Evolution of diabetes medical nutrition therapy.

Research supports the importance of medical nutrition therapy in achieving diabetes treatment goals. For persons requiring insulin therapy, the first priority is to integrate an insulin regimen into the patient's lifestyle. For type 2 diabetes, the priority is to focus on lifestyle strategies (that is, nutrition and exercise) that will improve metabolic outcomes at diagnosis and as the disease progresses. Patients with diabetes need nutrition recommendations that are supported by scientific evidence and that can be easily understood and translated into everyday life. To achieve positive outcomes, a coordinated team effort that provides continued education and support is essential.

Developmental effects of ambient UV-B light and landfill leachate in Rana blairi and Hyla chrysoscelis.

This study assessed the effects of ambient UV light on the development of two native species of anurans, Rana blairi and Hyla chrysoscelis, during their normal breeding season in Oklahoma. Additionally, the effects of ambient UV light and water contaminated with landfill leachate in Rana blairi were examined. Embryos were collected from the field and distributed equally among replicates of four filter treatments of ambient UV light in experimental tubs filled with either FETAX solution or landfill leachate diluted to 25, 10, and 5% concentrations. Three endpoints (mortality, teratogenesis, and growth) were compared between filter treatments. By itself, UV-B caused no significant effects. Leachate at 10 and 25% concentrations caused 100% mortality across all filter treatments. There was a significant interaction between filter treatment and water toxicity at leachate concentrations of 5% for both malformation and growth. Increased UV-B exposure decreased the malformation rate and increased growth in the leachate treatments.

Evaluation of a reproductive toxicity assay using Xenopus laevis: boric acid, cadmium and ethylene glycol monomethyl ether.

Cadmium (Cd), boric acid (BA) and ethylene glycol monomethyl ether (EGME) were evaluated for reproductive and developmental toxicity in Xenopus laevis. Eight reproductively mature adult male and eight superovulated female Xenopus laevis were exposed to at least five separate sublethal concentrations of each material via the culture water for a period of 30 days. Four respective pairs were mated and the offspring evaluated for developmental effects; an evaluation of reproductive status was performed on the remaining four specimens. Ovary pathology, oocyte count, oocyte maturity and maturation capacity (germinal vesicle breakdown, GVBD) and necrosis were evaluated in the female, whereas testis pathology, sperm count, dysmorphology and motility were studied in the male. Based on this assessment, each test material exerted reproductive toxicity in Xenopus laevis, but with varying potencies. Adult female exposure to Cd and EGME particularly, and to a lesser extent to BA, resulted in transgenerational toxicity to the developing progeny. Further, this model appears to be a useful tool in the initial assessment and prioritization of potential reproductive toxicants for further testing.

The effect of cadmium on oogenesis in Xenopus laevis.

Reproductive toxicity studies have historically centered on post-fertilization events. A thorough assessment of reproductive hazards to an organism should include all aspects of its life cycle. Cadmium is a teratogenic and carcinogenic heavy metal that occurs naturally in the environment but is also released anthropogenically. The effect of cadmium administration on oocyte development in Xenopus laevis was studied. Adult female Xenopus were injected in the dorsal lymph sac with cadmium chloride (CdCl2) at doses of 0.5, 0.75, 1.0, 3.0 or 5.0 mg/kg every other day for 21 days. Significant adverse effects of Cd on oocyte development were observed. The percentage of oocytes at all stages of oogenesis was decreased while the population of atretic oocytes increased dramatically (P < 0.0001). Numerous oocytes exhibited a speckled or mottled appearance and the incidence of completely atretic oocyte follicles increased. The observations indicate that Cd has the potential to significantly disrupt oogenesis and that examination of developing gametes may be a useful parameter for assessing the influence of environmental contaminants on reproductive capacity.

Effects of dietary fructose on plasma lipids in healthy subjects.

