Cytochrome P-450- and flavin-containing monooxygenase-catalyzed formation of the carcinogen N-hydroxy-2-aminofluorene and its covalent binding to nuclear DNA.

The metabolic N-oxidation of the carcinogen 2-aminofluorene was examined in vitro using fortified hepatic microsomes from a variety of species. Rat, dog, human, and pig liver microsomes catalyzed the formation of N-hydroxy-2-aminofluorene (N-OH-AF) from AF at rates of 1.6, 1.0, 1.2, and 3.5 nmol/min/mg protein, respectively. The involvement of both cytochrome P-450 and the flavin-containing monooxygenase was demonstrated with hepatic microsomes and with purified enzymes by using specific enzyme inhibitors. 2-[(2,4-Dichloro-6-phenyl)phenoxy]ethylamine, a potent cytochrome P-450 inhibitor, decreased microsomal N-OH-AF formation by 96, 83, 70, and 46% in the rat, dog, human, and pig, respectively; and further addition of methimazole, a high-affinity flavin-containing monooxygenase substrate, abolished the residual N-hydroxylating activity. Using the purified porcine flavin-containing monooxygenase, metabolic formation of N-OH-AF occurred at a rate of 4.9 nmol/min/nmol flavin adenine nucleotide and was insensitive to 2-[(2,4-dichloro-6-phenyl)phenoxy]ethylamine inhibitor. In addition, purified rat liver cytochrome P-450 (isolated from 5,6-naphthoflavone-induced animals) N-hydroxylated AF (1.1 nmol/min/nmol P-450) and was completely inhibited by 2-[(2,4-dichloro-6-phenyl)-phenoxy]ethylamine, but the reaction was insensitive for methimazole. To determine whether or not the metabolic formation of N-OH-AF could lead directly to covalently bound adduct(s) with DNA under these incubation conditions (30 min, pH 7.5), the binding of synthetic and metabolically formed [3H]-N-OH-AF to added calf thymus DNA and to DNA in isolated rat liver nuclei was investigated. In all cases, the amount of DNA-bound carcinogen accounted for 0.08 to 0.15% of the N-OH-AF present in the incubation mixtures. These data, when compared to the levels of AF bound to hepatic nuclear DNA reported in vivo, suggest that the nonenzymatic reaction of N-OH-AF with nuclear DNA may be sufficient to account for a substantial portion of the observed in vivo binding of this carcinogen.

Application of a hybrid CFD-PBPK nasal dosimetry model in an inhalation risk assessment: an example with acrylic acid.

The available inhalation toxicity information for acrylic acid (AA) suggests that lesions to the nasal cavity, specifically olfactory degeneration, are the most sensitive end point for developing a reference concentration (RfC). Advances in physiologically based pharmacokinetic (PBPK) modeling, specifically the incorporation of computational fluid dynamic (CFD) models, now make it possible to estimate the flux of inhaled chemicals within the nasal cavity of experimental species, specifically rats. The focus of this investigation was to apply an existing CFD-PBPK hybrid model in the estimation of an RfC to determine the impact of incorporation of this new modeling technique into the risk assessment process. Information provided in the literature on the toxicity and mode of action for AA was used to determine the risk assessment approach. A comparison of the approach used for the current U.S. Environmental Protection Agency (U.S. EPA) RfC with the approach using the CFD-PBPK hybrid model was also conducted. The application of the CFD-PBPK hybrid model in a risk assessment for AA resulted in an RfC of 79 ppb, assuming a minute ventilation of 13.8 l/min (20 m(3)/day) in humans. This value differs substantially from the RfC of 0.37 ppb estimated for AA by the U.S. EPA before the PBPK modeling advances became available. The difference in these two RfCs arises from many factors, with the main difference being the species selected (mouse vs. rat). The choice to conduct the evaluation using the rat was based on the availability of dosimetry data in this species. Once these data are available in the mouse, an assessment should be conducted using this information. Additional differences included the methods used for estimating the target tissue concentration, the uncertainty factors (UFs) applied, and the application of duration and uncertainty adjustments to the internal target tissue dose rather than the external exposure concentration.

Lessons learned in applying the U.S. EPA proposed cancer guidelines to specific compounds.

