Species-specific developmental toxicity in rats and rabbits: Generation of a reference compound list for development of alternative testing approaches.

For regulatory information requirements, developmental toxicity testing is often conducted in two mammalian species. In order to provide a set of reference compounds that could be used to explore alternative approaches to supersede testing in a second species, a retrospective data analysis was conducted. The aim was to identify compounds for which species sensitivity differences between rats and rabbits are not caused by maternal toxicity or toxicokinetic differences. A total of 330 compounds were analysed and classified according to their species-specific differences. A lack of concordance between rat and rabbit was observed in 24% of the compounds, of which 10% were found to be selective developmental toxicants in one of the species. In contrast to previously published analyses the presented comparison is based entirely on publically data allowing validating and comparing alternative approaches for developmental toxicity testing. Furthermore, this list could be useful to identify mechanisms leading to species differences.

Toxicokinetics of ethers used as fuel oxygenates.

The toxicokinetics and biotransformation of methyl-tert.butyl ether (MTBE), ethyl-tert.butyl ether (ETBE) and tert.amyl-methyl ether (TAME) in rats and humans are summarized. These ethers are used as gasoline additives in large amounts, and thus, a considerable potential for human exposure exists. After inhalation exposure MTBE, ETBE and TAME are rapidly taken up by both rats and humans; after termination of exposure, clearance by exhalation and biotransformation to urinary metabolites is rapid in rats. In humans, clearance by exhalation is slower in comparison to rats. Biotransformation of MTBE and ETBE is both qualitatively and quantitatively similar in humans and rats after inhalation exposure under identical conditions. The extent of biotransformation of TAME is also quantitatively similar in rats and humans; the metabolic pathways, however, are different. The results suggest that reactive and potentially toxic metabolites are not formed during biotransformation of these ethers and that toxic effects of these compounds initiated by covalent binding to cellular macromolecules are unlikely.

Biotransformation of MTBE, ETBE, and TAME after inhalation or ingestion in rats and humans.

The biotransformation of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) was studied in humans and in rats after inhalation of 4 and 40 ppm of MTBE, ETBE, and TAME, respectively, for 4 hours, and the biotransformation of MTBE and TAME was studied after ingestion exposure in humans to 5 and 15 mg in water. tert-Butyl alcohol (TBA), a TBA conjugate, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate were found to be metabolites of MTBE and ETBE. tert-Amyl alcohol (TAA), free and glucuronidated 2-methyl-2,3-butanediol (a glucuronide of TAA), 2-hydroxy-2-methyl butyrate, and 3-hydroxy-3-methyl butyrate were found to be metabolites of TAME. After inhalation, MTBE, ETBE, and TAME were rapidly taken up by both rats and humans; after termination of exposure, clearance from blood of the ethers by exhalation and biotransformation to urinary metabolites occurred with half-times of less than 7 hours in rats and humans. Biotransformation of MTBE and ETBE was similar in humans and rats after inhalation exposure. 2-Hydroxyisobutyrate was recovered as a major product in urine. All metabolites of MTBE and ETBE excreted with urine were eliminated with half-times of less than 20 hours. Biotransformation of TAME was qualitatively similar in rats and humans, but the metabolic pathways were different. In humans, 2-methyl-2,3-butanediol, 2-hydroxy-2-methyl butyrate, and 3-hydroxy-3methyl butyrate were recovered as major urinary products. In rats, however, 2-methyl-2,3-butanediol and its glucuronide were major TAME metabolites recovered in urine. After ingestion of MTBE and TAME, both compounds were rapidly absorbed from the gastrointestinal tract. Hepatic first-pass metabolism of these ethers was not observed, and a significant part of the administered dose was transferred into blood and cleared by exhalation. Metabolic pathways for MTBE and TAME and kinetics of excretion were identical after ingestion and inhalation exposures. Results of studies presented here suggest (1) that excretion of MTBE, ETBE, and TAME in rats and humans is rapid, (2) that biotransformation and excretion of MTBE and ETBE are identical in rats, and (3) that biotransformation and excretion of TAME is quantitatively different in rats and humans.

Toxicokinetics of methyl tert-butyl ether and its metabolites in humans after oral exposure.