BACKGROUND: About 9% of average dietary energy intake in the United States comes from fructose. Such a high consumption raises concern about the metabolic effects of this sugar. OBJECTIVE: The objective of this study was to determine the effect of dietary fructose on plasma lipids. DESIGN: The study was conducted in the General Clinical Research Center at Fairview-University of Minnesota Medical Center. The participants were 24 healthy adult volunteers (12 men and 12 women; 6 of each sex were aged <40 y and 6 of each sex were aged >/=40 y). All subjects received 2 isoenergetic study diets assigned by using a randomized, balanced crossover design. One diet provided 17% of energy as fructose. The other diet was sweetened with glucose and was nearly devoid of fructose. Each diet was fed for 6 wk. Both diets were composed of common foods and contained nearly identical amounts of carbohydrate, protein, fat, fiber, cholesterol, and saturated, monounsaturated, and polyunsaturated fatty acids. All meals were prepared in the metabolic kitchen of the General Clinical Research Center. RESULTS: The responses to the study diets differed by sex. In men, the fructose diet produced significantly higher fasting, postprandial, and daylong plasma triacylglycerol concentrations than did the glucose diet. The daylong plasma triacylglycerol concentration after 6 wk of the fructose diet was 32% greater in men than the corresponding concentration during the glucose diet (P: < 0.001). The fructose diet had no significant effect on fasting or postprandial plasma triacylglycerol concentrations in women. The fructose diet also had no persistent effect on fasting plasma cholesterol, HDL cholesterol, or LDL cholesterol in either men or women. CONCLUSIONS: Dietary fructose was associated with increased fasting and postprandial plasma triacylglycerol concentrations in men. Diets high in added fructose may be undesirable, particularly for men. Glucose may be a suitable replacement sugar.

A high carbohydrate versus a high monounsaturated fatty acid diet lowers the atherogenic potential of big VLDL particles in patients with type 1 diabetes.

The objective of the present study was to compare the effects of two diets on the atherogenic potential of two VLDL subfractions harvested from fasting subjects by measuring the number and composition of particles and the amount of esterified cholesterol accumulated in macrophages. A high (25%) monounsaturated fatty acid (Mono) diet and a high (61%) carbohydrate (CHO) diet were provided for 4 wk in a randomized crossover design to 19 normolipidemic, nonobese patients with type 1 diabetes. The two diets were matched for protein, polyunsaturated/saturated fatty acids, cholesterol and fiber content. The number of circulating big VLDL (S:(f) 100-400) particles was greater during the high Mono than during the high CHO diet based on the levels of apolipoprotein B (means +/- SEM): 31.4 +/- 7.4 versus 20.0 +/- 3.8 mg/L (P: < 0.025, paired t test). The following variables did not differ during the diet periods: number of small VLDL (S:(f) 20-100) particles, esterified cholesterol accumulated in THP-1 macrophages incubated with the same number of big and small VLDL particles and particle composition. We conclude that a high CHO diet might be preferable to a high Mono diet, on the basis of the premise that more big VLDL particles could increase the atherosclerotic risk in patients with diabetes.

Chronic treatment with phentermine combined with fenfluramine lowers plasma serotonin.

As expected on the basis of published research in both humans and animals, treatment with phentermine/fenfluramine lowers plasma 5-hydroxytryptamine [corrected], whereas treatment with phentermine had no significant effect. In light of these findings, future research should focus on mechanisms other than increased plasma 5-hydroxytryptamine [corrected] to explain how fenfluramine increases the risk of primary pulmonary hypertension and valvular heart disease.

Evaluation of the developmental toxicity of thalidomide using frog embryo teratogenesis assay-xenopus (FETAX): biotransformation and detoxification.

The developmental toxicity of thalidomide was evaluated using FETAX (Frog Embryo Teratogenesis Assay - Xenopus). Young X. Laevis embryos were exposed to this compound in each of two concentration-response experiments with and without differently induced exogenous metabolic activation systems (MASs) and/or inhibited MASs. Young male Sprague-Dawley rats were treated with either isoniazid or Aroclor 1254 to induce cytochrome P-450. Several of the rats were subsequently treated with diethyl maleate (DM) to deplete glutathione reserves. Specific aliquots of rat liver microsomes were treated with 3-amino-1,2,4-triazole (ATZ) or alpha-napthoflavone (alpha-N) to selectively inhibit P-450 activity. Bioactivation was indicated by increased developmental toxicity observed in MAS tests. Results obtained indicated that thalidomide was predominantly activated by P-450 isozyne CYP2E1, although weak cross-specificity between CYP1A1/A2 may have existed. Detoxification pathways for thalidomide were investigated by treatment of the MAS with cyclohexene oxide (CHO) and DM to inhibit the epoxide hydrolase and glutathione conjugation pathways, respectively. Results indicated that epoxide hydrolase was primarily responsible for the detoxification of bioactivated thalidomide. Teratogenesis Carcinog. Mutagen. 20:35-47, 2000.

Phase III interlaboratory study of FETAX. Part 3. FETAX validation using 12 compounds with and without an exogenous metabolic activation system.

