Toxicokinetic testing strategies to demonstrate bone marrow exposure in in vivo micronucleus study for genotoxicity assessment of agrochemicals.

Following adoption of the new OECD test guideline (TG) 474 for the in vivo mammalian erythrocyte micronucleus (MN) test (29 July 2016), demonstration of exposure of target tissue (bone marrow) is required, if the test result is negative i.e. no cytogenetic damage. It implies that for many active ingredients, relevant metabolites or significant impurities with existing in vivo MN tests resulting in negative genotoxicity findings, evidence of target tissue exposure may be lacking and is considered a data gap in regulatory reviews. We present here toxicokinetic (TK) testing strategies for the design and conduct of studies that would demonstrate evidence of delivery of the test substance to the bone marrow. To illustrate this, three examples are presented with methods utilized under each scenario. We also propose a decision tree that may help design suitable TK studies to establish evidence of bone marrow exposure.

Age dependent in vitro metabolism of bifenthrin in rat and human hepatic microsomes.

Bifenthrin, a pyrethroid insecticide, undergoes oxidative metabolism leading to the formation of 4'-hydroxy-bifenthrin (4'-OH-BIF) and hydrolysis leading to the formation of TFP acid in rat and human hepatic microsomes. In this study, age-dependent metabolism of bifenthrin in rats and humans were determined via the rates of formation of 4'-OH-BIF and TFP acid following incubation of bifenthrin in juvenile and adult rat (PND 15 and PND 90) and human (<5years and >18years) liver microsomes. Furthermore, in vitro hepatic intrinsic clearance (CLint) of bifenthrin was determined by substrate consumption method in a separate experiment. The mean Vmax(±SD) for the formation of 4'-OH-BIF in juvenile rat hepatic microsomes was 25.0±1.5pmol/min/mg which was significantly lower (p<0.01) compared to that of adult rats (86.0±17.7pmol/min/mg). However, the mean Km values for juvenile (19.9±6.6µM) and adult (23.9±0.4µM) rat liver microsomes were similar. On the other hand, in juvenile human hepatic microsomes, Vmax for the formation of 4'-OH-BIF (73.9±7.5pmol/min/mg) was significantly higher (p<0.05) than that of adults (21.6±0.6pmol/min/mg) albeit similar Km values (10.5±2.8µM and 8.9±0.6µM) between the two age groups. The trends in the formation kinetics of TFP acid were similar to those of 4'-OH-BIF between the species and age groups, although the differences between juveniles and adults were less pronounced. The data also show that metabolism of bifenthrin occurs primarily via oxidative pathway with relatively lesser contribution (~30%) from hydrolytic pathway in both rat and human liver microsomes. The CLint values for bifenthrin, determined by monitoring the consumption of substrate, in juvenile and adult rat liver microsomes fortified with NADPH were 42.0±7.2 and 166.7±20.5µl/min/mg, respectively, and the corresponding values for human liver microsomes were 76.0±4.0 and 21.3±1.2µl/min/mg, respectively. The data suggest a major species difference in the age dependent metabolism of bifenthrin. In human liver microsomes, bifenthrin is metabolized at a much higher rate in juveniles than in adults, while the opposite appears to be true in rat liver microsomes.

In vitro metabolism of [14C]-benalaxyl in hepatocytes of rats, dogs and humans.

The in vitro comparative animal metabolism study is now a data requirement under EU Directive 1107/2009 for registration of plant protection products. This type of study helps determine the extent of metabolism of a chemical in each surrogate species and whether any unique human metabolite(s) are formed. In the present study, metabolism of racemic [14C]-benalaxyl, a fungicide was investigated in cryopreserved rat, dog and human hepatocytes. The metabolites generated were identified/characterized by LC/MS/MS with radiometric detection and comparison with reference standards. [14C]-glucuronide conjugates of benalaxyl metabolites in rat, dog and human hepatocytes were confirmed via additional experiments in which known reference standards were incubated with dog liver microsomes in the presence of UDPGA. After 4 h of incubation, benalaxyl was extensively metabolized in all the species with the following trend: dog (100%) > human (86%) > rat (75%). In all species, the major metabolic pathways consisted of hydroxylation of the methyl group in the xylene moiety to 2-hydroxymethyl-benalaxyl, further oxidation to its carboxylic acid analogue (benalaxyl-2-benzoic acid), and hydrolysis of the methyl ester to yield benalaxyl acid or 2-hydroxymethyl benalaxyl acid. In addition, glucuronidation of phase I metabolites occurred in all species, to a higher extent in dog hepatocytes in which 2-hydroxymethyl-benalaxyl-glucuronide conjugate constituted the most significant metabolite. No major unique metabolite was observed in human hepatocytes. Also, benalaxyl did not undergo stereo-selective metabolism in rat or human hepatocytes.

Bioconcentration of two basic pharmaceuticals, verapamil and clozapine, in fish.

