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An important aspect of safety assessment of chemicals (industrial and agricultural chemicals and pharmaceuticals) is determining their potential reproductive and developmental toxicity. A number of guidelines have outlined a series of separate reproductive and developmental toxicity studies from fertilization through adulthood and in some cases to second generation. The Extended One-Generation Reproductive Toxicity Study (EOGRTS) is the most recent and comprehensive guideline in this series. EOGRTS design makes toxicity testing progressive, comprehensive, and efficient by assessing key endpoints across multiple life-stages at relevant doses using a minimum number of animals, combining studies/evaluations and proposing tiered-testing approaches based on outcomes. EOGRTS determines toxicity during preconception, development of embryo/fetus and newborn, adolescence, and adults, with specific emphasis on the nervous, immunological, and endocrine systems, EOGRTS also assesses maternal and paternal toxicity. However, EOGRTS guideline is complex, criteria for selecting doses is unclear, and monitoring systemic dose during the course of the study for better interpretation and human relevance is not clear. This paper discusses potential simplification of EOGRTS, suggests procedures for relevant dose selection and monitors systemic dose at multiple life-stages for better interpretation of data and human relevance.

Traditionally, toxicity testing is conducted at fixed dose rates (i.e., mg/kg/day) without considering life-changing events, e.g., stress, sickness, aging- and/or pregnancy-related changes in physical, physiological and biochemical parameters. In humans, life-changing events may cause systemic dose non-proportionality requiring modulation of drug dosage; similar changes occur in animals altering systemic dose during chronic/carcinogenic testing leading to "late-occurring" effects in some studies. For example, propylene monomethyl ether, an industrial chemical, initially induced sedation in rats and mice with recovery upon induction of hepatic CYPs after ~1 week. Sedation reappeared in rats but not in mice after ~12 months of exposure due to decreased CYP activity in rats, elderly mice were able to maintain slightly higher CYP activity avoiding recurrence of sedation. The systemic dose of two pharmaceuticals (doxazosin and brimonidine tartrate) increased up to 6-fold in ≥12-month old rats with no toxicity. In a rat reproductive toxicity study, systemic dose of 2,4-D, an herbicide, rapidly increased due to increased consumption of 2,4-D-fortified diet during pregnancy, lactation and neonatal growth, requiring adjustment to maintain the targeted systemic dose. Ideally, toxicological studies should be based on systemic dose with the option of modulating external dose rates to maintain the targeted systemic dose. Systemic dose can easily be monitored in selected core study animals at desired intervals considering recent developments in sampling and analysis at a fraction of the overall cost of a study.

Objective: To ascertain the frequency of correction of vitamin D deficiency (VDD) with single or multiple doses of oral (PO) and intramuscular (IM) administration of 2 high-dose preparations of vitamin D3 (VD3). Methods: This was a prospective intervention study conducted in an ambulatory care setting. One hundred participants with VDD (25-hydroxy vitamin D [25-OHD]

This article is a revision of the previous edition article by Alan L. Blankenship, volume 3, pp 503–507, © 2005, Elsevier Inc.

Humans are exposed to chemicals either voluntarily or involuntarily. Exposure to therapeutic agents is generally voluntary via various routes, whereas exposure to occupational and environmental chemicals is involuntary primarily via inhalation, dermal, and/or oral routes. Not all of the chemical that a human is exposed to is absorbed and becomes available at the site of action. The most common way to determine the actual absorbed dose is to measure concentrations of active chemical (parent and/or metabolite(s)) in the systemic circulation. The ADME (absorption, distribution, metabolism, and excretion) study determines fate of chemicals following exposure and is conducted routinely in animal species used for testing toxicity. Such studies also elucidate biological processes that may be saturated above certain dose levels resulting in nonproportional distribution of the systemic dose. Such dose nonproportionality in systemic dose may result in variable or excessive concentrations at the target site(s) causing toxicity that may not otherwise occur when those biological processes are not saturated. For pharmaceuticals, systemic dose data are generated routinely in nonclinical safety studies very early in the toxicity testing program and play a major role in the selection of clinical trial doses. Additionally, such data are also generated in almost all general toxicology studies needed for investigational new drug-enabling programs. Concentration of parent drug and/or metabolite(s) in systemic circulation is widely used and accepted as a better estimate of exposure than comparison of administered dose. On the other hand, estimation of the systemic dose in toxicological studies for nonpharmaceuticals, like plant protection products (PPPs), is relatively rare. Only one ADME study is required by the guideline, which is usually conducted in the middle of the toxicity testing program of a new PPP, when most of the other studies have already been initiated and some of the short-term studies completed. Additionally, findings of the ADME study have been used rarely to understand adverse effects observed in other short- and long-term studies in the current hazard-based testing relying mostly on identifying adverse effects, often at very high dose levels. These studies are designed typically to determine toxicity-based maximum tolerated dose (MTD) without any consideration of changes in systemic dose due to dose nonproportionality and/or margin of exposure. This article is focused on methods and benefits of determining systemic dose in toxicological studies, especially conducted for nonpharmaceuticals, and use of kinetically-derived maximum dose (KMD) instead of MTD for the dose selection. The role of systemic dose in late-occurring toxicity due to change in physiological and biochemical parameters with age is also discussed. Determining systemic dose in toxicology studies will increase the understanding of potential mode of action (MOA) reducing the use of animals by avoiding unnecessary additional MOA studies.

