Emergency do not consume/do not use concentrations for blended phosphates in drinking water.

The U.S. Congress [PL 107-188] amended the Safe Drinking Water Act and required each community water system serving more than 3,000 people to conduct vulnerability assessments. These assessments address potential circumstances that could compromise the safety and reliability of municipal water. The present evaluation concerns the concentrations of the blended phosphates (also known as polyphosphates, condensed complex phosphates, polyphosphate glassy balls, and pyrophosphates) intended to aid regulatory agencies in decisions to avoid contact with affected water. Polyphosphates are direct food additives and they are used to treat municipal drinking water, but depending upon the concentration and duration of exposure these substances can induce chemical burns. Ingested polyphosphates are degraded by phosphatase enzymes to monophosphates, substances that are over-the-counter bowel purgatives. High oral doses of the monophosphates can induce transient hyperphosphatemia in older and susceptible young people, which can lead to acute phosphate nephropathy. In some patients, the condition is fatal. Based on the acute diarrhea after the ingestion of a single oral dose of monobasic (NaH2PO(4)) and dibasic (Na2HPO(4)) monophosphates in adults, a do not consume concentration of 600 mg PO(4)/L can be derived. Based on mild local irritation after topical application of 1.0% sodium metaphosphate [(NaPO(3))6 • H2O] to intact skin of sensitive volunteers, a do not use concentration of 8,000 mg PO4/L can be assigned. Given the lack of eye irritation in rabbits after direct instillation of 0.2% (NaPO(3))6 • H2O, an acute ocular contact limit of 50 mg PO4/L serves as the overall do not use level.

Emergency Do Not Consume/do Not Use concentrations for potassium permanganate in drinking water.

Over the past decade, regulatory authorities and water purveyors have become increasingly concerned with accidental or intentional adulteration of municipal drinking water. Emergency response guidelines, such as the 'Do Not Consume' or use concentration limits derived herein, can be used to notify the public in such cases. Potassium permanganate (KMnO(4)) is used to control iron concentrations and to reduce the levels of nuisance materials that affect odor or taste of finished drinking water. Manganese (Mn) is recognized an essential nutrient, permanganate (MnO4 (-)) and manganous (Mn(+2)) ions are caustic, and the acute toxicity of KMnO(4) is defined by its oxidant/irritant properties and by the toxicity of Mn. Ingestion of small amounts (4-20 mg/kg) of aqueous KMnO(4) solutions that are above 200 mg/L causes gastrointestinal distress, while bolus ingestion has caused respiratory arrest following coagulative necrosis and hemorrhage in the esophagus, stomach, or liver. Dilute KMnO(4) solutions (1-100 mg/L) are used as a topical antiseptics and astringents, but >1:5000 (200 mg/L) dilutions can irritate or discolor sensitive mucous membranes and direct skin or ocular contact with concentrated KMnO(4) can perforate tissues. Based on clinical experience with 200 mg/L KMnO(4), a Do Not Consume concentration of 7 mg/L KMnO(4) (equivalent to 2 mg Mn/L) is recommended. Recognizing limited empirical data from which to calculate an ocular reference value, a skin contact 'Do Not Use' concentration of 30 mg Mn/L is recommended based on the skin irritation in some patients after a 10-min contact with 100 mg KMnO4/L.

Emergency do not consume/do not use concentrations for ferric chloride in drinking water.

The U.S. Congress [PL 107-188] amended the Safe Drinking Water Act and required each community water system serving more than 3,000 people to conduct vulnerability assessments. These assessments address potential circumstances that could compromise the safety and reliability of municipal water. Ferric chloride is used in coagulation and flocculation, and it is used to treat raw water with high viral loads, elevated dissolved solids or high bromide. Iron is an essential nutrient, but elevated concentrations of FeCl3 are corrosive as a result of hydrolysis to HCl. Based on a no-observed-adverse effect level (NOAEL) of 0.5% FeCl3 • 6H2O administered in drinking water to male and female F344 rats for up to 2 years, a do not consume concentration of 200 mg FeCl3 /L can be derived. Since instillation of 0.3 M (48.7 g/L) FeCl3 in saline to rodent vagina failed to elicit damage, a topical do not use concentration of 2000 mg FeCl3/L (600 mg Fe/L) can be assigned. The only FeCl3 data available to quantify ocular toxicity involved a pH 1 solution in rabbit eyes, but HCl instillation (pH 2.5) to rabbit eyes found permanent corneal ulceration after 10 min. The pH of FeCl3 in water at the do not use limit (2.4-2.6) is near the pH (2.0) considered corrosive by regulatory agencies. As direct eye contact with water at pH 4.5 or below increases complaints of ocular discomfort, emergency response plans that address FeCl3 in drinking water must account for Fe levels and the pH of the affected water.