The present study examined the bioconcentration of 2 basic pharmaceuticals: verapamil (a calcium channel blocker) and clozapine (an antipsychotic compound) in 2 fresh water fishes, fathead minnow and channel catfish. In 4 separate bioconcentration factor (BCF) experiments (2 chemicals × 1 exposure concentration × 2 fishes), fathead minnow and channel catfish were exposed to 190 µg/L and 419 µg/L of verapamil (500 µg/L nominal) or 28.5 µg/L and 40 µg/L of clozapine (50 µg/L nominal), respectively. Bioconcentration factor experiments with fathead consisted of 28 d uptake and 14 d depuration, whereas tests conducted on catfish involved a minimized test design, with 7 d each of uptake and depuration. Fish (n = 4-5) were sampled during exposure and depuration to collect different tissues: muscle, liver, gills, kidneys, heart (verapamil tests only), brain (clozapine tests only), and blood plasma (catfish tests only). Verapamil and clozapine concentrations in various tissues of fathead and catfish were analyzed using liquid chromatography-mass spectrometry. In general, higher accumulation rates of the test compounds were observed in tissues with higher perfusion rates. Accumulation was also high in tissues relevant to pharmacological targets in mammals (i.e. heart in verapamil test and brain in the clozapine test). Tissue-specific BCFs (wet wt basis) for verapamil and clozapine ranged from 0.7 to 75 and from 31 to 1226, respectively. Tissue-specific concentration data were used to examine tissue-blood partition coefficients.

Tissue-specific uptake and bioconcentration of the oral contraceptive norethindrone in two freshwater fishes.

The environmental presence of the oral contraceptive norethindrone (NET) has been reported and shown to have reproductive effects in fish at environmentally realistic exposure levels. The current study examined bioconcentration potential of NET in fathead minnow (Pimephales promelas) and channel catfish (Ictalurus punctatus). Fathead minnows were exposed to 50 µg/l NET for 28 days and allowed to depurate in clean water for 14 days. In a minimized 14-day test design, catfish were exposed to 100 µg/l NET for 7 days followed by 7-day depuration. In the fathead test, tissues (muscle, liver, and kidneys) were sampled during the uptake (days 1, 3, 7, 14, and 28) and depuration (days 35 and 42) phases. In the catfish test, muscle, liver, gill, brain, and plasma were collected during the uptake (days 1, 3, and 7) and depuration (day 14) stages. NET tissue levels were determined by gas chromatography-mass spectrometry (GC-MS). Accumulation of NET in tissues was greatest in liver followed by plasma, gill, brain, and muscle. Tissue-specific bioconcentration factors (BCFs) ranged from 2.6 to 40.8. Although NET has been reported to elicit reproductive effects in fish, the present study indicated a low potential to bioconcentrate in aquatic biota.

Bioconcentration of ibuprofen in fathead minnow (Pimephales promelas) and channel catfish (Ictalurus punctatus).

Pharmaceutical products and their metabolites are being widely detected in aquatic environments and there is a growing interest in assessing potential risks of these substances to fish and other non-target species. Ibuprofen is one of the most commonly used analgesic drugs and no peer-reviewed laboratory studies have evaluated the tissue specific bioconcentration of ibuprofen in fish. In the current study, fathead minnow (Pimephales promelas) were exposed to 250 µg L(-1) ibuprofen for 28 d followed by a 14 d depuration phase. In a minimized bioconcentration test design, channel catfish (Ictalurus punctatus) were exposed to 250 µg L(-1) for a week and allowed to depurate for 7 d. Tissues were collected during uptake and depuration phases of each test and the corresponding proportional and kinetic bioconcentration factors (BCFs) were estimated. The results indicated that the BCF levels were very low (0.08-1.4) implying the lack of bioconcentration potential for ibuprofen in the two species. The highest accumulation of ibuprofen was observed in the catfish plasma as opposed to individual tissues. The minimized test design yielded similar bioconcentration results as those of the standard test and has potential for its use in screening approaches for pharmaceuticals and other classes of chemicals.

Physiological effects and bioconcentration of triclosan on amphibian larvae.

We examined the acute effects of triclosan (TCS) exposure, a common antimicrobial found as a contaminant in the field, on survival and physiology of amphibian larvae. LC50 values were determined after 96h for North American larval species: Acris crepitans blanchardii, Bufo woodhousii woodhousii, Rana sphenocephala, and for a developmental model: Xenopus laevis. Amphibian larvae were most sensitive to TCS exposure during early development based upon 96-h LC50 values. Heart rates for X. laevis and North American larvae exposed to TCS were variable throughout development. Metabolic rates of X. laevis and R. sphenocephala larvae exposed to TCS were significantly affected in larvae exposed to [50% LC50] and [LC50]. Tissue uptake and tissue bioconcentration factor (BCF) of TCS were investigated in X. laevis, B. woodhousii woodhousii, and R. sphenocephala. In general, a significant increase was observed as exposure concentration increased. Tissue BCF values were dependent upon stage and species. While TCS concentrations used here are higher than environmental concentrations, exposure to TCS was dependent upon species and developmental stage, with early developmental stages being most sensitive to TCS exposure.