Plant protection products (PPPs) and the active substance(s) contained within them are rigorously and comprehensively tested prior to registration to ensure that human health is not impacted by their use. In recent years, there has been a widespread drive to have more relevant testing strategies (e.g., ILSI/HESI-ACSA and new EU Directives), which also take account of animal welfare, including the 3R (replacement, refinement, and reduction) principles. The toxicity potential of one such new active substance, sulfoxaflor, a sulfoximine insecticide (CAS #946578-00-3), was evaluated utilizing innovative testing strategies comprising: (1) an integrated testing scheme to optimize information obtained from as few animals as possible (i.e., 3R principles) through modifications of standard protocols, such as enhanced palatability study design, to include molecular endpoints, additional neurotoxicity and immunotoxicity parameters in a subchronic toxicity study, and combining multiple test guidelines into one study protocol; (2) generation of toxicokinetic data across dose levels, sexes, study durations, species, strains and life stages, without using satellite animals, which was a first for PPP development, and (3) addition of prospective mode of action (MoA) endpoints within repeat dose toxicity studies as well as proactive inclusion of specific MoA studies as an integral part of the development program. These novel approaches to generate key data early in the safety evaluation program facilitated informed decision-making on the need for additional studies and contributed to a more relevant human health risk assessment. This supplement also contains papers which describe in more detail the approach taken to establish the MoA and human relevance framework related to toxicities elicited by sulfoxaflor in the mammalian toxicology studies: developmental toxicity in rats mediated via the fetal muscle nicotinic acetylcholine receptor (nAChR) ( Ellis-Hutchings et al. 2014 ); liver tumors in rodents mediated via CAR/PXR ( LeBaron et al. 2014 ); and Leydig cell tumors in Fischer 344 rats ( Rasoulpour et al. 2014 ).

Life-stage-dependent toxicity and dose-dependent toxicokinetics (TK) were evaluated in Sprague Dawley rats following dietary exposure to 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D renal clearance is impacted by dose-dependent saturation of the renal organic anion transporter; thus, this study focused on identifying inflection points of onset of dietary nonlinear TK to inform dose selection decisions for toxicity studies. Male and female rats were fed 2,4-D-fortified diets at doses to 1600 ppm for 4-weeks premating,2 weeks during mating, and to test day (TD) 71 to parental (P1) males and to P1 females through gestation/lactation to TD 96. F1 offspring were exposed via milk with continuing diet exposure until postnatal day (PND) 35. As assessed by plasma area under the curve for the time-course plasma concentration, nonlinear TK was observed ≥ 1200 ppm (63 mg/kg/day) for P1 males and between 200 and 400 ppm (14-27 mg/kg/day) for P1 females. Dam milk and pup plasma levels were higher on lactation day (LD) 14 than LD 4. Relative to P1 adults, 2,4-D levels were higher in dams during late gestation/lactation and postweaning pups (PND 21-35) and coincided with elevated intake of diet/kg body weight. Using conventional maximum tolerated dose (MTD) criteria based on body weight changes for dose selection would have resulted in excessive top doses approximately 2-fold higher than those identified incorporating critical TK data. These data indicate that demonstration of nonlinear TK, if present at dose levels substantially above real-world human exposures, is a key dose selection consideration for improving the human relevance of toxicity studies compared with studies employing conventional MTD dose selection strategies.

Dietary administration is a relevant route of oral exposure for regulatory toxicity studies of agrochemicals as it mimics potential human intake of the chemical via treated crops and commodities. Moreover, dietary administration of test compounds during a developmental toxicity study can deliver a prolonged and stable systemic exposure to the embryo or fetus at all stages of development. In this study, strategies were employed to optimize rabbit test material consumption via diet. Comparative toxicokinetic profiles of gavage versus dietary administration were evaluated in pregnant or non-pregnant New Zealand White rabbits for 2 novel agrochemicals with different plasma half-lives of elimination (sulfoxaflor, t½ = 13.5 h and halauxifen, t½ = 1 h). Dietary administration of sulfoxaflor resulted in stable 24-h plasma concentrations, whereas gavage administration resulted in a 3-fold fluctuation in plasma levels between Cmax and Cmin Dietary administration of sulfoxaflor resulted in a 2-fold higher nominal and diurnal systemic dose when compared with gavage dosing due to Cmax-related maternal toxicity following gavage. Results with the shorter half-life molecule, halauxifen, were more striking with a 6-fold diurnal fluctuation by the dietary route compared with a 368-fold fluctuation between Cmax and Cmin by gavage. Furthermore, plasma halauxifen was detectable only up to 12 h following gavage but up to 24 h following dietary administration. Finally, the presence of these compounds in fetal blood samples was demonstrated, confirming that dietary exposure is appropriate for achieving fetal exposure. Collectively, the results of these studies support the use of dietary exposure in rabbit developmental toxicity studies.

Integrated toxicokinetics (TK) data provide information on the rate, extent and duration of systemic exposure across doses, species, strains, gender, and life stages within a toxicology program. While routine for pharmaceuticals, TK assessments of non-pharmaceuticals are still relatively rare, and have never before been included in a full range of guideline studies for a new agrochemical. In order to better understand the relationship between diurnal systemic dose (AUC24h) and toxicity of agrochemicals, TK analyses in the study animals is now included in all short- (excluding acute), medium- and long-term guideline mammalian toxicity studies including reproduction/developmental tests. This paper describes a detailed procedure for the implementation of TK in short-, medium- and long-term regulatory toxicity studies, without the use of satellite animals, conducted on three agrochemicals (X11422208, 2,4-D and X574175). In these studies, kinetically-derived maximum doses (KMD) from short-term studies instead of, or along with, maximum tolerated doses (MTD) were used for the selection of the high dose in subsequent longer-term studies. In addition to leveraging TK data to guide dose level selection, the integrated program was also used to select the most appropriate method of oral administration (i.e., gavage versus dietary) of test materials for rat and rabbit developmental toxicity studies. The integrated TK data obtained across toxicity studies (without the use of additional/satellite animals) provided data critical to understanding differences in response across doses, species, strains, sexes, and life stages. Such data should also be useful in mode of action studies and to improve human risk assessments.

TK Modeler 1.0 is a Microsoft® Excel®-based pharmacokinetic (PK) modeling program created to aid in the design of toxicokinetic (TK) studies. TK Modeler 1.0 predicts the diurnal blood/plasma concentrations of a test material after single, multiple bolus or dietary dosing using known PK information. Fluctuations in blood/plasma concentrations based on test material kinetics are calculated using one- or two-compartment PK model equations and the principle of superposition. This information can be utilized for the determination of appropriate dosing regimens based on reaching a specific desired C(max), maintaining steady-state blood/plasma concentrations, or other exposure target. This program can also aid in the selection of sampling times for accurate calculation of AUC(24h) (diurnal area under the blood concentration time curve) using sparse-sampling methodologies (one, two or three samples). This paper describes the construction, use and validation of TK Modeler. TK Modeler accurately predicted blood/plasma concentrations of test materials and provided optimal sampling times for the calculation of AUC(24h) with improved accuracy using sparse-sampling methods. TK Modeler is therefore a validated, unique and simple modeling program that can aid in the design of toxicokinetic studies.