FETAX (Frog Embryo Teratogenesis Assay-Xenopus) is a 96-h whole-embryo developmental toxicity screening assay that can be used in ecotoxicology and in detecting mammalian developmental toxicants when an in vitro metabolic activation system is employed. A standardized American Society for Testing and Materials (ASTM) guide for the conduct of FETAX has been published, along with a companion atlas that helps in embryo staging and in identifying malformations. As part of the ASTM process, an interlaboratory validation study was undertaken to evaluate the repeatability and reliability of FETAX and to evaluate the potential teratogenic hazard of 12 compounds. Three different laboratories participated in the study. All three participating laboratories had extensive experience with the assay. FETAX intralaboratory and interlaboratory variability, as judged by coefficients of variation, were very low. Potential teratogenic hazard was evaluated using two major criteria from FETAX experiments employing metabolic activation systems (MAS). These were the teratogenic index TI (TI = 96-h lc(50)/96-h ec(50) (malformation)) and the minimum concentration that inhibits growth (MCIG). A compound was considered teratogenic by this criterion when the MCIG was significantly different from controls at concentrations below the 30% level of the MAS 96-h lc(50). Based on the results of this and other studies, a decision table was constructed in order to evaluate additional studies. Severity of malformations caused, especially near the MAS 96-h ec(50) (malformation), were also evaluated. Four compounds were non-teratogenic but two compounds were clearly teratogenic. The remaining six compounds were ranked as equivocal teratogens. The results were discussed in light of the difficulty of producing an adequate decision table. FETAX proved to yield repeatable and reliable data as long as care was taken during range-finding and technicians were adequately trained. The MAS was essential in using FETAX to predict developmental hazard in mammals, and still requires further development.

Lead inhibition of DNA-binding mechanism of Cys(2)His(2) zinc finger proteins.

The association of lead with chromatin in cells suggests that deleterious metal effects may in part be mediated through alterations in gene function. To elucidate if and how lead may alter DNA binding of cysteine-rich zinc finger proteins, lead ions were analyzed for their ability to alter the DNA binding mechanism of the Cys(2)His(2) zinc finger protein transcription factor IIIA (TFIIIA). As assayed by DNase I protection, the interaction of TFIIIA with the 50-bp internal control region of the 5S ribosomal gene was partially inhibited by 5 microM lead ions and completely inhibited by 10 to 20 microM lead ions. Preincubation of free TFIIIA with lead resulted in DNA-binding inhibition, whereas preincubation of a TFIIIA/5S RNA complex with lead did not result in DNA-binding inhibition. Because 5S RNA binds TFIIIA zinc fingers, this result is consistent with an inhibition mechanism via lead binding to zinc fingers. The complete loss of DNase I protection on the 5S gene indicates the mechanism of inhibition minimally involves the N-terminal fingers of TFIIIA. Inhibition was not readily reversible and occurred in the presence of an excess of beta-mercaptoethanol. Inhibition kinetics were fast, progressing to completion in approximately 5 min. Millimolar concentrations of sulfhydryl-specific arsenic ions were not inhibitory for TFIIIA binding. Micromolar concentrations of lead inhibited DNA binding by Sp1, another Cys(2)His(2) finger protein, but not by the nonfinger protein AP2. Inhibition of Cys(2)His(2) zinc finger transcription factors by lead ions at concentrations near those known to have deleterious physiological effects points to new molecular mechanisms for lead toxicity in promoting disease.

Pharmacologic induction of weight loss to treat type 2 diabetes.