Weight-of-evidence versus strength-of-evidence in toxicologic hazard identification: Di(2-ethylhexyl)phthalate (DEHP).

Toxicokinetic and mode of action data for DEHP reduce the concern for its potential carcinogenic hazard to human health. Chronic, high dose ingestion of DEHP and related peroxisome proliferators (PP) by mice and rats precipitate the following: activation of peroxisome proliferator activated receptor (PPARalpha) and its binding to peroxisome proliferator response elements (PPREs) within promoters of PP-responsive genes, peroxisome proliferation, increased microsomal fatty acid oxidation, increased hepatic hydrogen peroxide, hepatomegaly, hyperplasia and subsequent neoplasia. Neither peroxisome proliferation nor increased liver cancer occur in patients treated with pharmacologic doses of PP. Species differences in endogenous PPARalpha expression and differential activity of the peroxisome proliferator response element (PPRE) contribute to the failure of humans to respond in a manner qualitatively similar to that of rats or mice. Where it can be demonstrated that a mechanism for rodent tumor formation has no relevance for humans, then a substance which elicits a carcinogenic response in the test species via that mechanism should not be classified as anything other than an animal carcinogen. Systemic noncarcinogenic endpoints are available for definition of a DEHP reference dose. Considerable difficulty is encountered in the revision of promulgated regulations and in public risk communication when a material is no longer considered a carcinogenic hazard to humans.

Distribution, teratogenicity, and embryonic delivered dose of retinoid Ro 23-9223.

Ro 23-9223 is a highly lipophilic aromatic retinoid with antiproliferative and sebum supressive effects in preclinical disease models of acne. To investigate the relation between Ro 23-9223 developmental toxicity, drug distribution, and transplacental transfer, groups of pregnant hamsters were given oral doses of 50-500 mg/kg Ro 23-9223 on days 8 and 9 of gestation. The teratogenic phenotype induced at doses greater than 125 mg/kg per day was similar to that found after exposure to doses of 13-cis-retinoic acid (isotretinoin, Accutane) greater than 37.5 mg/kg per day. Oral bioavailability of Ro 23-9223 was very low compared to 13-cis-retinoic acid. The highest concentrations of Ro 23-9223 were found in maternal liver, lung, adipose tissue, cardiac muscle, and placenta, whereas only little of the compound crossed the blood-brain barrier. Based on embryo AUC, Ro 23-9223 had a 30- to 50-fold greater embryo:maternal concentration ratio than 13-cis-retinoic acid plus its bioactive metabolites following similar doses of the two retinoids. In preclinical pharmacology studies, oral doses of Ro 23-9223 (5 mg/kg per day) and 13-cis-retinoic acid (10 mg/kg per day) produced comparable gland size reductions in the hamster ear sebaceous gland reduction assay. Under these conditions, Ro 23-9223 plasma AUC was 40 times smaller than that of 13-cis-retinoic acid plus its bioactive metabolites. Assuming that the near linear dose-exposure relationship of Ro 23-9223 extends beyond the dose range of this study, embryo AUCs of Ro 23-9223 and 13-cis-retinoic acid (plus metabolites) would be near identical following pharmacologically equivalent doses. A comparison of embryo retinoid AUCs suggests a 4-fold lower teratogenic potency of Ro 23-9223 compared to with 13-cis-retinoic acid. Despite high embryo levels in hamsters, the data suggest an improved therapeutic index for Ro 23-9223 compared with 13-cis-retinoic acid in a preclinical acne disease model.

Embryonic delivered dose of isotretinoin (13-cis-retinoic acid) and its metabolites in hamsters.