Vitamin D, parathyroid hormone levels and calcium absorption was assessed before and after cholecalciferol using Strontium as a surrogate. Increase in 25OHD, lowering of iPTH with no effect on Sr absorption was seen, suggesting the possibility that maximal Ca absorption had already been achieved in these volunteers. This paper discusses the determination of calcium (Ca) absorption, using strontium (Sr) as a surrogate, before and after a single IM injection of vitamin D3 (600,000 IU). Baseline serum 25-hydroxyvitamin D (25OHD), Sr, Ca, P, and intact parathyroid hormone (iPTH) were determined in 53 fasting volunteers, followed by administrating (PO) 0.03 mM (4.8 mg/kg) SrCl2 and collecting blood at 0.5, 1 and 4 h to determine the absorption (AUC0→t ) of Sr. Following the initial absorption test, volunteers received a single IM injection of 600,000 IU vitamin D3. Two months later, the fasting serum and the Sr absorption test were repeated, as described above. The IM injection of vitamin D3 caused a significant increase in fasting 25OHD (from 43.5 ± 19 to 66.1 ± 19.1 nmol/L (p

It is well recognized that expression of enzymes varies during development and growth. However, an in-depth review of this acquired knowledge is needed to translate the understanding of enzyme expression and activity into the prediction of change in effects (e.g. kinetics and toxicity) of xenobiotics with age. Age-related changes in metabolic capacity are critical for understanding and predicting the potential differences resulting from exposure. Such information may be especially useful in the evaluation of the risk of exposure to very low (µg/kg/day or ng/kg/day) levels of environmental chemicals. This review is to better understand the ontogeny of metabolizing enzymes in converting chemicals to either less-toxic metabolite(s) or more toxic products (e.g. reactive intermediate[s]) during stages before birth and during early development (neonate/infant/child). In this review, we evaluated the ontogeny of major "phase I" and "phase II" metabolizing enzymes in humans and commonly used experimental animals (e.g. mouse, rat, and others) in order to fill the information gap.

These studies were conducted to determine subchronic toxicity and genotoxicity of the biocide diiodomethyl-p-tolysulfone (DIMPTS) in rats and dogs. Male and female Sprague-Dawley rats and Beagle dogs were administered DIMPTS for 90-days via the diet at 0, 5, 20, and 80 mg/kg/day to rats and via capsules at 0, 2, 10, and 60 mg/kg/day to dogs. In rats, the only treatment-related finding was squamous metaplasia of the salivary gland duct in the 80 mg/kg/day group. In dogs, female body weights in the high-dose group were significantly lower than controls. Altered clinical pathology parameters were considered secondary to inflammatory changes observed in some of the dogs. Treatment-related alterations were found in the thyroid glands, salivary glands, GI-tract in the mid- and/or high-dose groups. DIMPTS was negative in the four in vitro and one in vivo genotoxicity assays. The toxicological effects noted in the two mammalian species are consistent with the principal toxic effects of iodine, and are proposed to arise from release of iodide from the DIMPTS molecule with toxic sequelae.

The biocide diiodomethyl-p-tolylsulfone (DIMPTS) caused dystocia, decreased neonatal survival and hypothyroidism in rat reproduction studies resembling the effects caused by iodine. One molecule of DIMPTS contains two iodine moieties that are hydrolyzed upon ingestion and systemically absorbed, suggesting iodine toxicity as a probable mode of action for the effects observed in rats. This study compared the effects induced by DIMPTS and an equimolar concentration of its de-iodinated analogue, methyl-p-tolylsulfone (MPTS). Groups of 20 female Sprague Dawley rats were fed diets supplying 80 mg DIMPTS/kg/day, 32 mg MPTS/kg/day or control feed from prior to breeding through lactation and gonadal function, mating performance, conception, gestation, parturition, lactation, survival, growth and development of pups evaluated through postnatal day 7. Serum thyroid hormones and iodine levels in milk and sera were also determined. Females given DIMPTS had increased incidence of vulvar discharge and dystocia, decreased litter size, decreased body weights and feed consumption, increased thyroid weights, thyroid follicular cell hypertrophy with decreased colloid, decreased triidothyronine, and increased thyroid stimulating hormone levels. DIMPTS pups had decreased neonatal survival and body weights. These effects were associated with elevated levels of iodine in milk and sera. In contrast, MPTS did not produce similar effects in adult females or their offspring. These data support the hypothesis that the dystocia, altered neonatal survival and hypothyroidism following repeated dietary administration of DIMPTS were due to excessive iodine released from DIMPTS during absorption and metabolism.

N-(2-aminoethyl)ethanolamine (AEEA) caused aneurysms of the great vessels in rats exposed in utero and during the first days post partum, exacerbated by postnatal treatment of the lactating dams (Moore et al., 2012. Birth Defects Res B Dev Reprod Toxicol [95:116-122]). The purpose of this work was to examine the systemic availability of AEEA during gestation and early lactation. The absorption of AEEA was determined following oral administration to nonpregnant and pregnant female Wistar rats. A single dose administered by gavage (0.5 or 50 mg/kg) on gestation day 18 was rapidly and extensively (>90%) absorbed from the gastrointestinal tract (absorption t1/2 = 0.1–0.2 hr). Elimination from the plasma followed a biphasic pattern, with a rapid elimination phase (t1/2 α = 1.6–1.8 hr) followed by a slower phase (t1/2 β = 16.7–17.3 hr). Following repeated gavage administration during gestation day 17 to 19, 14C-AEEA–derived radioactivity readily partitioned into the fetus and was evenly distributed therein, but cleared approximately twofold slower from the fetal blood and tissues than the maternal blood and chorioallantoic placenta. When administered to lactating dams during lactation days 1 to 12, 14C-AEEA–derived radioactivity preferentially partitioned into the milk reaching levels that were between 1.6- and 2.5-fold higher than the maternal blood. Although the concentration of AEEA equivalents in the maternal blood remained quite consistent, the concentration in the milk fell by almost 40% between lactation days 4 and 12, probably reflecting an increase in milk production over this same period. We confirm exposure of the offspring to AEEA both in utero and during lactation, but that AEEA does not appear to specifically concentrate in the great vessels.

