.61 +/- 0.33). Interlitter differences in ovarian blood flow during midgestation were found to be a result of differences in litter size and distribution of embryo/fetuses between the two uterine horns. The fact that the majority of changes observed in maternal organ flow coincide with placental development, rapid augmentation of total uterine flow, and/or maternal hormonal changes suggests that these patterns may be important indicators of the dynamic physiology of pregnancy.

Blood flow during pregnancy in the rat: II. Dynamics of and litter variability in uterine flow.

Normal blood flow patterns were characterized in individual CD rats, nonpregnant (NP) or on day 6, 7, 8, 10, 11, 12, 13, 16, 18, or 20 of gestation using the radioactive microsphere technique. Five animals were evaluated at each stage of pregnancy. Weights and flow values were determined for several maternal organs and uterine tissue samples. Embryo/fetal (E/F) sex was determined from day 11 on by measuring the prevalence of nuclei with sex chromatin in amnion smears. There was a marked increase in absolute flow to the uterus and all uterine contents during gestation (0.28 +/- 0.13 ml/min to the NP, diestrous uterus; 9.07 +/- 0.97 ml/min on day 20 of pregnancy). However, relative blood flow (ml/min/g tissue) decreased by day 20 to one-third NP values. Thus, though blood flow greatly increased, it did so at a rate lower than total tissue growth (including the uterus, placental tissues, and the E/F itself). There was a rapid redistribution of blood flow from the decidua parietalis (DEC) to the chorioallantoic placenta (CAP) on days 11-13, with nearly equal flow being delivered to the CAP and DEC of the "average" embryo on day 12 of gestation. By day 16 the DEC functionally had atrophied, and nearly 100% of the flow was delivered to the CAP. Male E/Fs tended to weigh more than female; however, these differences were statistically significant only on days 13, 18, and 20. E/F sex was not found to be strongly related to any of the variables evaluated in this study except E/F weight. Significant interlitter variability in E/F weight and blood flow consistently was observed at all gestational stages. Differences in litter size and E/F distribution within the two uterine horns did not account for the majority of this variability.