All-trans-retinoic acid (all-trans-RA) is required in normal embryogenesis and both deficiency and excess are teratogenic. Isotretinoin (13-cis-RA) is teratogenic in all species examined; based on administered dose, humans appear most sensitive, followed by (in order or decreasing sensitivity) monkey, rabbit, hamster, mouse, and rat. Identification of the teratogenic threshold in these species is difficult because RAs are normal physiologic constituents. The rabbit no-observed-adverse-effect-level (NOAEL) and lowest-observed-adverse-effect-level (LOAEL) administered doses (3 and 15 mg/kg/day, respectively, on gestation Days 8-11) are less than the corresponding values in hamster (7.5 and 37.5 mg/kg/day, respectively, on gestation Days 7 and 8), but drawing conclusions from administered dose alone ignores differences in absorbed, metabolized, and embryonic delivered dose. Therefore, distribution and metabolism studies of 13-cis-RA at the NOAEL and LOAEL in pregnant hamsters were performed and plasma and tissue concentrations of parent compound and metabolites were compared to those found in rabbits. Metabolites of 13-cis-RA common to all species include three RAs (all-trans-RA, all-trans-4-oxoRA, 13-cis-4-oxoRA) and the glucuronide conjugate of 13-cis-RA (13-cis-RAG). As in rabbits, we found 13-cis-4-oxoRA also to be the major metabolite of 13-cis-RA in hamster plasma, peripheral tissues, and embryo. Of maternal tissues, peak 13-cis-RA concentrations were highest in liver. Total concentration of RA (13-cis-RA + 13-cis-4-oxoRA + all-trans-RA + all-trans-4-oxoRA) per gram of wet tissue was greatest in maternal liver, followed by that in lung, adipose tissue, muscle, kidney, and brain. At the NOAEL, total RA plasma Cmax in hamster was 6 times that in rabbit; at the LOAEL, hamster plasma total RA Cmax was 4 times that in rabbit. Hamster absorbed and metabolized dose (as AUC of plasma total RA) at the NOAEL and LOAEL was 2.6 and 2.4 times that in rabbit, respectively. In the embryo, hamster total RA Cmax was 2.7 times (at NOAEL) and 2.6 times (at LOAEL) that in rabbit. However, embryonic delivered dose (total RA AUC in hamster and rabbit embryo, respectively) at the NOAEL (2.08 and 2.14 microg . hr.g-1) and LOAEL (5.34 and 5.54 microg . hr . g-1) was virtually identical. Embryonic AUCs in hamster and rabbit for all-trans-RA and all-trans-4-oxoRA, metabolites which transactivate directly the nuclear RA receptors (RARs), were also very similar at the NOAEL (0.66 and 0.81 microg . hr g-1) and at the LOAEL (1.14 and 1.32 microg . hr g-1). Based on embryonic delivered dose, we suggest that 13-cis-RA is an equipotent teratogen in hamster and rabbit.

Receptor-selective retinoid agonists and teratogenic activity.

Retinoid mechanism of action is dependent upon interaction with the retinoid nuclear RAR and RXR subfamilies of receptors. Study of ligands selective for the different receptors or which modify that interaction may provide insight into which receptors play roles in or contribute to retinoid teratogenesis. The retinoids considered here include in the RAR alpha-selective arylcarboxamidobenzoic acid CD 336 (Am580), the RAR beta/gamma-selective naphthalenecarboxylic acid CD 135 and the adamantyl-phenylcarboxamidobenzoic acid CD 394, the RAR-selective tetrahydrotetra-methylanthracenylbenzoic acid (TTAB) SRI 3961, the carboxyphenylretinamide SRI 7167-67, and the RXR-selective diarylisopropylidene SR 11217. CD 135 has a 3-fold higher affinity for RAR beta when compared with RAR gamma, whereas CD 394 has a 3-fold higher affinity for RAR gamma when compared with RAR beta. A separate investigation into potential amelioration of retinoid teratogenesis by concomitant administration of the cyclohexanetrione (Ro 31-0521) was also conducted. When pregnant hamsters were given an oral bolus of CD 336 or CD 135 during the early primitive streak stage of gestation, these retinoids proved 60-100 times more potent teratogens than all-trans-retinoic acid. Intubation of CD 394 resulted in production of terata similar to that seen after an equivalent dose of all-trans-retinoic acid. Administration of SRI 3961 found this compound 8000 times more potent than all-trans-retinoic acid, while SRI 7167-67 failed to show any evidence for developmental toxicity even after exposure to 105 mg/kg. Studies with the RXR-selective SR 11217 found it to be far less potent than all-trans-retinoic acid. These data point to the conclusion that those retinoids which have no affinity for retinoid nuclear receptors also have little potential for induction of developmental toxicity at doses which do not also provoke maternal intoxication. Comparing in vitro transcriptional activation of wild-type human RAR for the supertoxic TTNBP (Ro 13-7410) and TTAB (SRI 3961) with their relative teratogenic potency in hamster found that the more toxic congener also had the lower in vitro EC50 transactivation value (at ratios approximating their differential toxicities measured as administered dose). The RAR beta/gamma-selective CD 135 (TTNN) was not as efficient as TTNBP (Ro 13-7410) or TTAB (SRI 3961) in hRAR transactivation and CD 135 was less toxic than either Ro 13-7410 or SRI 3961. Although the RXR-selective SR 11217 failed to elicit terata after moderate doses, malformations consistent with those induced by high doses of retinoic acid could be produced following a single large bolus of SR 11217. Under the conditions here, simultaneous administration of Ro 31-0521 with all-trans-retinoic acid appeared to reduce the total percentage of abnormal fetuses seen after exposure to retinoic acid alone, but fetal body weights remained depressed and the numbers of dead embryos remained elevated, suggesting only limited influence of the cyclohexanetrione on retinoid developmental toxicity.