OBJECTIVE: Most individuals with type 2 diabetes are overweight, and weight loss for them is an important therapeutic objective. However, usual weight-loss strategies have generally not produced sustained weight loss. Pharmacologic agents to assist weight loss might be useful, but no long-term data on their effectiveness and safety in patients with type 2 diabetes are available. We therefore initiated a 2-year placebo-controlled trial of the weight-loss medications fenfluramine and phentermine in type 2 diabetic subjects. RESEARCH DESIGN AND METHODS: A total of 44 overweight (> 120% ideal body weight) subjects with type 2 diabetes were enrolled in a randomized, placebo-controlled, double-blind trial of fenfluramine and phentermine. All subjects received intensive nutrition counseling, an exercise prescription, and instruction in behavior modification. Subjects were randomly assigned to 20 mg fenfluramine three times a day and 37.5 mg phentermine daily (n = 23) or dual placebos (n = 21). Diabetes medications were adjusted as necessary to achieve glycemic goals. Changes in weight, glycemia, lipemia, and blood pressure were assessed every 2 months, as were adverse events. In September 1997, when fenfluramine was withdrawn from the U.S. market, fenfluramine was stopped in all subjects. Thus the length of drug treatment varied, but 16 subjects (8 in each group) reached 12 months of treatment. Only data obtained before the withdrawal of fenfluramine are included in this report. RESULTS: A study termination, diabetes medications had been reduced in 1 subject in the placebo group (5%) and 11 subjects in the drug treatment group (52%) (P = 0.005). Drug treatment resulted in significant reductions in body weight, BMI, and HbA1c at all time points through 6 months. Changes in weight at 6 months were -2.7 +/- 1.4 kg (mean +/- SEM) with placebo treatment and -9.6 +/- 1.5 kg with drug treatment (P = 0.003). Even though more subjects in the drug treatment group required reductions in diabetes medications, at 6 months, changes in HbA1c were -0.3 +/- 0.2% with placebo treatment and -1.6 +/- 0.3% with drug treatment (P = 0.002). Fasting plasma glucose and triglycerides were significantly reduced at some time points with drug treatment. No serious adverse events attributable to study medications were observed. CONCLUSIONS: Premature study termination decreased the power of our study at later time points. However, our data suggest that weight loss medications are an effective treatment for type 2 diabetes during active weight loss. Whether the benefit persists after weight loss has stopped remains to be determined.

Animals as sentinels of human health hazards of environmental chemicals.

A workshop titled "Using Sentinel Species Data to Address the Potential Human Health Effects of Chemicals in the Environment," sponsored by the U.S. Army Center for Environmental Health Research, the National Center for Environmental Assessment of the EPA, and the Agency for Toxic Substances and Disease Registry, was held to consider the use of sentinel and surrogate animal species data for evaluating the potential human health effects of chemicals in the environment. The workshop took a broad view of the sentinel species concept, and included mammalian and nonmammalian species, companion animals, food animals, fish, amphibians, and other wildlife. Sentinel species data included observations of wild animals in field situations as well as experimental animal data. Workshop participants identified potential applications for sentinel species data derived from monitoring programs or serendipitous observations and explored the potential use of such information in human health hazard and risk assessments and for evaluating causes or mechanisms of effect. Although it is unlikely that sentinel species data will be used as the sole determinative factor in evaluating human health concerns, such data can be useful as for additional weight of evidence in a risk assessment, for providing early warning of situations requiring further study, or for monitoring the course of remedial activities. Attention was given to the factors impeding the application of sentinel species approaches and their acceptance in the scientific and regulatory communities. Workshop participants identified a number of critical research needs and opportunities for interagency collaboration that could help advance the use of sentinel species approaches.

Differences in the metabolism of postprandial lipoproteins after a high-monounsaturated-fat versus a high-carbohydrate diet in patients with type 1 diabetes mellitus.

There is little information comparing the effects of a high-monounsaturated (Mono)-fat versus a high-carbohydrate (CHO) diet in patients with type 1 diabetes mellitus. In the present study, the effects of these diets on a number of metabolic parameters were compared. Seventeen normolipidemic, nonobese patients with type 1 diabetes were provided with the diets for 4 weeks each in a randomized, crossover design. The percentages of Mono fat of the two diets were 25 Mono versus 9 CHO, with a corresponding total fat content of 40% versus 24% and a total CHO content of 45% versus 61%. At the end of each dietary period, parameters of glycemic control, coagulation factors, and fasting and postprandial lipoproteins were assessed. There were no differences in weight, glycemia, insulin dose, fasting lipid profile, or coagulation factors between the two diets. However, the metabolism of postprandial lipoproteins after a fat load differed; viz, after the Mono diet compared with the CHO diet, mean plasma triglyceride levels over 10 hours were higher (P=.0025, by repeated-measures ANOVA). The levels of triglyceride (P=.0045) and retinyl esters (P=.0046) in chylomicrons (Sf>400) and chylomicron remnants (Sf 100 to 400) (P=.0047 and P=.043, respectively), and the total particle number (apolipoprotein B levels) in chylomicron remnants (P=.001) and small, very low density lipoprotein (Sf 20 to 100, P=.016) were also higher. Our data suggest that in patients with type 1 diabetes, a CHO diet might be preferable to a Mono diet, since adherence to the former results in a lower number of circulating postprandial lipoprotein particles that are potentially atherogenic.