Toxicokinetic (TK) information can substantially enhance the value of the data generated from toxicity testing, and is an integral part of pharmaceutical safety assessment. It is less widely used in the chemical, agrochemical and consumer products industries, but recognition of its value is growing, as reflected by increased reference to the use of TK information in new and draft OECD test guidelines. To help promote increased consideration of the important role TK can play in chemical risk assessment, we have gathered practical examples from the peer-reviewed literature, as well as in-house industry data, that highlight opportunities for the use of TK in the selection of dose levels. Use of TK can help to ensure studies are designed to be of most relevance to assessing potential risk in humans, and avoid the use of excessively high doses that could result in unnecessary suffering in experimental animals. Greater emphasis on the potential contribution of TK in guiding study design and interpretation should be incorporated in regulatory data requirements and associated guidance.

This study determined the metabolism of 3 drinking water disinfection by-products (halogenated acetic acids [HAAs]), bromodichloroacetic acid (BDCAA), chlorodibromoacetic acid (CDBAA), and tribromoacetic acid (TBAA), using rat, mouse, human liver microsomes, and recombinant P450. Metabolism proceeded by reductive debromination forming a di-HAA; the highest under nitrogen >>2% oxygen > atmospheric headspaces. Vmax for the loss of tri-HAA was 4 to 5 times higher under nitrogen than atmospheric headspace. Intrinsic metabolic clearance was TBAA>CDBAA>>BDCAA. At the high substrate concentrations, tri-HAA consumption rate was 2 to 3 times higher than the formation of di-HAA. Liberation of Br− from TBAA corresponded to the expected amount produced after DBAA formation, indicating retention of Br− by additional metabolite/metabolites. Subsequent experiments with CDBAA detected negligible formation of chlorodibromomethane (CDBM) and failed to account for the missing tri-HAA. Carbon monoxide and especially diphenyleneiodonium ([DPI] P450 reductase inhibitor) blocked CDBAA metabolism. Other chemical inhibitors were only partially able to block CDBAA metabolism. Most effective were inhibitors of CYP 2E1 and CYP 3A4. Immunoinhibition studies using human liver microsomes and anti-human CYP 2E1 antibodies were successful in reducing CDBAA metabolism. However, CDBAA metabolism in wild-type (WT) and CYP 2E1 knockout (KO) mouse liver microsomes was similar, suggesting significant interspecies differences in CYP isoform in tri-HAA metabolism. Additional assessment of CYP isoform involvement was complicated by the finding that recombinantly expressed rat and human P450 reductase was able to metabolize CDBAA, which may be a contributing factor in interspecies differences in tri-HAA metabolism.

A previously developed PBPK model for ethylene glycol and glycolic acid was extended to include glyoxylic acid, oxalic acid, and the precipitation of calcium oxalate that is associated with kidney toxicity in rats and humans. The development and evaluation of the PBPK model was based upon previously published pharmacokinetic studies coupled with measured blood and tissue partition coefficients and rates of in vitro metabolism of glyoxylic acid to oxalic acid, glycine and other metabolites using primary hepatocytes isolated from male Wistar rats and humans. Precipitation of oxalic acid with calcium in the kidneys was assumed to occur only at concentrations exceeding the thermodynamic solubility product for calcium oxalate. This solubility product can be affected by local concentrations of calcium and other ions that are expressed in the model using an ion activity product estimated from toxicity studies such that calcium oxalate precipitation would be minimal at dietary exposures below the NOAEL for kidney toxicity in the sensitive male Wistar rat. The resulting integrated PBPK predicts that bolus oral or dietary exposures to ethylene glycol would result in typically 1.4-1.6-fold higher peak oxalate levels and 1.6-2-fold higher AUC's for calcium oxalate in kidneys of humans as compared with comparably exposed malemore » Wistar rats over a dose range of 1-1000 mg/kg. The converse (male Wistar rats predicted to have greater oxalate levels in the kidneys than humans) was found for inhalation exposures although no accumulation of calcium oxalate is predicted to occur until exposures are well in excess of the theoretical saturated vapor concentration of 200 mg/m{sup 3}. While the current model is capable of such cross-species, dose, and route-of-exposure comparisons, it also highlights several areas of potential research that will improve confidence in such predictions, especially at low doses relevant for most human exposures.« less

This study was conducted to determine species differences in the metabolism of ethylbenzene (EB) in liver and lung. EB (0.22-7.0mM) was incubated with mouse, rat and human liver and lung microsomes and the formation of 1-phenylethanol (1PE), acetophenone (AcPh), 2-ethylphenol (2EP), 4-ethylphenol (4EP), 2,5-ethylquinone, and 3,4-ethylquinone were measured. Reactive metabolites (2,5-dihydroxyethylbenzene-GSH [2EP-GSH] and 3,4-dihydroxyethylbenzene-GSH [4EP-GSH]) were monitored via glutathione (GSH) trapping technique. None of the metabolites were formed at detectable levels in incubations with human lung microsomes. Percent conversion of EB to 1PE ranged from 1% (rat lung; 7.0mM EB) to 58% (mouse lung; 0.22 mM EB). More 1PE was formed in mouse lung than in mouse liver microsomes, although formation of 1PE by rat liver and lung microsomes was similar. Metabolism of EB to 1PE was in the order of mouse > rat > human. Formation of AcPh was roughly an order of magnitude lower than 1PE. Conversion of EB to ring-hydroxylated metabolites was much lower (0.0001% [4EP-GSH; rat lung] to 0.6% [2EP-GSH; mouse lung]); 2EP-GSH was typically 10-fold higher than 4EP-GSH. Formation of 2EP-GSH was higher by lung (highest by mouse lung) than liver microsomes and the formation of 2EP-GSH by mouse liver microsomes was higher than rat and human liver microsomes. Increasing concentrations of EB did lead to a decrease in amount of some formed metabolites. This may indicate some level of substrate- or metabolite-mediated inhibition. High concentrations of 2EP and 4EP were incubated with microsomes to further investigate their oxidation to ethylcatechol (ECat) and ethylhydroquinone (EHQ). Conversion of 2EP to EHQ ranged from 6% to 9% by liver (mouse > human > rat) and from 0.1% to 18% by lung microsomes (mouse >> rat >> human). Conversion of 4EP to ECat ranged from 2% to 4% by liver (mouse > human approximately rat) and from 0.3% to 7% by lung microsomes (mouse >> rat >> human). Although ring-oxidized metabolites accounted for a relatively small fraction of overall EB metabolism, its selective elevation in mouse lung microsomes is nonetheless consistent with the hypothesized mode of action for observed preferential toxicity of EB to the lung in this species.