An expert panel was convened to evaluate the U.S. Environmental Protection Agency's "Proposed Guidelines for Carcinogen Risk Assessment" through their application to data sets for chloroform (CHCl3) and dichloroacetic acid (DCA). The panel also commented on perceived strengths and limitations encountered in applying the guidelines to these specific compounds. This latter aspect of the panel's activities is the focus of this perspective. The panel was very enthusiastic about the evolution of these proposed guidelines, which represent a major step forward from earlier EPA guidance on cancer-risk assessment. These new guidelines provide the latitude to consider diverse scientific data and allow considerable flexibility in dose-response assessments, depending on the chemical's mode of action. They serve as a very useful template for incorporating state-of-the-art science into carcinogen risk assessments. In addition, the new guidelines promote harmonization of methodologies for cancer- and noncancer-risk assessments. While new guidance on the qualitative decisions ensuing from the determination of mode of action is relatively straightforward, the description of the quantitative implementation of various risk-assessment options requires additional development. Specific areas needing clarification include: (1) the decision criteria for judging the adequacy of the weight of evidence for any particular mode of action; (2) the role of mode of action in guiding development of toxicokinetic, biologically based or case-specific models; (3) the manner in which mode of action and other technical considerations provide guidance on margin-of-exposure calculations; (4) the relative roles of the risk manager versus the risk assessor in evaluating the margin of exposure; and (5 ) the influence of mode of action in harmonizing cancer and noncancer risk assessment methodologies. These points are elaborated as recommendations for improvements to any revisions. In general, the incorporation of examples of quantitative assessments for specific chemicals would strengthen the guidelines. Clearly, any revisions should retain the emphasis present in these draft guidelines on flexibility in the use of scientific information with individual compounds, while simultaneously improving the description of the processes by which these mode-of-action data are organized and interpreted.

Limiting the uncertainty in risk assessment by the development of physiologically based pharmacokinetic and pharmacodynamic models.

Analysis of the default cancer risk assessment methodology suggests that the confidence interval usually associated with the prediction of an upper bound on risk underestimates the uncertainty in the risk estimate. This underestimate of uncertainty is based on the use of a large number of policy decisions or professional judgements that are incorporated into the methodology as exact values with no estimate of error. An alternative approach is to develop a comprehensive biologically based risk assessment that provides scientific data to substitute for many of the policy decisions of the default methodology.

Summary of panel discussion on the 'advantages/limitations/uncertainties in the use of physiologically based pharmacokinetic and pharmacodynamic models in hazard identification and risk assessment of toxic substances'.

A panel of scientists discussed a variety of issues related to the development of physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models for toxicological risk assessment. The panel concluded that although there are a variety of potential technical problems associated with the use of these models for hazard identification and risk assessment, PBPK/PD modeling represents an important technical advance in risk assessment methodology that should continue to be developed and applied. In addition to the technical issues that were addressed, the necessity of providing additional education for toxicologists in the skills necessary for the development and evaluation of PBPK/PD models was stressed.

Modeling the reactivity of acrylic acid and acrylate anion with biological nucleophiles.

Based on the results of prior in-vitro reactivity experiments, the pathway for the Michael addition of two representative nucleophiles (methylamine and imidazole) to acrylate anion (AA-) was explored with the semiempirical quantum model, AM1. The results of the calculations indicate that there is no viable reaction pathway for the addition of nucleophiles to AA-. An alternative route for the formation of the Michael products via the non-ionized form of acrylic acid (AA) was explored and found to be theoretically possible. The alternative route is plausible, but is considered to be insignificant in vivo based upon the rapid metabolism and excretion of AA (excretion half-life of 1-8 h after oral dosing).

The regional hydrolysis of ethyl acrylate to acrylic acid in the rat nasal cavity.

Cytotoxicity is primarily limited to the olfactory epithelium of the dorsal meatus region of the nasal cavity of rodents following inhalation exposure to acrylic monomers. To investigate the biochemical basis for this effect, three regions of the Fischer F344N rat nasal cavity were evaluated for carboxylesterase activity for the representative acrylic ester, ethyl acrylate. Prior studies have indicated that the rodent olfactory epithelium is sensitive to the cytotoxic effects of short chain organic acids. In this study, no regional difference in carboxylesterase activity was observed between sensitive and non-sensitive regions of olfactory epithelium. Respiratory epithelium (resistant to cytotoxicity) was found to be have a much lower rate of carboxylesterase activity than olfactory epithelium. These results suggest that the regional distribution of cytotoxicity observed in the rat nasal cavity at high concentrations of inhaled acrylic monomers may be due in part to the amount of released organic acid following deposition. However, the observation of the same esterase activity in sensitive and nonsensitive olfactory regions suggests that nasal air flow patterns and regional deposition may also be critical factors.

