David L. Eisenbrandt

Assessment of Possible Carcinogenicity of Oxyfluorfen to Humans Using Mode of Action Analysis of Rodent Liver Effects

Oxyfluorfen is a herbicide that is not genotoxic and produces liver toxicity in rodents, following repeated administration at high dose levels. Lifetime rodent feeding studies reported in 1977 with low-purity oxyfluorfen (85%) showed no increase in any tumor type in rats (800 ppm, high dose) and only a marginally increased incidence of hepatocellular tumors in male CD-1 mice at the highest dose (200 ppm). To evaluate the potential carcinogenicity of the currently registered oxyfluorfen (> 98% purity), we conducted a series of short-term liver mode of action (MOA) toxicology studies in male CD-1 mice administered dietary doses of 0, 40, 200, 800, and 1600 ppm for durations of 3, 7, 10, or 28 days. MOA endpoints examined included liver weight, histopathology, cell proliferation, nuclear receptor-mediated gene expression, and other peroxisome proliferator-specific endpoints and their reversibility. Minimal liver effects were observed in mice administered doses at or below 200 ppm for up to 28 days. Increased liver weight, single-cell necrosis, cell proliferation, and peroxisomal acyl-CoA oxidase (ACO) were observed at 800 ppm after 28 days, but there was no increase in peroxisomes. Expression of Cyp2b10 and Cyp4a10 transcripts, markers of constitutive androstane receptor and peroxisome proliferator activated receptor α nuclear receptor activation, respectively, were increased at 800 and 1600 ppm after 3 or 10 days. Collectively, these data along with the negative genotoxicity demonstrate that oxyfluorfen (> 98% purity) has the potential to induce mouse liver tumors through a nongenotoxic, mitogenic MOA with a clear threshold and is not predicted to be carcinogenic in humans at relevant exposure levels.

A Tiered Approach to Life Stages Testing for Agricultural Chemical Safety Assessment

Aproposal has been developed by the Agricultural Chemical Safety Assessment (ACSA) Technical Committee of the ILSI Health and Environmental Sciences Institute (HESI) for an improved approach to assessing the safety of crop protection chemicals. The goal is to ensure that studies are scientifically appropriate and necessary without being redundant, and that tests emphasize toxicological endpoints and exposure durations that are relevant for risk assessment. The ACSA Life Stages Task Force proposes a tiered approach to toxicity testing that assesses a compounds potential to cause adverse effects on reproduction, and that assesses the nature and severity of effects during development and adolescence, with consideration of the sensitivity of the elderly. While incorporating many features from current guideline studies, the proposed approach includes a novel rat reproduction and developmental study with enhanced endpoints and a rabbit development study. All available data, including toxicokinetics, ADMEdata, and systemic toxicity information, are considered in the design and interpretation of studies. Compared to existing testing strategies, the proposed approach uses fewer animals, provides information on the young animal, and includes an estimation of human exposure potential for making decisions about the extent of testing required.

Characterization of Nuclear Receptor-Mediated Murine Hepatocarcinogenesis of the Herbicide Pronamide and its Human Relevance

The key events responsible for mouse liver tumors induced by a pesticide (viz., pronamide) were investigated in a series of studies employing molecular, biochemical, cellular, and apical endpoints. Based on these studies, it was demonstrated that the liver tumors were mediated by a mode of action (MoA) involving nuclear receptors (NRs) through the following key events: (1) CAR and PPAR-α receptor activation, (2) increased hepatocellular proliferation, eventually leading to (3) hepatocellular tumors. Specifically, gene expression analysis indicated robust, simultaneous coactivation of the CAR and PPAR-α NRs, as indicated by the induction of hepatic Cyp2b10 and Cyp4a10 transcripts, in response to dietary administration of pronamide to mice. The presence of hepatocellular hypertrophy and peroxisome proliferation was indicative of the activation of these two NRs at carcinogenic dose levels. Demonstrated induction of Cyp2b10 gene and protein, however, was not accompanied by enhancement of the corresponding enzyme activity (7-pentoxyresorufin-O-dealkylase (PROD)), suggesting that pronamide administration resulted in mechanism-based (suicide) inhibition of the enzyme in vivo. This was confirmed with an in vitro assay for suicide inhibition, where pronamide and/or its metabolites irreversibly inhibited Cyp2b10-mediated PROD activity. Analysis of hepatocellular proliferation via BrdU incorporation indicated a clear dose- and duration-related induction of S-phase DNA synthesis only in animals treated at and above the carcinogenic dose level. The available MoA data were evaluated for weight-of-evidence based upon the Bradford Hill criteria, followed by a human relevance framework. The conclusion from this evaluation is that pronamide-induced mouse liver tumors occur via an NR-mediated MoA involving CAR and PPAR-α activation and this MoA is not relevant to humans based on qualitative/quantitative differences between mice and humans.