Comparative disposition, receptor affinity, and teratogenic activity of sulfon arotinoids.

To investigate the relationship between sulfon arotinoid biotransformation and teratogenic activity, the potency of the ethyl (Ro 15-1570) and methyl (Ro 14-9706) arotinoid sulfones and their in vivo disposition in pregnant hamsters were studied. Administration of Ro 15-1570 was teratogenic, but Ro 14-9706 showed no such activity. Total absorbed doses of the ethyl and methyl sulfones (measured as maternal plasma AUC) were very similar. Total delivered dose of Ro 14-9706 to liver and lung was 120-160% that of Ro 15-157, and Ro 14-9706 was transferred in greater amounts to the embryo as well. Placenta AUC for parent sulfon arotinoids was 160-250% that in the embryo. Plasma analyses by HPLC suggested that the ethyl sulfone was oxidized and appeared in maternal plasma as the corresponding sulfinic (Ro 14-9572) and sulfonic (Ro 14-3899) acids, amounting to 10% and 16%, respectively, of the mean maternal ethyl sulfone Cmax value. The concentrations of sulfinic and sulfonic metabolites were always less than the analytical limit of detection in placenta and embryo after maternal ethyl sulfone intubation. Neither the sulfinic nor the sulfonic acid were ever detected in maternal circulation, placenta, or embryo after methyl sulfone intubation. Comparisons of their binding affinities found that neither the ethyl nor the methyl arotinoid sulfone could act as a ligand for cellular retinoic acid-binding protein (CRABP), nor could these compounds bind retinoid nuclear receptors (RAR). Transcriptional activation of RARs was weak and similar for both compounds. The sulfinic and sulfonic acid arotinoids bind and transactivate RARs, and bind CRABP with efficiencies similar to all-trans-retinoic acid. Furthermore, they are active in cultured limb bud chondrocytes. The results suggest that the methyl sulfone (in accord with its lack of activity in cultured limb bud chondrocytes) is of no toxicologic significance in hamster embryo--even after relatively high delivered dose. Teratogenicity of the ethyl sulfone (which shows marked inhibition of chondrogenesis in cultured limb bud) does not appear to depend on measurable concentrations of these sulfinic/sulfonic acid metabolites in the hamster embryo.

Selenium kinetics, placental transfer, and neonatal exposure in cynomolgus macaques (Macaca fascicularis).

Forty pregnant cynomolgus macaques were treated daily from gestational day 20 to 50 by nasogastric intubation of 0, 25, 150, or 300 micrograms selenium as L-selenomethionine/kg body weight. In each group, 7-8 pregnancies were terminated by hysterotomy at gestational day 100 +/- 2 and the fetuses were examined, while 2-3 pregnancies in each group were allowed to proceed to term. Selenium and soluble glutathione peroxidase were measured in: maternal, neonatal, and fetal plasma and erythrocytes; fetal kidney, liver, muscle, and placenta; and maternal breast milk. The area under the multidose maternal plasma selenium concentration:time curve, the maximum maternal plasma selenium concentration, and the maternal urinary selenium excretion rates were proportional to the L-selenomethionine dose. Selenium concentrations in all fetal and neonatal, tissues were also proportional to maternal L-selenomethionine dose. Glutathione peroxidase was affected only in maternal erythrocytes, fetal kidney, and neonatal plasma. The selenium concentration in fetal plasma was an average 33% of that in maternal plasma. Although selenium concentrations in macaque milk were doubled by the highest dose, intrauterine selenium accumulation accounted for the majority of the neonatal selenium body burden. Despite the elevated selenium concentrations in fetal tissues, neonatal blood, and milk, no deleterious effects on neonates were observed. These results suggest that primate fetuses are well protected against selenium toxicity arising from high maternal L-selenomethionine intakes.