Quantitation of an epithelial S-phase response in the rat forestomach and glandular stomach following gavage dosing with ethyl acrylate.

Gavage dosing of the irritant, ethyl acrylate (EA), has been found to induce hyperplasia in the rat forestomach, but no signs of toxicity in the glandular stomach or in organs remote from the site of dosing. To quantitatively describe this effect as a background for subsequent modeling studies, pulse measurements of the number of S-phase cells were made following a single gavage dose of EA. The time-course of the S-phase response in the forestomach epithelium following a high dose (200 mg/kg or a 4% solution in corn oil) indicated that the number of S-phase nuclei was decreased relative to control animals immediately following gavage dosing with a minimum at 6 hr, but that the number of S-phase nuclei increased significantly above control values by 20 hr and remained significantly elevated until at least 48 hr following the gavage dose. A single-dose dose-response study with gavage doses of 0, 2, 10, 20, 50, 100, or 200 mg/kg EA and S-phase analysis at 24 hr following gavage dosing indicated that a significant increase in S-phase nuclei was evident at doses of 20 mg/kg or higher. Dosing with EA for 2 weeks at dose levels of 0, 10, 50, or 200 mg/kg caused a prolonged elevation of S-phase nuclei only at the 200 mg/kg dose level during the 24 hr following the last gavage dose. Lower doses did not induce a significant increase in the S-phase nuclei. In contrast to the forestomach, the S-phase response of the glandular stomach was transient following a single 200 mg/kg gavage dose, and only a marginal response was observed following multiple 200 mg/kg doses. No effects were observed at lower doses. Comparison of these results to prior determinations of the effect of EA on the concentration of nonprotein sulfhydryls (primarily glutathione) in the forestomach and glandular stomach indicate a correlation of the stimulation in S-phase activity in the forestomach with the repletion and overshoot of tissue nonprotein sulfhydryl levels.

Rate and route of oxidation of acrylic acid to carbon dioxide in rat liver.

Results of in vivo metabolism studies with acrylic acid (AA) have indicated that 60-80% of the administered dose is excreted as CO2 within 2-8 hr of oral dosing of rats; however, the pathway of AA metabolism to CO2 in mammals has not been determined. To define this route, rat hepatocytes were isolated and incubated with [1-14C]AA in a sealed vial modified to trap evolved 14CO2. Rapid oxidation of AA to CO2 was observed. Similar incubations conducted with rat liver homogenates fortified with ATP, ADP, coenzyme A, carnitine, and malate also resulted in oxidation of AA. Mitochondria isolated from liver homogenates were incubated with AA under the same conditions and yielded higher rates of AA oxidation than homogenates. Addition of equimolar amounts of propionic acid, 3-hydroxypropionic acid, or 3-mercaptopropionic acid significantly inhibited the oxidation of AA by mitochondria. HPLC analysis of the mitochondrial incubation mixtures indicated that a single major metabolite, which coeluted with 3-hydroxypropionate, accumulated in the solution. The results indicate that AA is rapidly incorporated into a mitochondrial pathway for propionic acid catabolism that results in the release of CO2 and possible bioincorporation as acetate. This pathway appears to be the principal route of detoxification of AA in mammals.

Metabolism of acrylic acid to carbon dioxide in mouse tissues.

Acrylic acid (AA) is acutely irritating at sites of initial contact but causes little systemic toxicity probably due to its rapid metabolism to CO2 and acetyl-CoA via a secondary pathway of propionic acid catabolism. In this study, the rate of AA oxidation in 13 tissues of C3H mice was measured by incubating tissue slices with [1-14C]AA and collecting, 14CO2. Oxidation of AA followed pseudo-Michaelis-Menten kinetics in the liver, kidney, and skin. Pseudo-Km values were similar among these tissues and averaged 0.67 mM. The maximal rate of AA oxidation in kidney, liver, and skin was 2890 +/- 436 (mean +/- SE, N = 3), 616 +/- 62, and 47.9 +/- 5.8 nmol/hr/g, respectively. The remaining organs oxidized AA at rates less than 40% of the rate in liver. Rates of metabolism in tissues from male and female mice were similar. 3-Hydroxypropionic acid was the only metabolite detected by high-performance liquid chromatographic analysis following incubation of tissues with [1-14C]AA. Kidney and liver also oxidized [2,3-14C]AA and [1-14C]acetate well, thus providing for the complete metabolism of AA carbons to CO2. These results demonstrate that the rate of AA metabolism varies significantly among mouse tissues and suggest that the kidneys and liver are major sites of detoxification of AA.