Methyl isobutyl ketone (MIBK) is a solvent used in numerous products and processes and may be present in the air of the workplace as a vapor. The American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value-time-weighted average (TLV-TWA) and TLV-short term exposure limit (TLV-STEL) for MIBK are 50 and 75 ppm, respectively. These workplace air concentration limits were set to protect workers from irritation, neurasthenic symptoms and possible adverse effects to their livers and kidneys. A recent revision of the ACGIH limit value has been proposed, to reduce the current TLV-TWA to 30 ppm. This article predicts the kinetics and accumulation of MIBK in humans exposed repeatedly in various exposure scenarios (8, 12, and 24h/day for 7 days) to the current ACGIH TLV-TWA of 50 ppm. The kinetic parameters of the model were derived from published human time-course blood MIBK data from a single 2h inhalation exposure to 48.9 ppm MIBK. The model correctly simulated single exposure experimental data with a rapid rise in blood concentration to 1.06 microg/ml within 1h and approachedor=99% steady-state blood level in 4h of exposure. MIBK was predicted to be rapidly eliminated from blood after terminating the exposure, reaching 0.53 microg/ml and 0.13 microg/ml within 0.5 and 2h post-exposure, respectively. Within 4h after the termination of exposure, blood concentration would be expected to1% of the steady-state concentration. On the basis of these results, it is concluded that accumulation of MIBK in workers due to repeated inhalation exposure is not likely to occur at the current TLV-TWA concentration of 50 ppm.

Studies were conducted to assess the effects of black carbon, clay type and aging (1–1.5 yr) on desorption and bioavailability of hexachlorobenzene (HCB) in spiked artificial sediments. Tenax (a super sorbent)-mediated desorption was used to examine the effects of these parameters on the physicochemical availability of HCB. The Tenax-mediated desorption of HCB from the four aged artificial sediments exhibited biphasic kinetics. The fast desorbing fractions ranged from 64.8% to 22.3%, showing reductions of 4.0–18.9% compared with freshly-spiked sediments. Statistical analysis on the fast desorbing fractions showed that all three treatment effects (i.e., montmorillonite clay, black carbon content, and aging) were significant. Two sediments with higher black carbon content exhibited much greater aging effects (i.e., greater reduction in fast desorbing fraction) than the other two sediments without the addition of black carbon. For both freshly-spiked and aged sediments, the desorption resistant sediment-bound HCB (i.e., slow desorbing fraction) correlated reasonably well to previously reported rat fecal elimination of HCB, which is a measure of the non-bioavailable fraction of sediment-bound HCB. A similar correlation was also observed between fast desorbing fraction and previously reported accumulation of HCB in the rat body (carcass + skin). These observations suggest that physicochemical availability, as defined by the desorption of HCB from sediments, provides a reasonable prediction of the oral bioavailability of sediment-bound HCB to rats. These results showed that montmorillonite clay, black carbon and aging reduced physicochemical availability and ultimately bioavailability of sediment-bound HCB.

This study determined the oral and dermal ADME of 2-amino-2-methyl-1-propanol (AMP), a substituted aliphatic alcohol used in a number of industrial and consumer products. Groups of 4 male Fischer 344 rats received either a single bolus oral or dermal dose of 18 mg/kg (14)C-AMP in water. The dermal dose was applied to an area of 12 cm(2) on the back of the rats for 6h under semi-occluded conditions and fitted with rodent jackets to prevent grooming. Time-course blood and excreta were collected, radioactivity determined and blood and urine analyzed for AMP and metabolites. The orally administered (14)C-AMP was rapidly absorbed and eliminated in urine. Elimination of radioactivity from blood was biphasic with a rapid alpha phase (t(1/2 alpha) approximately 1h) followed by a slower beta phase (t(1/2 beta)=41+/-4h plasma and 69+/-34 h RBC). Total urinary elimination accounted for 87-93% of the dose, most (72-77%) within the first 48 h. Fecal elimination accounted for only 3-10%. Only 3-4% of the dose was found in tissues 168h post-dosing. The total dermal absorption of (14)C-AMP was 42% that included approximately 8% of the dose remaining at the application site 162 h after washing. Less than 1% of the applied dose remained in the stratum corneum and approximately 6% of the dose was found in tissues. Urinary elimination was 43% of the administered dose, most ( approximately 17%) within 48 h, and approximately 2% was eliminated in feces. It took much longer to reach plasma C(max) after dermal application (8.5+/-4.7 h in plasma and 4.0+/-2.8h in RBC) than the oral dose (0.3h) and the AUC(0-->alpha) for dermal dose was approximately 8-fold lower than with the oral dose. Again, elimination of the radioactivity from blood was biphasic with apparent t(1/2 alpha) of 9+/-6 and 2+/-1h for plasma and RBC, respectively. However, the alpha phase was "flipped-flopped" due to relatively slow dermal penetration and rapid elimination of the systemically absorbed dose, which was corrected to approximately 0.3 h after separating alpha elimination phase from the absorption. The slope of the beta phase became parallel to the oral route upon cessation of the absorption from the dose site skin, between 18 and 42 h post-washing. No metabolite of AMP was detected either in blood or excreta of any rat. Results of this study suggests that toxicologically significant concentrations of AMP are unlikely to be achieved in the systemic circulation and/or target tissues in humans as a result of dermal application of products containing AMP. Additionally, systemically absorbed dose will be rapidly eliminated from the body with little remaining at the application site.