Pronamide: Weight of evidence for potential estrogen, androgen or thyroid effects.

Based on the exposure potential to humans and environment, pronamide was one of 52 chemicals on the first list evaluated under US EPAs Endocrine Disruptor Screening Program (EDSP). The purpose of EDSP is to screen chemicals for their potential to interact with estrogen-, androgen-, or thyroid-signaling pathways. A battery of 11 Tier 1 assays was completed for pronamide in accordance with EDSP test guidelines. In addition, Other Scientifically Relevant Information, which included existing data from regulatory guideline studies and published literature, was used in a weight-of-evidence (WoE) evaluation of potential endocrine activity. The WoE conclusion is that pronamide does not interact directly with estrogen, androgen, or thyroid receptors or post-receptor events. Across in vivo studies, the liver is consistently and reproducibly the target organ for pronamides effects. Pronamide activates hepatocytic nuclear receptors (including constitutive androstane receptor), induces hepatic enzymes, produces hepatocellular hypertrophy and increases liver weights. These changes are coupled with increased metabolic activity and a subsequent increased metabolism and/or clearance of both steroid and thyroid hormones. Thus, while pronamide alters some endocrine-sensitive endpoints in EDSP Tier 1 assays, effects on liver metabolism likely explain altered hormone levels and indirect endocrine changes.

Integration of novel approaches demonstrates simultaneous metabolic inactivation and CAR-mediated hepatocarcinogenesis of a nitrification inhibitor

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Pronamide: Human relevance of liver-mediated rat leydig cell tumors

Dietary exposure to pronamide resulted in higher incidences of Leydig cell tumors (LCT) at 1000ppm in a 2-year cancer bioassay, but there were no testes effects at 40 or 200ppm, and no testes effects at 12-months at any concentration. A 90-day mode-of-action (MoA) study was conducted at concentrations of 0, 200, 1000 and 2000ppm. Standard parameters and stereological and proliferation analyses of LCs, targeted testis and liver gene expression, in vitro metabolism of testosterone by liver microsomes, and quantification of serum hormones and testosterone metabolites were evaluated. Increased testosterone metabolism due to increases in hepatic microsomal activity, alterations in serum hormone levels, and other data suggest that LCTs were mediated through a perturbation of the HPG-axis. Data suggest that this occurs after a threshold of exposure is reached, indicating a nonlinear/threshold dose-response. Pronamide-induced rat LCTs mediated by alterations to the HPG-axis have low relevance to humans due to quantitative differences in sensitivity between rats and humans to LCTs. Pronamide displayed no genotoxicity or direct endocrine effects. A margin of exposure approach for risk assessment and derivation of the chronic reference dose based on a point of departure of 200ppm is most appropriate and protective of human health.

Nitrapyrin: A scientific advisory group review of the mode of action and carcinogenicity in B6C3F1 mice

Nitrapyrin has been registered as a nitrogen stabilizer in the United States for many years based on a robust set of regulatory data. These data demonstrated that nitrapyrin was not genotoxic and that there were no tumors elicited in rats or mice that were relevant for human risk assessment. A repeat carcinogenicity study in B6C3F1 mice, conducted at two substantially higher-dose levels (0, 125 or 250 mg/kg/day) than the original study (0, 5, 25 or 75 mg/kg/day) identified liver, stomach, epididymal and Harderian gland tumors. In order to assess the relevance of these findings for human risk assessment, a Scientific Advisory Group (SAG) examined relevant microscopic changes in these tissues and also evaluated genotoxicity and mechanistic data. The SAG determined that the maximum tolerated dose had been exceeded in mice given 125 or 250 mg/kg/day, based on 26-33% decreased body weight gains (males-250 mg/kg/day), hepatocellular necrosis and compensatory hepatocellular proliferation (males and females-125 and 250 mg/kg/day). The SAG believed that the increased incidences of hepatocellular foci of alteration and hepatocellular neoplasms represented an epigenetic response to hepatocellular necrosis and increased mitogenesis. Increased incidences of proliferative lesions in the forestomach mucosa were likely secondary to the irritant effects of nitrapyrin. Neither the liver nor forestomach effects were interpreted to be a direct carcinogenic effect. Higher incidences of Harderian gland adenomas (females) and undifferentiated sarcomas in the epididymis represented normal biological variations in incidence and were unrelated to nitrapyrin. Therefore, it was the SAGs opinion that nitrapyrin exposure that does not produce target organ toxicity in exposed individuals would not be expected to increase the risk of cancer.

Mode of action and human relevance of pronamide-induced rat thyroid tumors.