Embryogenesis in cultured whole rat embryos after combined exposures to 3,3',5-triiodo-L-thyronine (T3) plus all-trans-retinoic acid and to T3 plus 9-cis-retinoic acid.

Retinoid-induced malformations of the jaw, ears, face, skull, eyes, and heart in humans and rodents are well known. Data on nuclear receptors and developmental toxicity bioassays indicate that thyroid hormones can modulate the biologic activity of retinoids. The present investigation concerned the potential for interactions of all-trans-retinoic acid (RA) with 3,3'5-triiodo-L-thyronine (T3) and of 9-cis-retinoic acid (9-cis-RA) with T3 in the morphogenesis of cultured whole rat embryos. Varying concentrations of retinoids or T3 were microinjected into the amniotic fluid or placed in the culture medium alone or in combinations of T3 with each retinoid. At 200 ng/ml, T3 increased the incidence of branchial arch defects produced by either RA or 9-cis-RA but did not elicit branchial arch defects alone except at concentrations significantly compromising survival (2,000 ng/ml; 32% mortality). Similarly high culture medium concentrations of T3 alone were associated with failure of neural tube closure in the rhombencephalon (rhombencephalic schisis). At this concentration, other dysmorphia were minimal and at 670 ng/ml T3, no dysmorphogenic or embryotoxic effects could be detected. Modulation of T3 effects by the yolk sac placenta was suggested by failure of microinjected T3 to elicit dysmorphia at very high amniotic fluid concentrations. RA (300 ng/ml) or 9-cis-RA (600 ng/ml) alone elicited no or minimal rhombencephalic schisis at the highest concentrations studied. RA plus T3 produced a much greater than additive effect on rhombencephalic schisis, whereas 9-cis-RA plus T3 produced a less than additive effect. Conversely, much greater than additive effects on anterior schisis were observed for 9-cis-RA plus T3 whereas combined effects of RA and T3 were approximately additive. For most other dysmorphia, the combined effects of each retinoid with T3 were greater than additive and were particularly striking for cephalic defects.

Incorporation of a micronucleus study into a developmental toxicology and pharmacokinetic study of L-selenomethionine in nonhuman primates.

Concomitant to a developmental toxicology study of selenium in long-tailed macaques (Macaca fascicularis), a transplacental bone marrow micronucleus assay was conducted in the fetuses of treated animals. Selenium was administered as L-selenomethionine by nasogastric intubation at 0, 150 or 300 micrograms/kg-day to pregnant macaques daily throughout organogenesis (gestation days 20-50). Pregnancy was terminated on gestation day 100 +/- 2 and fetuses were obtained by hysterotomy. Selenium concentrations in maternal blood were monitored throughout pregnancy and selenium concentrations in fetal blood were measured at hysterotomy. Maternal circulating selenium did not exceed 4 ppm in plasma or 3.7 ppm in erythrocytes. Selenium in cord blood was < or = 0.1 ppm in plasma and < or = 1.1 ppm in erythrocytes at 300 micrograms/kg-day. Fetal bone marrow smears were prepared from the humerus and micronucleated polychromatic erythrocytes were scored. No increase of micronucleus frequency was detected in any dose group, although signs of maternal selenosis were obvious. This finding is compared to the previous observation that micronuclei were induced in the bone marrow of adult nonpregnant macaques treated at 600 micrograms/kg-day, a lethal dose yielding blood selenium levels to 7.3 ppm in plasma and 5.7 ppm in erythrocytes after 15 days of daily treatment, when death occurred. These data demonstrate that measurement of circulating xenobiotics can be useful for the interpretation of genetic toxicology results.

Effects of excess selenomethionine on selenium status indicators in pregnant long-tailed macaques (Macaca fascicularis).

