A perspective on In vitro developmental neurotoxicity test assay results: An expert panel review.

For decades, there has been increasing concern about the potential developmental neurotoxicity (DNT) associated with chemicals. Regulatory agencies have historically utilized standardized in vivo testing to evaluate DNT. Owing to considerations including higher-throughput screening for DNT, reduction in animal use, and potential cost efficiencies, the development of alternative new approach methods (NAMs) occurred; specifically, the advent of the DNT in vitro test battery (DNT IVB). SciPinion convened an expert panel to address specific questions related to the interpretation of in vitro DNT test data. The consensus of the expert panel was that the DNT IVB might be used during initial screening, but it is not presently a complete or surrogate approach to determine whether a chemical is a DNT in humans. By itself, the DNT IVB does not have the ability to capture nuances and complexity of the developing nervous system and associated outcomes including behavioral ontogeny, motor activity, sensory function, and learning/memory. Presently, such developmental landmarks cannot be adequately assessed in the DNT IVB or by other NAMs. The expert panel (all who serve as co-authors of this review) recommended that additional data generation and validation is required before the DNT IVB can be considered for application within global regulatory frameworks for decision-making.

No change in calcium absorption in adult Pakistani population before and after vitamin D administration using strontium as surrogate.

UNLABELLED: 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. INTRODUCTION: This paper discusses the determination of calcium (Ca) absorption, using strontium (Sr) as a surrogate, before and after a single IM injection of vitamin D(3) (600,000 IU). METHODS: 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) SrCl(2) and collecting blood at 0.5, 1 and 4 h to determine the absorption (AUC(0 → t )) of Sr. Following the initial absorption test, volunteers received a single IM injection of 600,000 IU vitamin D(3). Two months later, the fasting serum and the Sr absorption test were repeated, as described above. RESULTS: The IM injection of vitamin D(3) caused a significant increase in fasting 25OHD (from 43.5 ± 19 to 66.1 ± 19.1 nmol/L (p < 0.001)) and a trend toward lower serum iPTH (from 59.8 ± 27.8 to 53 ± 31 ng/L). Fasting serum Ca and P remained unchanged. A higher 25OHD level failed (p = 0.32) to translate into a higher rate of Sr absorption. AUC(0 → 4 h) were almost identical before and after the IM injection of vitamin D(3). CONCLUSION: A single vitamin D(3) injection of 600,000 IU significantly increase mean 25OHD concentration and tended to lower iPTH concentrations in volunteers with initially low 25OHD status, suggesting to utilize this simple form of treatment to improve vitamin D status and to have a possible biological effect on Ca homeostasis. However, we found no obvious effect on Sr absorption, suggesting the possibility that maximal vitamin D-dependent Ca absorption had already been achieved in these volunteers at a lower vitamin D status.

Extension of a PBPK model for ethylene glycol and glycolic acid to include the competitive formation and clearance of metabolites associated with kidney toxicity in rats and humans.

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 male 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 200mg/m(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.

Pharmacokinetics and bioavailability of diisopropanolamine (DIPA) in rats following intravenous or dermal application.

This study was conducted to determine the relative dermal bioavailability (absorption), distribution, metabolism, and excretion (ADME) of diisopropanolamine (DIPA), an alcohol amine used in a number of industrial and personal care products. Groups of 4 female Fischer 344 rats received either a single bolus i.v. dose of 19.0mg/kg (14)C-DIPA in water or a dermal application of 19.5mg/kg (14)C-DIPA in acetone to an area of 1cm(2) on the back and covered with a bandage. Time-course blood and excreta were collected and radioactivity determined. Urine was analyzed for DIPA and monoisopropanolamine (MIPA). Following i.v. administration, DIPA was rapidly cleared from the plasma and excreted into urine in a biexponential manner (t(1/2alpha), 0.4h; t(1/2beta), 2.9h). The levels of radioactivity in plasma dropped below the limit of detection 12h post-dosing. A total of 97+/-4% of the dose was actively excreted in urine by kidney, most ( approximately 71%) within 6h of dosing, virtually all as parent compound; renal clearance exceeded the glomerular filtration rate. Following dermal application, approximately 20% of the dose was absorbed in 48 h with the steady-state penetration rate of approximately 0.2%/h. Most (14.4%) of the applied radioactivity was excreted in urine at a relatively constant rate due to the presence of large amount of the (14)C-DIPA at the application site. Fecal elimination was <0.2% of the dose. The absorbed DIPA did not accumulate in tissues; only approximately 0.1% of the administered dose was found in liver and kidney. The absolute systemic dermal bioavailability (dose corrected AUC(dermal)/AUC(i.v.)) of (14)C-DIPA was 12%. The ADME of DIPA contrasts that of its diethanol analogue, diethanolamine, which displays a broad spectrum of toxicity in rats and mice. Toxicologically significant concentrations of DIPA are unlikely to be achieved in the systemic circulation and/or tissues as a result of repeated dermal application of products containing DIPA due to slow absorption from the skin, rapid unchanged elimination in urine, and majority of the products contain