Pronamide, a selective, systemic, pre- and post-emergence herbicide, caused an increased incidence of thyroid follicular cell adenomas in a rat carcinogenicity study. Thyroid tumors, as well as liver and pituitary changes, were limited only to the high-dose group. The evidence for and against specific potential modes of action (MoAs) for rat thyroid follicular cell adenomas and their relevance to humans is discussed. Pronamide is not mutagenic and therefore, direct DNA reactivity is not relevant as a MoA. The hypothesized MoA for this effect is altered homeostasis of the hypothalamic-pituitary-thyroid (HPT) axis mediated by the induction of hepatic enzymes, including uridine diphosphate glucuronosyltransferase (UGT). Evaluation of data from a series of regulatory guideline and MoA studies aimed at identifying the causative and associated key events supported a UGT-mediated MoA in the development of thyroid follicular tumors. This MoA for pronamide-induced thyroid tumors in rats, which involves increased thyroid hormone metabolism/clearance, altered thyroid hormone homeostasis and HPT stimulation is not considered relevant to humans based on quantitative species differences, making rats markedly more sensitive than humans to thyroid perturbations.

Chapter 103 – Sulfuryl Fluoride

Publisher Summary This chapter discusses the hazard identification, toxicity, and metabolism of sulfuryl fluoride. It is an odorless, colorless gas with a melting point of –135.82 °C and a boiling point of –55.38 °C. The vapor pressure is 1.3 × 104 torr at 25 °C. The solubility in water is 0.75 g/kg at 25 °C. Sulfuryl fluoride is used to control a wide variety of pests, including termites, cockroaches, clothes moths, bedbugs, rodents, and carpet beetles. Initial concentrations in fumigated structures are typically 2000–4000 ppm, although other concentrations may be used depending upon the target pest to be controlled, temperature, and the length of the exposure period. Sulfuryl fluoride is restricted to use only by certified applicators. Because sulfuryl fluoride is a gas, structures are completely sealed prior to fumigation and a small amount of chloropicrin is introduced into the structure to warn people and animals that the structure is being fumigated. If it comes into direct contact with skin, it can result in frostbite. Animals exposed to lethal doses of sulfuryl fluoride had tremors and convulsions. The dermal route does not appear to play a significant role in the toxicity of sulfuryl fluoride. Individuals have entered structures during fumigation with sulfuryl fluoride and were found dead or died shortly after exposure. The cause of death appeared to be severe pulmonary edema with congestion. The fumigation site cannot be reoccupied until aeration is complete. Only an approved detection device of sufficient sensitivity, such as the INTERSCAN, MIRAN, or Spectros ExplorIR gas analyzer, can be used to confirm a concentration of sulfuryl fluoride is 1 ppm or less.

Inter-Species Pharmacokinetic Comparison of Organic Acid Herbicides. Is the Dog a Relevant Species for Evaluation of Human Health Risk?

3,5,6-Trichloro-2-pyridinyloxyacetic acid (triclopyr) and 2,4-dichlorophenoxyacetic acid (2,4-D) are widely utilized herbicides that have undergone extensive toxicity and pharmacokinetic analysis. Based on this large data-base, the dog appears to exhibit the lowest No-Observed-Effect Levels (NOEL) relative to other species that have undergone toxicological evaluation. It is proposed that this unique sensitivity of the dog to organic acid herbicides is primarily due to the dog’s low capacity, relative to other species (including humans), to excrete organic acids. The excretion of organic acids, like triclopyr and 2,4-D, in the kidney is the net result of filtration, secretion and reabsorption processes. Although renal clearance mechanisms for organic acids are ubiquitous in mammalian species there are qualitative differences between species that account for observed discrepancies in clearance capacity. The pharmacokinetics and metabolism of triclopyr and 2,4-D are fairly consistent across species (including the dog), these herbicides are rapidly absorbed following oral administration and quickly cleared from the body into the urine predominantly unmetabolized. Aliometric parameter scaling was used to compare the triclopyr and 2,4-D pharmacokinetic parameters across species. The results of this analysis clearly illustrated the decreased capacity of the dog to effectively clear these organic acids relative to other species, including humans. In addition, this decreased clearance capacity of the dog is not compound specific, but is associated with organic acids that share common pharmacokinetic properties: as (1) extensive but reversible plasma protein-binding, (2) weak acids that are highly ionized at physiological pH, (3) limited metabolism of the parent acid, and (4) urinary excretion primarily involving a saturable, active secretion. The most likely mechanism responsible for the lower clearance in the dog, is a low capacity to actively secrete organic acids by the kidney. Other potential mechanisms for the lower clearance such as increased reabsorption from the tubules can not be excluded. This comparative species evaluation of the pharmacokinetics and renal clearance of triclopyr and 2,4-D questions the relevance of using dog toxicity data for the extrapolation of human health risk for these organic acids. Based on the lack of a pharmacokinetic concordance between the dog and other mammalian species it is suggested that the dog is not a reasonable surrogate for humans. Therefore, from a risk assessment perspective, it would seem prudent to utilize toxicity data from other species (e.g. rodents) for extrapolation of human health risk for these organic acid herbicides.