Ground and surface water developmental toxicity at a municipal landfill: description and weather-related variation.

Contaminated groundwater poses a significant health hazard and may also impact wildlife such as amphibians when it surfaces. Using FETAX (Frog Embryo Teratogenesis Assay-Xenopus), the developmental toxicity of ground and surface water samples near a closed municipal landfill at Norman, OK, were evaluated. The groundwater samples were taken from a network of wells in a shallow, unconfined aquifer downgradient from the landfill. Surface water samples were obtained from a pond and small stream adjacent to the landfill. Surface water samples from a reference site in similar habitat were also analyzed. Groundwater samples were highly toxic in the area near the landfill, indicating a plume of toxicants. Surface water samples from the landfill site demonstrated elevated developmental toxicity. This toxicity was temporally variable and was significantly correlated with weather conditions during the 3 days prior to sampling. Mortality was negatively correlated with cumulative rain and relative humidity. Mortality was positively correlated with solar radiation and net radiation. No significant correlations were observed between mortality and weather parameters for days 4-7 preceding sampling.

Evaluation of the developmental toxicities of coumarin, 4-hydroxycoumarin, and 7-hydroxycoumarin using FETAX.

The developmental toxicities of coumarin and hydroxycoumarin metabolites were evaluated using FETAX. Young X. laevis embryos were exposed to coumarin, 4-hydroxycoumarin, and 7-hydroxycoumarin in each of two separate concentration-response experiments with and without an exogenous metabolic activation system (MAS) and/or inhibited MAS. The MAS was treated with carbon monoxide (CO), cimetidine (CIM), or ellipticine (ELL) to selectively modulate cytochrome P-450 activity. The MAS was also treated with cyclohexene oxide (CHO) to selectively modulate epoxide hydrolase activity. Without the MAS or inhibited MAS, coumarin and 7-hydroxycoumarin were nearly equitoxic, whereas 4-hydroxycoumarin was nearly 2-fold less developmentally toxic than coumarin on an equimolar basis. Addition of the MAS and CIM-MAS increased the developmental toxicities of coumarin and, particularly, 4-hydroxycoumarin. Addition of the CHO-MAS greatly increased the developmental toxicity of coumarin and, especially, 4-hydroxycoumarin. Addition of the ELL- or CO-inhibited MAS did not increase the developmental toxicity of coumarin. However, addition of the intact MAS did not alter the developmental toxicity of 7-hydroxycoumarin. Results from these studies suggested that P-450; specifically ELL-inhibited P-450 (arylhydrocarbon hydroxylase) may have been responsible for increasing the developmental toxicity of coumarin. Furthermore, the increased toxicity of coumarin or 4-hydroxycoumarin following co-incubation with CHO-treated microsomes indicated that highly toxic epoxide intermediates may be produced from oxidative P-450 metabolism and that epoxide hydrolase may play a role in detoxification of the reactive intermediates.

Phase III interlaboratory study of FETAX, Part 2: interlaboratory validation of an exogenous metabolic activation system for frog embryo teratogenesis assay--Xenopus (FETAX).

Interlaboratory validation of an exogenous metabolic activation system (MAS) developed for the alternative, short-term developmental toxicity bioassay, Frog Embryo Teratogenesis Assay-Xenopus (FETAX) was performed with cyclophosphamide and caffeine. Seven study groups within six separate laboratories participated in the study in which three definitive concentration-response experiments were performed with and without the MAS in a side-by-side format for each chemical. Since both chemicals had been previously tested in FETAX, the test concentrations were provided to each laboratory prior to testing. Interlaboratory coefficient of variation (CV) values for unactivated cyclophosphamide (no MAS) were 15%, 15%, 29%, and 25% for the 96-hr LC50, 96-hr EC50 (malformation), Minimum Concentration to Inhibit Growth (MCIG), and Teratogenic Index (TI) values, respectively. Addition of the MAS increased the CV values of each endpoint at least 3.9-fold. Interlaboratory CV values for unactivated caffeine were 31%, 18%, 31%, and 46% for the 96-hr LC50, 96-hr EC50 (malformation), MCIG, and TI values, respectively. Addition of the MAS decreased the CV values of each respective endpoint by at least 1.6-fold. Results indicated that bioactivated toxicants may be prone to greater variability in response amongst laboratories than compounds, which are detoxified. Even though more variability was noted with activated cyclophosphamide, results were within interlaboratory variation expected for other aquatic-based bioassays. Thus, results from these studies warrant the continued use and further refinement of FETAX for alternative developmental toxicity assessment.