Oral absorption, metabolism and excretion of 1-phenoxy-2-propanol in rats.

1. This study was designed to determine the absorption, metabolism and excretion of 1-phenoxy-2-propanol in Fischer 344 rats following oral administration in an effort to bridge data with other propylene glycol ethers. 2. Rats were administered a single oral dose of 10 or 100 mg kg(-1) 14C-1-phenoxy-2-propanol as a suspension in 0.5% methyl cellulose ether in water (w/w). Urine was collected at 0-12, 12-24 and 24-48 h and faeces at 0-24 and 24-48 h post-dosing and the radioactivity was determined. Urine samples were pooled by time point and dose level and analysed for metabolites using LC/ESI/MS and LC/ESI/MS/MS. 3. The administered doses were rapidly absorbed from the gastrointestinal tract and excreted. The major route of excretion was via the urine, accounting for 93 +/- 5% of the low and 96 +/- 3% of the high dose. Most of the urinary excretion of radioactivity occurred within 12 h after dosing; 85 +/- 2% of the low and 90 +/- 1% of the high dose. Total faecal excretion remained < 10%. Rats eliminated the entire administered dose within 48 h after dosing; recovery of the administered dose ranged from 100 to 106%. Metabolites tentatively identified in urine were conjugates of phenol (sulphate, glutathione) with very low levels (< 2%) of hydroquinone (glucuronide), conjugates of parent compound (glucuronide, sulphate) and a ring-hydroxylated metabolite of parent. There was no free parent compound or phenol in non-acid-hydrolysed urine. In acid-hydrolysed urine, 61% of the dose was identified as phenol and 13% as 1-phenoxy-2-propanol. Although the parent compound was stable to acid hydrolysis, some of the phenol in acid hydrolysed urine may have arisen from degradation of acid-labile metabolite(s) as well as hydrolysis of phenol conjugates. 4. Rapid oral absorption, metabolism and urinary excretion of 1-phenoxy-2-propanol in rats were similar to other propylene glycol ethers.

Kinetics of monochloroacetic acid in adult male rats after intravenous injection of a subtoxic and a toxic dose.

Distribution, metabolism, and excretion of monochloroacetic acid (MCA) were examined in adult male rats at a subtoxic (10 mg/kg) and a toxic (75 mg/kg) dose. Rats were injected i.v. with [14C]MCA and housed individually. Urine and feces were collected. Animals were euthanized at different time intervals after dosing and tissues procured. Radioactivity in aliquots showed very rapid distribution of MCA to tissues. Concentrations of MCA in plasma, liver, heart, lungs, and brown fat paralleled each other, whereas those in brain and thymus did not. There was no dose proportionality in tissue concentrations. Elimination of MCA from plasma required modeling by two compartments. Most of the radioactivity found in plasma was parent MCA. Elimination rate constant (K(10)) and distribution rate constant (K(12)) were greatly reduced at the toxic dose. Elimination of the toxic dose was further retarded due to increased retention of MCA in the peripheral compartment as indicated by increased mean residence times in most tissues. A very large fraction of dose was found in the gastrointestinal tract, almost all of which was reabsorbed. Attempts to reduce toxicity by blocking the enterohepatic circulation with activated charcoal or cholestyramine failed. Radioactivity found in bile was associated with one metabolite more polar than the parent compound. A very large fraction of dose (73 and 59%) was found in urine, 55 to 68% of which was parent MCA. The rate-determining step in the toxicity of MCA was identified as its detoxification by the liver. A therapeutic approach in MCA intoxications is suggested.