The desorption kinetics of hexachlorobenzene (HCB) in four freshly spiked artificial sediments were determined using a polymeric adsorbent Tenax-mediated desorption. The sediments included a standard sediment (SS) prepared as per Organisation for Economic Cooperation and Development 218 guidelines and three derived artificial sediments prepared by supplementing the SS sediment with various levels of black carbon (lamp black soot) and/or montmorillonite clay. The desorption kinetics exhibited biphasic behavior, i.e., a fast desorbing fraction followed by a slow desorbing fraction. The addition of either lamp black soot or montmorillonite clay resulted in the reduction of the fast desorbing fraction (Ffast) of HCB in three derived sediments compared with SS sediment. Both black carbon and montmorillonite clay treatment effects on the fast desorbing fraction were statistically significant for the four artificial sediments. The black carbon treatment (i.e., addition of 0.5% wt/wt lamp black soot) effect was an average reduction of Ffast by approximately 11%, whereas the montmorillonite treatment (i.e., addition of 15% wt/wt montmorillonite clay) effect was an average reduction of Ffast by approximately 17%. The presence of soot black carbon particles reduced the desorption rate of HCB in sediments since black carbon exhibits very high sorption capacity and extremely slow diffusion rate compared with those of the natural organic matter in sediment.

Many new molecular entities targeted for pharmaceutical applications face serious development challenges because of poor water solubility. Although particle engineering technologies such as controlled precipitation have been shown to enhance aqueous dissolution and bioavailability of poorly water soluble active pharmaceutical ingredients, the data available are the results of laboratory-scale experiments. These technologies must be evaluated at larger scale to ensure that the property enhancement is scalable and that the modified drugs can be processed on conventional equipment. In experiments using ketoconazole as the model drug, the controlled precipitation process was shown to produce kg-scale modified drug powder with enhanced dissolution comparable to that of lab-scale powder. Ketoconazole was demonstrated to be stable throughout the controlled precipitation process, with a residual methanol level below the ICH limit. The modified crystalline powder can be formulated, and then compressed using conventional high-speed tableting equipment, and the resulting tablets showed bioavailability more than double that of commercial tablets. When appropriately protected from moisture, both the modified powder and tablets prepared from the modified powder showed no change in dissolution performance for at least 6 months following storage at accelerated conditions and for at least 18 months following storage at room temperature.

The guidelines for risk assessment of plant protection products (PPPs) and other non-pharmaceuticals were developed over three decades ago and have generally not been updated to incorporate advancements in toxicology and exposure sciences. These guidelines recommend using maximum-tolerated-dose (MTD) even when human relevance of such high doses is mostly limited due to orders of magnitude margin-of-exposure. Conducting animal studies at such high doses often requires further mode-of-action (MoA) studies elucidating human relevance. In order to improve data, ILSI/HESI-ACSA technical committee proposed a tiered approach with emphasis on determining systemic dose of parent and/or metabolite(s) in test animals as biological effects are reflective of systemic rather than administered dose. Any deviation from linearity in systemic dose (saturation of absorption or elimination) in animal studies may have profound toxic effect(s) not expected to occur in likely human exposure scenarios and should be avoided. Toxicity studies should ideally be conducted at kinetically linear doses or slightly above the point of departure from linearity or kinetically-derived maximum dose (KMD) as the systemic dose nonlinearity is a more sensitive parameter occurring much earlier than the MTD endpoints. Therefore, determining systemic dose, especially KMD, in study animals is an improvement to hazard assessment of PPPs and other non-pharmaceuticals allowing toxicologists to better understand findings in animals at systemically linear as well as nonlinear doses to likely human exposures which can easily be accomplished using core study animals as outlined below. Determining systemic dose in studies will also increase the understanding of initial potential MoA of a PPPs and other non-pharmaceuticals and reduce the use of animals by avoiding unnecessary additional MoA studies.

Haber's Rule and associated time to coma after monochloroacetic acid (MCA) exposure in male Sprague-Dawley (SD) rats and time to death after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in female Sprague-Dawley rats and male A/J mice were investigated at isoeffective or nearly isoeffective doses. Animals exposed to MCA received either single bolus intravenous (iv) doses or a loading dose rate via the iv route followed by a maintenance dose rate through subcutaneously implanted osmotic mini pumps. For TCDD, rats received a loading dose rate via bolus oral gavage followed by maintenance dose rates through iv injection every fourth day until death. Mice received both loading and maintenance (once a week) dose rates via oral gavage. Different dosing regimens were employed to demonstrate that the key to Haber's Rule lies not in the route of administration but in conducting experiments under conditions of kinetic steady state. Single doses of MCA produced inconsistent time responses but a reasonably constant c x t product (7657+/-391 mg/kg x min) which was not anticipated although it should have been expected because MCA's elimination half-life (2 h) is twice as long as its time to coma ( approximately 1h). Generation of kinetic steady state by infusion of MCA after iv injection of a loading dose rate resulted in a consistently decreasing time response with increasing dose which diminished the variability in the c x t (dose x time)=k relationship (8032+/-136 mg/kg x min). Both acute and chronic toxicity of TCDD under conditions of kinetic steady state yielded consistent time responses with inverse proportionality between dose and time leading to robust c x t=k products in both rats (1060+/-82 microg/kg x day) and mice (80+/-2 mg/kg x day).