Forty pregnant long-tailed macaques were treated daily for 30 d with 0, 25, 150, or 300 micrograms selenium as L-selenomethionine/kg body weight. Erythrocyte and plasma selenium and glutathione peroxidase specific activities, hair and fecal selenium, and urinary selenium excretion were increased by and were linearly related to L-selenomethionine dose. Hair selenium was most sensitive to L-selenomethionine dose, with an 84-fold increase in the 300 micrograms selenium/(kg-d) group relative to controls (r = 0.917). Daily urinary selenium excretion (80-fold, r = 0.958), plasma selenium (22-fold, r = 0.885), erythrocyte selenium (24-fold, r = 0.920), and fecal selenium (18-fold, r = 0.911) also responded strongly to L-selenomethionine. Erythrocyte and plasma glutathione peroxidase specific activities increased 154% and 69% over controls, respectively. Toxicity was associated with erythrocyte selenium > 2.3 micrograms/mL, plasma selenium > 2.8 micrograms/mL, and hair selenium > 27 micrograms/g. Plasma, erythrocyte, and hair selenium concentrations may be useful for monitoring and preventing the toxicity of L-selenomethionine administered to humans in cancer chemoprevention trials.

Absorption, distribution and elimination of selenium as L-selenomethionine in non-human primates.

20 adult female macaques (Macaca fascicularis) were given oral doses of L-selenomethionine (L-SeMet) equivalent to 0, 25, 150, 300 and 600 micrograms selenium (Se)/kg body weight, and plasma, erythrocyte, hair, faecal and urine Se concentrations were determined. The macaques were scheduled for 30 daily oral doses of L-SeMet, but systemic toxicity necessitated dose reduction in several animals; two macaques given 600 micrograms Se/kg body weight/day for 10-15 days died, and the concentration of Se in their tissues was determined and compared with Se concentrations in tissues collected from one untreated animal. Circulating and urinary Se concentrations in control macaques were within the normal human ranges. Plasma, erythrocyte, hair and urinary Se concentrations were generally dependent on the dose of L-SeMet administered. Plasma Se reflected more immediately exposure to L-SeMet, whereas erythrocyte Se concentrations increased and decreased more slowly. In some cases, erythrocyte Se was still increasing or showed a plateau after L-SeMet treatment was discontinued. Plasma Se concentrations of 6.7-7.3 ppm were observed in the two animals that died due to acute toxicity to L-SeMet. Neither plasma nor erythrocyte GPx activity was influenced by a single L-SeMet dose, but an increase in erythrocyte GPx activity occurred with continuous exposure. Total tissue Se increased 13-28-fold in macaques given 600 micrograms Se/kg body weight/day for 10-15 days, with the liver and kidneys containing the the highest Se concentrations.

Inner ear malformations induced by isotretinoin in hamster fetuses.

Inner ear malformations induced in anotic hamster fetuses following maternal treatment with 50 mg/kg isotretinoin (13-cis-retinoic acid) on gestational day 8 are described. Computer-assisted three dimensional reconstruction was used. Two general types of defective vestibulocochlear development were seen. Defects were bilateral and correlated with extent of middle ear deficiency and severity of mandibular defects. In the more severely affected fetuses the inner ear was limited to an epithelial sac with occasional small projections, no apparent innervation and a correspondingly reduced otic capsule. In most of the fetuses examined the inner ear was less severely affected and was characterized by a reduction in the number of semicircular ducts and alterations in the size and shape of the cochlear duct. These defects are similar to those seen in a child with the isotretinoin embryopathy. Pathogenesis may result from a direct effect on otic epithelium or from faulty inductive interactions with the rhombencephalon or with periotic neural crest cells.

Structure-affinity relationships of retinoids with embryonic cellular retinoic acid-binding protein.