Glucose measurement in patients with diabetes mellitus with dermal interstitial fluid.

Although measurement of capillary blood glucose remains the standard method of self-monitoring for persons with diabetes mellitus, a less-invasive method of monitoring would be desirable. Measurement of dermal interstitial fluid glucose might meet this need. To test this possibility, plasma glucose, capillary blood glucose (current standard), and dermal interstitial fluid glucose were measured in 17 subjects with type I diabetes during a 5-hour pre- and postprandial period when plasma glucose was changing rapidly. The objective was to assess the ability of dermal interstitial fluid glucose to accurately predict plasma glucose over a wide range of potential glucose concentrations. Dermal interstitial fluid glucose was highly correlated with plasma glucose (r = 0.95, p < 0.0001). The mean absolute and percent differences between dermal interstitial fluid glucose and plasma glucose were 1.2 mmol/L (21 mg/dl) and 10.6%, respectively. The kinetics of dermal interstitial fluid glucose and plasma glucose were similar. There was no significant difference between dermal interstitial fluid glucose and plasma glucose in mean glucose excursion, peak glucose concentration, or time to peak glucose concentration. The correlation between dermal interstitial fluid glucose and plasma glucose was as strong as the correlation between capillary blood glucose and plasma glucose. In conclusion, dermal interstitial fluid glucose can be used to estimate plasma glucose, and has the potential to be used for monitoring patients with diabetes mellitus.

Evaluation of the developmental toxicity of benzo[a]pyrene and 2-acetylaminofluorene using Xenopus: modes of biotransformation. Stover Group.

The developmental toxicities of benzo[a]pyrene (BAP) and 2-acetylaminofluorene (AAF) were evaluated using FETAX (Frog Embryo Teratogenesis Assay-Xenopus). Young X. laevis embryos were exposed to these two compounds in each of two separate concentration-response experiments with and without an exogenous metabolic activation system (MAS) and/or inhibited MAS. The MAS was treated with cimetidine (CIM), ellipticine (ELL), or alpha-napthoflavone (alpha-N) to selectively modulate cytochrome P-450 activity. Bioactivation of both of these compounds was indicated by increased developmental toxicity observed in MAS tests. Results obtained in treated MAS tests indicated that BAP was predominantly activated by Cytochrome P-450 isozyme CYP1A1. AAF bioactivation was shown to be only partly mediated by CYP1A1/2. Detoxification pathways for these two compounds were investigated by treatment of the MAS with cyclohexene oxide (CHO) and diethyl maleate (DM) to inhibit the epoxide hydroxylase and glutathione conjugation pathways, respectively. Results indicated that epoxide hydroxylase was primarily responsible for the detoxification of BAP, with glutathione conjugation playing a secondary role. Detoxification of AAF by these two pathways was not indicated.

FETAX interlaboratory validation study: phase III--Part 1 testing.

The Frog Embryo Teratogenesis Assay-Xenopus (FETAX) is a 96-h whole embryo developmental toxicity screening assay that can be used in ecotoxicology and in detecting mammalian developmental toxicants when an in vitro metabolic activation system is employed. A standardized American Society for Testing and Materials (ASTM) guide for the conduct of FETAX has been published, along with a companion atlas that aids in embryo staging and identifying malformations. As part of the ASTM process, a three-phase interlaboratory validation study was undertaken to evaluate the repeatability and reliability of FETAX. Seven different participants collaborated in the study. In Phase I, FETAX proved to be more repeatable and reliable than many bioassays. However, some excessive variation was observed in a few laboratories. An initial lack of assay experience by some technicians caused variation. Phase II showed far less intra- and interlaboratory variability than Phase I. Non-teratogens showed the most consistent results, while more variability was observed for the two teratogens tested. Interlaboratory coefficient of variation values for all endpoints ranged from 7.3 to 54.7. Phase III--Part 1, using coded samples and test concentration ranges selected by each laboratory, showed results similar to Phase I. Analysis of the causes of variation suggested that some technicians judged some embryos to be malformed while others consistently judged similar embryos as normal. Concentration ranges tested by some of the laboratories varied greatly and a new protocol for selecting concentrations for initial testing was written to reduce variation from this source. Testing to date suggests that FETAX is as repeatable and reliable as other standard bioassays.