To determine potential nitrosation of triethanolamine (TEA) to N-nitrosodiethanolamine (NDELA) at different physiological conditions of the GI tract, in vitro NDELA formation was examined in aqueous reaction mixtures at several pHs (2-10) adjusted with acetic, sulphuric or hydrochloric acids or in cultures of mouse cecal microflora incubated. In vivo NDELA formation was also determined in blood, ingesta, and urine of female B6C3F1 mice after repeated dermal, most relevant human route, or single oral exposure to 1000 mg/kg TEA in the presence of high oral dosages of NaNO(2). Appropriate diethanolamine (DEA) controls were included to account for this impurity in the TEA used. Samples were analyzed for NDELA using GC/MS. The highest degree of nitrosation of TEA to NDELA ( approximately 3%) was observed in the in vitro cultures at pH 4 and acetic acid with lower amounts obtained using sulphuric acid ( approximately 1.3%) and hydrochloric acid ( approximately 1.2%). At pH 7,1% of the TEA was nitrosated to NDELA and at pH 2 (HCl) or pH 10 (NaOH) no NDELA was found above the limit of detection. In incubated cultures containing cecal microflora and nutrient broth, only 0.68% of TEA was nitrosated to NDELA. No NDELA was formed in rats repeatedly dermally dosed with TEA at the limits of detection in blood (0.001 microg/ml, ppm), ingesta (0.006 microg/ml, ppm), and urine (0.47 microg/ml, ppm). Levels of NDELA measured in blood and ingesta after a single oral dose of TEA and NaNO(2) were less than those in DEA controls. These findings in toto confirm the lack of any significant formation of NDELA from TEA in vivo.

Disinfection of drinking water typically produces a mixture of mono-, di-, and tri-halogenated acetic acids (HAAs). In this study, we investigated the toxicokinetics of HAA mixtures in naive and glutathione transferase zeta 1 (GSTzeta)-depleted male F344 rats administered orally or iv to Mixture-1 (monobromo [MBAA]- dichloro- [DCAA], chlorodibromo- [CDBAA], tribromo- [TBAA] acetic acids) or Mixture-2 (bromochloro- [BCAA], dibromo- [DBAA], trichloro- [TCAA] bromodichloro- [BDCAA] acetic acids) at a dose of 25 micromol/kg HAA. Serial blood samples were collected at various times up to 36 h, and the plasma concentrations of each HAA quantified by GC-ECD. Rats were pretreated for 7 d with drinking water containing 0.2 g/l DCAA to deplete the GSTzeta (GSTZ1-1) activity in the liver. An additional group of GSTzeta-depleted rats were orally dosed with each mixture and euthanized at 0.25, 0.5, 1, 2, and 4 h to determine tissue distribution of mixture components. In both mixtures, GSTzeta depletion primarily affected the toxicokinetics of di-HAAs (DCAA, BCAA, and DBAA), with the total body clearance (Cl b) decreasing 3- to 10-fold. Interestingly, DCAA pretreatment appeared to increase the elimination of Mixture-2 tri-HAAs (TCAA and BDCAA). After oral administration, DCAA exhibited a complex time-course plasma profile with secondary peaks appearing long after completion of the initial absorption phase. This phenomenon coincided with elevated DCA levels in the lower portion of the GI tract compared to CDBAA and TBAA. Comparison of the results with previous studies employing similar or higher doses of individual HAAs indicated the primary difference in HAA toxicokinetics when administered as mixture was a reduction in Cl b. These results suggest competitive interactions between tri- and di-HAAs beyond what would be predicted from individual HAA studies. For di-HAAs, the total dose is important, as clearance is dose dependent due to competition for GSTzeta. When considering HAA dosimetry, importance should be placed on both the components of the mixture and prior exposure history to di-HAAs.

We studied the pharmacokinetics of dichloroacetate (DCA) in naive rats and rats depleted of glutathione S-transferase-zeta (GST-zeta), at doses approaching human daily exposure levels. We also compared in vitro metabolism of DCA by rat and human liver cytosol. Jugular vein-cannulated male Fischer-344 rats received graded doses of DCA ranging from 0.05 to 20 mg/kg (intravenously or by gavage), and we collected time-course blood samples from the cannulas. GST-zeta activity was depleted by exposing rats to 0.2 g/L DCA in drinking water for 7 days before initiation of pharmacokinetic studies. Elimination of DCA by naive rats was so rapid that only 1-20 mg/kg intravenous and 5 and 20 mg/kg gavage doses provided plasma concentrations above the method detection limit of 6 ng/mL. GST-zeta depletion slowed DCA elimination from plasma, allowing kinetic analysis of doses as low as 0.05 mg/kg. DCA elimination was strongly dose dependent in the naive rats, with total body clearance declining with increasing dose. In the GST-zeta-depleted rats, the pharmacokinetics became linear at doses less than or equal to 1 mg/kg. Virtually all of the dose was eliminated through metabolic clearance; the rate of urinary elimination was < 1 mL/hr/kg. At higher oral doses (less than or equal to 5 mg/kg in GST-zeta-depleted and 20 mg/kg in naive rats), secondary peaks in the plasma concentration appeared long after the completion of the initial absorption phase. Oral bioavailability of DCA was 0-13% in naive and 14-75% in GST-zeta- depleted rats. Oral bioavailability of DCA in humans through consumption of drinking water was predicted to be very low and < 1%. The use of the GST-zeta-depleted rat as a model for assessing the kinetics of DCA in humans is supported by the similarity in pharmacokinetic parameter estimates and rate of in vitro metabolism of DCA by human and GST-zeta-depleted rat liver cytosol.

Excretion and tissue retention of a coplanar and a non-coplanar hexachlorobiphenyl (HxCB) were determined 48 h after a single intraperitoneal (ip) dose of 8 mg/kg radiolabeled [14C]-HxCBs to weanling male and female Long-Evans rats. The objective was to understand the involvement of initial target organs of chlorobiphenyl (CB) accumulation following acute exposure in immature animals. During the short interval, both HxCBs remained sequestered predominantly in mesenteric fat (compared to subcutaneous fat) and less than 1% of the doses were excreted. Excretion was 4- to 8-fold lower than adult rats. Coplanar CB 169 (3,3',4,4',5,5'-HxCB) did not accumulate appreciably in the brain, but was retained at 3-fold higher levels in the liver than was non-coplanar CB 153 (2,2',4,4',5,5'-HxCB). Accumulation of 14C-CB 153 in brains was 4- to 9-fold higher than that of 14C-CB 169 and was adequate to detect non-uniform distribution in serial cryostat sections by phosphor imaging autoradiography. The autoradiographs showed a higher CB 153-derived radioactivity associated with fiber tracts throughout the brain. Specifically, the corpus callosum, internal and external capsules, medial lemniscus, tegmentum of the mesencephalon and metencephalon, and cerebellar peduncles showed significantly higher 14C-CB 153 than the other structures. The 14C-CB 153 was not found in the ventricular system and vascular spaces. These results suggest for the first time that an ortho-substituted PCB congener accumulated preferentially in brain in a structure-specific manner when compared to a non-ortho-substituted PCB congener.