Applying simulation modeling to problems in toxicology and risk assessment--a short perspective.

The goals of this perspective have been to examine areas where quantitative simulation models may be useful in toxicology and related risk assessment fields and to offer suggestions for preparing manuscripts that describe these models. If developments in other disciplines serve as a bell-wether, the use of mathematical models in toxicology will continue to increase, partly, at least, because the new generations of scientists are being trained in an electronic environment where computation of all kinds is learned at an early age. Undoubtedly, however, the utility of these models will be directly tied to the skills of investigators in accurately describing models in their research papers. These publications should convey descriptions of both the insights obtained and the opportunities provided by these models to integrate existing data bases and suggest new and useful experiments. We hope these comments serve to facilitate the expansion of good modeling practices as applied to toxicological problems.

Structure of triornicin, a new siderophore.

Spectroscopic analysis of the tumor inhibitory factor triornicin produced by epicoccum purpurascens indicated that it was of similar structure to the known siderophore desferricoprogen, which is also produced by the fungus. The 1H and 13C NMR spectra indicated the replacement of an (E)-5-hydroxy-3-methyl-2-pentenoyl moiety of the desferricoprogen structure with an acetyl function. Cleavage of triornicin with basic methanol produced two fragments. The first was identified as a natural siderophore, dimerumic acid, which was also produced by basic cleavage of desferricoprogen. The second compound was identified as N alpha, N delta-diacetyl-N delta-hydroxyornithine. The structure of these fragments serves to define the structure of triornicin as a new siderophore.

The histopathological and biochemical response of the stomach of male F344/N rats following two weeks of oral dosing with ethyl acrylate.

Male F344/N rats were dosed with ethyl acrylate (EA) either by daily gavage or in the drinking water for 2 weeks. The gavage dose levels were 0, 2, 10, 20, 50, 100, and 200 mg/kg; the drinking water dose concentrations were 0, 200, 1,000, 2,000, and 4,000 ppm (corresponding to 0, 23, 99, 197, and 369 mg/kg/day, respectively). In those animals dosed by gavage, irritation of the forestomach increased in incidence and severity over the 20-200 mg/kg dose range. In those animals dosed with EA in the drinking water, a much lower incidence of forestomach irritation and less severe lesions were observed at corresponding dose levels. No lesions were observed in the glandular stomach from either of the 2 modes of oral administration. Following 2 weeks of gavage dosing with EA, the total non-protein sulfhydryl (NPSH) content of the forestomach and glandular stomach, and the NPSH concentration of the liver were determined 2-24 hr after the last gavage dose. Animals dosed at 200 mg/kg reached approximately 11% of the initial NPSH content in the forestomach at 6 hr after dosing. NPSH depletion of this magnitude has been associated with cytotoxicity of other tissues in other studies. By contrast, either the glandular stomach nor liver were depleted of NPSH to levels generally associated with toxicity. These observations are consistent with the conclusion that bolus dosing of EA induces severe depletion of critical cellular thiols in the forestomach with toxic consequences, but not in the glandular stomach or liver. Changing the mode of oral administration for EA to continued small doses in the drinking water allowed efficient detoxification and did not induce sulfhydryl depletion or comparable forestomach toxicity at the same daily body burden.

The disposition and metabolism of acrylic acid and ethyl acrylate in male Sprague-Dawley rats.