Differential and non-uniform tissue and brain distribution of two distinct 14C-hexachlorobiphenyls in weanling rats.

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.

Toxicity and tissue distribution of 2,2',4,4'- and 3,3',4, 4'-tetrachlorobiphenyls in houseflies.

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.

Short-term distribution, metabolism, and excretion of 2,2',5-tri-, 2, 2',4,4'-tetra-, and 3,3',4,4'-tetrachlorobiphenyls in prepubertal rats.

The excretion and tissue retention of three 14C-labeled lower chlorinated biphenyls were examined in prepubertal male and female Sprague-Dawley rats following IV administration. Urine and feces were collected individually at different time intervals up to 72 h for pharmacokinetic analyses. After 72 h, different organs were removed and extracted in acetone:hexane (1:1, v/v) to determine radioactivity. Within the first 10 h after dosing, 2,2', 5-trichlorobiphenyl (PCB 18) was rapidly excreted in urine (8-18% of the administered dose), whereas only 0.6-0.8% of 2,2',4, 4'-tetrachlorobiphenyl (PCB 47) and 0.3-0.8% 3,3',4, 4'-tetrachlorobiphenyl (PCB 77) were found in urine during this time period. The half-life of elimination was shortest for PCB 18 (37.5 to 49.2 h). The half-lives for PCB 47 and PCB 77 were 351 to 672 h and 152 to 186 h, respectively. The cumulative total excretion (urinary + fecal) of PCB 18 within 72 h was 51-62%, of PCB 77 was 22-25%, and of PCB 47 was 7-10%. No parent PCBs were detected in urine. PCB 47 accumulated preferentially in adipose tissues (subcutaneous fat > mesenteric fat); relatively high levels of PCB 47 were also found in adrenals, ovaries, lungs, liver, and skin. The highest concentration of PCB 77 was found in serum, followed by adipose tissues. Very low concentrations of PCB 18 were found in most tissues; the highest being found in serum, followed by ovaries and adrenal glands. This study suggests that prepubertal rats retain higher short-term serum levels and have lower excretion rates than adult rats.

Use of a medium-term liver focus bioassay to assess the hepatocarcinogenicity of 1,2,4,5-tetrachlorobenzene and 1,4-dichlorobenzene.

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).

Toxicities of 2,2',4,4'- and 3,3',4,4-tetrachlorobiphenyls to house flies at different ages and enzyme levels.

Two tetrachlorobiphenyls (2,2',4,4'-tetraCB and 3,3',4,4'-tetraCB) had different effects on the longevity of adult female house flies (Musca domestica), depending on enzyme levels at dosing. Twenty-four-hour acute toxicities of the two tetrachlorobiphenyls were also compared with one trichlorobiphenyl (2,2',5-triCB) at different enzyme levels. 2,2',4,4'-tetraCB killed more than 90% of the flies within 12 h at the highest dose (1,667 ppm, micrograms/g); however, the toxicity of the moderate dose (1,250 ppm) was age-dependent and greater in 1- and 15-day-old flies and lower in 5-day-old flies. Daily mortality patterns of the lower doses (390 and 833 ppm) were similar to the control. LT50s (time for 50% death) were also different in different age groups at the moderate dose. The toxicity of 3,3',4,4'-tetraCB was similar to the control in all age groups, with a slight increase in the early mortalities and a decrease in the LT50s at the highest (1,200 ppm) dose. The twenty-four-hour lethality of 2,2',4,4'-tetraCB was very high, even at the lower doses in 1- and 15-day-old flies. Lower doses were least toxic at day 5, when the levels of cytochrome P450 enzymes were at the highest. On the other hand, the acute toxicity of 2,2',5-triCB increased from 5 and 15% in 1- and 15-day-old flies, respectively, to about 50% in 5-day-old flies, suggesting bioactivation of 2,2',5-triCB. The acute toxicity of 3,3',4,4'-tetraCB was negligible in all ages of house flies.

Influence of enzyme levels on the toxicity and in vitro metabolism of 2,2',5-trichlorobiphenyl in houseflies.

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.