Separation and quantitation of cellular retinoic acid-binding protein (CRABP) in embryonic and fetal hamster tissues was accomplished with high-performance size-exclusion chromatography. Binding affinity of 26 retinoids was established by in vitro displacement of high specific activity all-trans-[3H2]retinoic acid from fetal CRABP. The CRABP concentration in presomite-to-early somite (Day 8) hamster embryos was 1.9 pmol/mg cytosolic protein and increased to 7.5 pmol/mg protein in Day 13 fetuses; CRABP concentrations subsequently declined as gestation progressed. CRABP was located primarily in fetal brain and skin (5.8 +/- 0.3 and 2.2 +/- 0.1 pmol/mg protein, respectively), whereas only trace concentrations were found in fetal liver, placenta, and maternal uterus. Retinoids that could displace all-trans-retinoic acid from CRABP had a free acid at the polar terminus (or were carboxylate esters that were readily hydrolyzed to the corresponding free acid) and had a hydrophobic ring at the distal position. The ligand specificity of the CRABP studied here suggests that this protein was analogous to the CRABP I isoform. The in vitro binding affinities of teratogenic retinoids that competed for embryonic CRABP failed to correlate directly with relative teratogenic potency. In some instances, the latter observation can be related to extensive in vivo biotransformation of retinoids to multiple teratogenic metabolites and to retinoid persistence in the embryo. Three analogs containing a free carboxy terminus, SRI 5898-21, SRI 7323-78, and SRI 6153-40, were identified with high teratogenic potency but failed to bind fetal hamster CRABP. The structure-activity and binding data of the analogs studied here indicate that many, if not most, teratogenic retinoids (or their acidic metabolites) bind with embryonic/fetal CRABP, but the present data question the role for CRABP in their teratogenic mechanism of action.

Temporal distribution of retinoic acid and cellular retinoic acid-binding protein (CRABP) in the fetal hamster.

The temporal relationship between the distribution of retinoic acid, a known human and rodent teratogen, and that of cellular retinoic acid-binding protein (CRABP) was investigated from Day 11 to Day 14 of hamster prenatal development. The 11,12-(3)H2 and 15(-14C) forms of all-trans-retinoic acid were used for quantitative distribution studies and autoradiography, respectively, and were evaluated 15 min after a single intravenous injection. Radioactivity was detected in all fetal tissues examined (brain, liver, heart, spinal cord, limb, and skin), and at Day 14, approximately 66% of the total radioactivity was present as parent all-trans-retinoic acid. High concentrations of total radioactivity were observed by autoradiography in the midbrain and hindbrain (mesencephalon, metencephalon, and myelencephalon) and spinal cord, but not in the forebrain. At the earliest time studied, limb buds showed relatively high concentrations of radioactivity. Levels of radioactivity were also high in portions of the developing face, nose, and tongue. Immunohistochemical analyses indicated that the amount of CRABP in Day 14 tissues was the highest in spinal cord followed by limb and skin; heart and liver contained only relatively small amounts of this protein. From Day 11 to Day 14, the amount of CRABP, as measured by high-performance size-exclusion liquid chromatography, in the whole body decreased as gestation progressed. Microscopic immunohistochemical localization of CRABP found the highest concentration in the ventral midbrain and in the ventral and lateral sides of the hindbrain and spinal cord; CRABP was also abundant in tongue, limb, and skin. The distribution of CRABP-positive cells in the central nervous system was similar to the distribution of retinoic acid. The data presented here indicate that fetal CRABP appears to play a role in differential accumulation of retinoic acid in certain structures of the developing hamster. The patterns of tissue retinoid and CRABP distribution observed here are consistent with the patterns of congenital malformations induced by prenatal retinoid exposure.

Developmental toxicity of L-selenomethionine in Macaca fascicularis.

Forty pregnant long-tailed macaques were dosed via nasogastric intubation with 0, 25, 150, or 300 micrograms/kg of L-selenomethionine (Se) daily during organogenesis [Gestational Day (GD) 20-50]. Clinical examination of the dams, maternal body weights, sonographic evaluations, clinical chemistry screens, and measures of serum progesterone and urinary estrone conjugates were used as indicators of maternal and fetal status in all animals. The pregnancies of two to three dams from each dose group were followed until term (approximately GD 165); the remainder (N = 7/dose group) were scheduled for hysterotomy on GD 100 +/- 2. A standard teratologic evaluation was performed including visceral and skeletal examinations. Fetal liver, kidney, skin, and smooth, cardiac, and skeletal muscles were examined by light microscopy; heart muscle was also evaluated by transmission electron microscopy. Neonates delivered at term remained with the dams and were removed periodically for morphometric, neurologic, behavioral, and ophthalmologic assessments on Days 1, 8, 15, 22, and 30 of age. Dose-dependent maternal toxicity as evidenced by anorexia, vomiting, and a significant reduction in body weight increased with increasing duration of Se exposure. One growth-retarded fetus was recovered on GD 131 from a compromised dam exposed to 25 micrograms/kg-day; one early embryonic death (GD 35) and two fetal deaths [GD 68 (followed by maternal death) and GD 123] occurred among animals dosed with 300 micrograms/kg-day. Pregnancy loss among treated animals was not significantly different from concurrent or historical controls. No statistically significant treatment-related effects were observed at necropsy on GD 100 +/- 2. One infant exposed to 150 micrograms/kg-day prenatally exhibited a unilateral cortical cataract, which may have been a spontaneous occurrence. The limited developmental effects observed and reported teratogenesis in nonmammalian species suggest that comparative pharmacokinetic studies are required before the full public health significance of elevated Se is understood.