Following oral dosing of [2,3-14C]acrylic acid (AA; 4, 40, or 400 mg/kg) and [2,3-14C]ethyl acrylate (EA; 2, 20, or 200 mg/kg), the dosed radioactivity was rapidly excreted, with 50-75% of the dose for both compounds eliminated within 24 hr. The primary excretory metabolite for both compounds is carbon dioxide, accounting for 44-68% of the dose. HPLC analysis of the urine of AA- and EA-dosed animals indicated the presence of 3-hydroxypropionic acid. The detection of this metabolite suggests the incorporation of AA into propionic acid metabolism and may explain the rapid evolution of carbon dioxide from AA and EA. HPLC analysis of urine from EA-dosed rats revealed the presence of two metabolites derived from glutathione conjugation, N-acetyl-S-(carboxyethyl)cysteine and N-acetyl-S-(carboxyethyl)cysteine ethyl ester. The excretion of the N-acetyl cysteine derivatives of EA, expressed as a percentage of the dosed compound, decreased in a dose-dependent manner that may be attributed to the depletion of glutathione in organs primarily responsible for glutathione conjugation. No significant decrease in hepatic nonprotein sulfhydryl (NPSH) content was observed following oral dosing with EA at 2-200 mg/kg. However, the depletion of NPSH content at the dosing site, forestomach, and glandular stomach, decreased significantly between 0.02 and 0.2% EA in the dose solution (2 and 20 mg/kg). This observation would suggest that the dosing site represents a significant site of conjugation for relatively low doses of EA. Treatment with the carboxylesterase inhibitor, tri-o-cresyl phosphate (TOCP), 18 hr prior to acrylate dosing potentiated the depletion of hepatic nonprotein sulfhydryls, emphasizing the dominance of hydrolysis as a systemic detoxifying mode in this species. In contrast to EA, AA did not significantly decrease NPSH content in the liver, blood, or forestomach at oral doses of less than 8% AA in the dose solution (400 mg/kg), although a significant depletion of NPSH was observed in the glandular stomach at doses greater than 0.08% (4 mg/kg). No conjugation involving the double bond of AA could be detected in in vitro reactions with glutathione or in the in vivo metabolites, suggesting a secondary effect of AA on NPSH content in these organs. The weights of the forestomach and glandular stomach increased with AA dose, reflecting gross edema and inflammation. With EA this effect on organ weight was only demonstrated in the forestomach, and the response was increased when hydrolysis of EA was inhibited with TOCP.(ABSTRACT TRUNCATED AT 400 WORDS)

A physiologically based pharmacokinetic and pharmacodynamic model to describe the oral dosing of rats with ethyl acrylate and its implications for risk assessment.

A physiologically based pharmacokinetic and pharmacodynamic model has been developed to describe the absorption, distribution, and metabolism of orally dosed ethyl acrylate. The model describes the metabolism of ethyl acrylate in 14 tissues based on in vitro metabolic studies conducted with tissue homogenates. The routes of metabolism included in the model are carboxylesterase-catalyzed ester hydrolysis, conjugation with glutathione, and binding to protein. To adequately describe the rate and extent of glutathione depletion following gavage dosing, the steady-state rate of glutathione synthesis in the organs of interest was included. In vivo validation of the model was conducted by comparing the predictions of the model to the results of a variety of gavage dosing experiments with ethyl acrylate, including (1) the time course of glutathione depletion in a variety of tissues up to 98 hr following dosing at three dose levels, (2) the rate and extent of radiolabeled carbon dioxide excretion, and (3) protein binding in the forestomach. The very rapid metabolism predicted by the model was consistent with the observation that ethyl acrylate was metabolized too rapidly in vivo to be detected by common analytical techniques for tissue metabolite analysis. The validation data indicated that the model provides a reasonable description of the pharmacokinetics and the pharmacodynamic response of specific rat tissues following gavage dosing of ethyl acrylate. A dose surrogate, or measure of delivered dose, for ethyl acrylate was calculated and correlated with the incidence and severity of contact site toxicity (edema, inflammation, ulceration, and hyperplasia). The model provides a quantitative tool for evaluating exposure scenarios for their potential to induce contact-site toxicity, and it provides a quantitative approach for understanding the lack of toxicity in tissues remote from the dosing site.

Induction of the mitochondrial permeability transition in vitro by short-chain carboxylic acids.

We recently reported that acrylic acid (AA) induces the MPT in vitro, which we suggested might be a critical event in the acute inflammatory and hyperplastic response of the olfactory epithelium. The purpose of the present investigation was to determine if induction of the MPT is a general response to short-chain carboxylic acids or if there are critical physical chemical parameters for this response. Freshly isolated rat liver mitochondria were incubated in the presence of varying concentrations of selected carboxylic acids. All of the acids that we tested caused a concentration-dependent induction of the MPT, which was blocked by cyclosporine A. Although the C4 carboxylic acids were slightly more potent than the C5 acids, there was no correlation with the degree of saturation, the octanol/water coefficient (log P), or the dissociation constant (pK(a)) of the acids that we tested. We conclude that induction of the MPT in vitro is a general response to short-chain carboxylic acids having a pK(a) of 4 to 5.