Insects selectively retain different polychlorinated biphenyls (PCBs) which are then contributed to the food chain. To quantitate specific differences, adult female houseflies (Musca domestica L.) were topically dosed with 0.5 microgram of two structurally distinct PCB congeners (14C-2,2',4,4'-tetraCB or 14C-3,3',4,4'-tetraCB). Total radioactivity in the acetone rinse of intact flies, several tissues, and excrement was determined at 11 time points over a 48-h period. Ninety-seven percent of the applied 2,2',4,4'-tetraCB disappeared from the surface following an initial rapid absorption of 79% within 3 h. The absorbed 2,2',4,4'-tetraCB was immediately found within the thoracic cuticle, then spreading laterally to the abdominal cuticle and head, through the wax layers, or penetrating to the alimentary canal and ovaries. Penetration of 3,3',4, 4'-tetraCB was markedly slower; even though 87% of the applied dose was absorbed within 48 h, only 19% of the dose penetrated into the fly body within the first 3 h. This PCB very slowly distributed into the tissues. Toxicities of the sublethal doses were determined by monitoring changes in activity of houseflies following dosing with 0. 5 microg of radioactive PCBs. Flies treated with 2,2',5-triCB and 3, 3',4,4'-tetraCB remained very active, whereas 2,2',4, 4'-tetraCB-treated flies were less active, consistent with the previously reported toxicity of this congener.

1,2,4,5-Tetrachlorobenzene (TeCB) and 1,4-dichlorobenzene (DCB) are important environmental contaminants that have been used extensively for a variety of industrial applications. Limited data are available in the literature regarding the carcinogenicity of TeCB. DCB has been shown to cause renal adenocarcinomas in rats and hepatic adenomas and carcinomas in mice at high doses in a 2-year study. In the studies presented here, we report that TeCB can promote the formation of preneoplastic foci and DCB cannot in a medium-term initiation/promotion assay. These results suggest that TeCB is a liver tumor promoter and that DCB is not at fairly low doses (0.1 and 0.4 mmol/kg per day).

2,4-Dichlorophenoxyacetic acid (2,4-D) was assessed for systemic toxicity, reproductive toxicity, developmental neurotoxicity (DNT), developmental immunotoxicity (DIT), and endocrine toxicity. CD rats (27/sex/dose) were exposed to 0, 100, 300, 600 (female), or 800 (male) ppm 2,4-D in diet. Nonlinear toxicokinetic behavior was shown at high doses; the renal clearance saturation threshold for 2,4-D was exceeded markedly in females and slightly exceeded in males. Exposure was 4 weeks premating, 7 weeks postmating for P1 males and through lactation for P1 females. F1 offspring were examined for survival and development, and at weaning, pups were divided in cohorts, by sex and dose, and by systemic toxicity (10), DNT (10), DIT (20), and reproductive toxicity (≥ 23). Remaining weanlings were evaluated for systemic toxicity and neuropathology (10-12). Body weight decreased during lactation in high-dose P1 females and in F1 pups. Kidney was the primary target organ, with slight degeneration of proximal convoluted tubules observed in high-dose P1 males and in high-dose F1 males and females. A slight intergenerational difference in kidney toxicity was attributed to increased intake of 2,4-D in F1 offspring. Decreased weanling testes weights and delayed preputial separation in F1 males were attributed to decreased body weights. Endocrine-related effects were limited to slight thyroid hormone changes and adaptive histopathology in high-dose GD 17 dams seen only at a nonlinear toxicokinetic dose. 2,4-D did not cause reproductive toxicity, DNT, or DIT. The "No Observed Adverse Effect Level" for systemic toxicity was 300 ppm in both males (16.6 mg/kg/day) and females (20.6 mg/kg/day), which is approximately 6700- to 93 000-fold higher than that reported for 2,4-D exposures in human biomonitoring studies.

Humans are exposed to chemicals either voluntarily or involuntarily through several routes. Therapeutic drugs are introduced into the human system via a number of routes including, but not limited to, oral, inhalation, intravenous (i.v.), topical, and subcutaneous. For occupational and environmental chemicals, the major routes of human exposure are inhalation, dermal, and oral. To determine the extent of exposure to chemicals, the concentration of the active molecules is measured in a biological medium. Determination of absolute and/or relative bioavailability of occupational and environmental chemical exposure through different routes is critical in understanding the risk to the general population of a low-level exposure to these chemicals. This unit describes typical protocol designs to generate data for the calculation of absorption, distribution, metabolism, and elimination (ADME) and absolute and relative bioavailability of chemicals when exposed through i.v., oral, and dermal routes.

Adult female houseflies (Musca domestica) were topically dosed with 10, 15, and 20 micrograms of 2,2',5-trichlorobiphenyl (PCB-18) in acetone at 1, 5, and 15 days following emergence. These doses caused a significant decrease in the mean survival time in 5-day-old flies. LT50's (time for 50% death) were dramatically reduced in 5-day-old flies, whereas no significant difference was found at any treatment level in 15-day-old flies. Abdomenal microsomal enzyme levels were determined by the rate of O-dealkylation of (p-nitrophenyl)ethyl ether for 1-, 5-, 11-, and 15-day-old female houseflies. The highest levels were found in 5-day-old flies and the lowest in 15-day-old flies. The greatest metabolism of PCB-18 by housefly microsomes also occurred in 5-day-old flies. The enzyme levels, metabolism, and toxicity suggest that PCB-18 is bioactivated to a product(s) which reduces the mean survival time of houseflies.