Percutaneous retinoid absorption and embryotoxicity.

A single application of 17 micrograms/kg or 8.7 mg/kg all-trans-[10,11-3H2]-retinoic acid dissolved in acetone to shaved dorsal hamster skin resulted in rapid absorption and dose-dependent rates of elimination. An equation describing a two-compartment open model with a very brief lag time and first-order uptake and elimination was used to describe the central plasma compartment kinetics. Unchanged all-trans-retinoic acid represented less than or equal to 4% of the total circulating radio-activity. Peak circulating concentrations of parent all-trans-retinoic acid were less than those observed after an equivalent oral dose, but prolonged absorption from the skin appears to contribute to high total bioavailability of topical retinoid. Topical administration to intact skin of up to three consecutive doses of 10.5 mg/kg/d all-trans-retinoic acid or a single 5 mg/kg dose of etretinate (Ro 10-9359) during a critical stage of embryogenesis in hamsters caused erythema and/or dose-dependent epidermal hyperplasia at the site of application, but failed to induce a significant teratogenic response. Topical application of 0.01-1.0 mg/kg arotinoid Ro 13-6298 resulted in dose-dependent mucocutaneous toxicity and an increase in the numbers of dead embryos and malformed offspring. The marked skin toxicity and attenuated concentrations in maternal blood, compared to the oral route, limit the amounts of retinoid that can reach the hamster embryo. It is thus more important to compare the retinoid systemic values (absorbed dose) than it is to compare the oral or topical (applied) dose, when interpreting the results of conventional teratogenicity bioassays. The data suggest that in the human it is skin toxicity that limits the amounts of retinoid that can be applied and subsequently reach the embryo. In the rodent, overt skin toxicity under continued dosing could increase the amounts of retinoid penetrating the skin and reaching the embryo.

Route-dependent pharmacokinetics, distribution, and placental permeability of organic and inorganic selenium in hamsters.

Inorganic selenium (Se) salts (selenite and selenate oxyanions) and the organic selenoamino acids (selenomethionine and seleniferous grains) are teratogenic and embryolethal in domestic and wild birds. Selenium bioaccumulation has been held responsible for reproductive failure among waterfowl at the Kesterson Reservoir (California), the Ouray and Stewart Lake Wildlife Refuges (Utah), and the Carson Sink (Nevada). Anecdotal field and controlled laboratory reports have implicated Se exposure in mammalian embryotoxicity (including human), but developmental toxicity studies in hamsters failed to demonstrate an adverse response, except at maternally toxic doses (Ferm et al., Reprod. Toxicol., in press). Uptake, distribution, and elimination of Se after a single bolus equimolar dose (60 mumol/kg) of selenate or selenomethionine by oral or intravenous administration were compared using day 8 pregnant hamsters. Intravenous selenate was eliminated ten times more rapidly from maternal plasma than oral selenate, but concentrated in liver, kidney, and placenta to the same degree. Intravenous (iv) L-selenomethionine achieved lower maximum circulating total [Se], but it was eliminated more slowly than iv selenate. Larger areas under the plasma and peripheral tissue [Se]:time curve (AUC) after oral or parenteral selenomethionine than after equimolar selenate were consistent with previous studies in rodents and in humans. Embryonic [Se] plateaued at 3 nmol/g after selenate, but embryonic [Se] after selenomethionine continued to accumulate (80 nmol/g) as gestation progressed. The lack of a teratogenic response in hamsters at doses of either selenate or selenomethionine less than those associated with maternal intoxication cannot be attributed to lack of Se accumulation in early embryonic and placental tissue.