Dosimetric adjustment factors for methyl methacrylate derived from a steady-state analysis of a physiologically based clearance-extraction model.

Cells within the epithelial lining of the nasal cavity metabolize a variety of low-molecular-weight, volatile xenobiotics. In common with terminology developed for other metabolizing organs, the nose extracts these chemicals from the airstream, thereby clearing some portion of the total nasal airflow. In this article, a physiologically based clearance-extraction (PBCE) model of nasal metabolism is used to predict extraction for steady-state conditions. This model, developed by simplification of existing physiologically based pharmacokinetic (PBPK) nasal models, has three tissue regions in two flow paths. A dorsal flow stream sequentially passes over a small area of respiratory epithelium and then over the entire olfactory epithelial surface within the nose. A ventral airstream, consisting of most of the total flow, passes over the larger portion (>80%) of the respiratory epithelium. Each underlying tissue stack has a mucus layer, an epithelial tissue compartment, and a blood exchange region. Metabolism may occur in any of the subcompartments within the tissue stacks. The model, solved directly for a steady-state condition, specifies the volumetric airflow over each stack. Computational fluid dynamic (CFD) solutions for the rat and human for the case with no liquid-phase resistance provided a maximum value for regional extraction, E(max)'. Equivalent air-to-liquid phase permeation coefficients (also referred to as the air-phase mass transfer coefficient) were calculated based on these E(max)' values. The PBCE model was applied to assess expected species differences in nasal extraction and in localized tissue metabolism of methyl methacrylate (MMA) in rats and in humans. Model estimates of tissue dose of MMA metabolites (in micromol metabolized/h/ml tissue) in both species were used to evaluate the dosimetric adjustment factor (DAF) that should be applied in reference concentration (RfC) calculations for MMA. For human ventilation rates equivalent to light exercise, the DAF was estimated to be 3.02 at 28.4 ppm, the benchmark concentration for nasal lesions. Depending on specific assumptions about distribution of esterase activities in human tissues, the range of DAF values was 1.56-8.00. The DAF for heavy exercise with a ventilation rate of 42 L/min was still 2.98. Estimated DAFs were concentration dependent, varying between 2.4 and 4.76 in the inhaled concentration range from 1 and 400 ppm. Present default methods utilize a DAF of 0.145. These steady-state calculations with this PBCE model should be useful in risk assessment calculations for a variety of vapors and gases that are converted to toxic metabolites in cells in the respiratory tract.

Evidence for estradiol promotion of neoplastic lesions in the rat vagina after initiation with N-methyl-N-nitrosourea.

The hypothesis that estrogens are tumor promoters was tested by tumor induction in the rat vagina. Ovariectomized rats were given a single dose of N-methyl-N-nitrosourea (MNU) by vaginal instillation, followed one week later by long-term release Silastic implants containing estradiol (E2). After 16 months, a significant incidence (4/9) of benign vaginal stromal polyps was found in the MNU-E2 group, but no vaginal polyps were seen in groups given either MNU or E2 alone. A number of non-neoplastic changes were also seen and were due to E2 treatment either with or without MNU. The incidence of stromal polyps and their restriction to animals receiving the initiator-promoter regimen alone suggests that estrogens promote tumorigenesis in the rat vagina.

Production and isolation of siderophores from the soil fungus Epicoccum purpurascens.

A large number of iron transport agents, siderophores, which stimulated the growth of Arthrobacter flavescens JG-9, were isolated during a study of the antitumor activity associated with the metabolic products of the fungus Epicoccum purpurascens. The production of the siderophores was significantly enhanced in a variety of media by culture of the fungus in the near absence of ferric iron. A novel method of purification involving a carboxylic ion-exchange resin separated the siderophores into four subgroups. The first subgroup, which contained the majority of the activity, was subsequently resolved in a similar manner with the carboxylic resin into seven individual siderophores. Of these, two were characterized as ferricrocin and coprogen whereas the others appeared to represent new compounds. One of the latter was given the name triornicin and exhibited slight antitumor activity in mice injected with Ehrlich ascites tumor cells.