Methyl tert-butyl ether (MTBE) is widely used as an additive to gasoline, to increase oxygen content and reduce tailpipe emission of pollutants. Widespread human exposure to MTBE may occur due to leakage of gasoline storage tanks and a high stability and mobility of MTBE in ground water. To compare disposition of MTBE after different routes of exposure, its biotransformation was studied in humans after oral administration in water. Human volunteers (3 males and 3 females, identical individuals, exposures were performed 4 weeks apart) were exposed to 5 and 15 mg 13C-MTBE dissolved in 100 ml of water. Urine samples from the volunteers were collected for 96 h after administration in 6-h intervals and blood samples were taken in intervals for 24 h. In urine, MTBE and the MTBE-metabolites tert-butanol (t-butanol), 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were quantified, MTBE and t-butanol were determined in blood samples and in exhaled air in a limited study of 3 male volunteers given 15 mg MTBE in 100 ml of water. MTBE blood concentrations were 0.69 +/- 0.25 microM after 15 mg MTBE and 0.10 +/- 0.03 microM after 5 mg MTBE. MTBE was rapidly cleared from blood with terminal half-lives of 3.7 +/- 0.9 h (15 mg MTBE) and 8.1 +/- 3.0 h (5 mg MTBE). The blood concentrations of t-butanol were 1.82 +/- 0.63 microM after 15 mg MTBE and 0.45 +/- 0.13 microM after 5 mg MTBE. Approximately 30% of the MTBE dose was cleared by exhalation as unchanged MTBE and as t-butanol. MTBE exhalation was rapid and maximal MTBE concentrations (100 nmol/l) in exhaled air were achieved within 10-20 min. Clearance of MTBE by exhalation paralleled clearance of MTBE from blood. T-butanol was cleared from blood with half-lives of 8.5 +/- 2.4 h (15 mg MTBE) and 8.1 +/- 1.6 h (5 mg MTBE). In urine samples, 2-hydroxyisobutyrate was recovered as major excretory product, t-butanol and 2-methyl-1,2-propane diol were minor metabolites. Elimination half-lives for the different urinary metabolites of MTBE were between 7.7 and 17.8 h. Approximately 50% of the administered MTBE was recovered in urine of the volunteers after both exposures, another 30% was recovered in exhaled air as unchanged MTBE and t-butanol. The obtained data indicate that MTBE-biotransformation and excretion after oral exposure is similar to inhalation exposure and suggest the absence of a significant first-pass metabolism of MTBE in the liver after oral administration.

Biotransformation and kinetics of excretion of tert-amyl-methyl ether in humans and rats after inhalation exposure.

tert-Amyl methyl ether (TAME) may be widely used as an additive to gasoline in the future. The presence of this ether in gasoline reduces the tail pipe emission of pollutants. Therefore, widespread human exposure to TAME may occur. To contribute to the characterization of potential adverse effects of TAME, its biotransformation was compared in humans and rats after inhalation exposure. Human volunteers (three males and three females) and rats (five males and five females) were exposed to 4 (3.8 +/- 0.2) and 40 (38.4 +/- 1.7) ppm TAME for 4 h in a dynamic exposure system. Urine samples were collected for 72 h in 6-h intervals and blood samples were taken at regular intervals for 48 h in humans. In urine, the TAME metabolites tert-amyl alcohol (t-amyl alcohol), 2-methyl-2, 3-butane diol, 2-hydroxy-2-methylbutyric acid, and 3-hydroxy-3-methylbutyric acid were quantified. TAME and t-amyl alcohol were determined in blood samples. After the end of the exposure period, blood concentrations of TAME were 4.4 +/- 1.7 microM in humans and 9.6 +/- 1.4 microM in rats after 40 ppm TAME, and 0.6 +/- 0.1 microM in humans and 1.4 +/- 0.8 microM in rats after 4 ppm. TAME was rapidly cleared from blood in both rats and humans. The blood concentrations of t-amyl alcohol were 9.2 +/- 1.8 microM in humans and 8.1 +/- 1.5 microM in rats after 40 ppm TAME, and 1.0 +/- 0.3 microM in humans and 1.8 +/- 0.2 microM in rats after 4 ppm TAME. t-Amyl alcohol was also rapidly cleared from blood. In urine of humans, 2-methyl-2,3-butane diol, 2-hydroxy-2-methylbutyric acid, and 3-hydroxy-3-methylbutyric acid were recovered as major excretory products in urine. In rats, 2-methyl-2,3-butane diol and its glucuronide were major TAME metabolites. t-Amyl alcohol and its glucuronide were minor TAME metabolites in both species. All metabolites of TAME excreted with urine in rats were rapidly eliminated, with elimination half-lives of less than 6 h. Metabolite excretion in humans was slower and elimination half-lives of the different metabolites were between 6 and 40 h in humans. The obtained data indicate differences in TAME biotransformation and excretion between rats and humans. In rats, TAME metabolites are rapidly excreted. In humans, metabolic pathways are different and metabolite excretion is slower. Recovery of TAME metabolites in urine was higher in humans as compared to rats, suggesting more intensive biotransformation of TAME in humans.

Biotransformation and kinetics of excretion of ethyl tert-butyl ether in rats and humans.

Ethyl tert-butyl ether (ETBE) may be used in the future as an additive to gasoline to increase oxygen content and reduce tailpipe emissions of pollutants. Therefore, widespread human exposure may occur. To contribute to the characterization of potential adverse effects of ETBE, its biotransformation was compared in humans and rats after inhalation exposure. Human volunteers (3 males and 3 females) and rats (5 males and 5 females) were exposed to 4 (4.5+/-0.6) and 40 (40.6+/-3.0) ppm ETBE for 4 h in a dynamic exposure system. Urine samples from rats and humans were collected for 72 h at 6-h intervals, and blood samples were taken in regular intervals for 48 h. In urine, ETBE and the ETBE-metabolites tert-butanol (t-butanol), 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were quantified; ETBE and t-butanol were determined in blood samples. After the end of the exposure period to inhalation of 40-ppm ETBE, blood concentrations of ETBE were found at 5.3+/-1.2 microM in rats and 12.1+/-4.0 microM in humans. The ETBE blood concentrations, after inhalation of 4-ppm ETBE, were 1.0+/-0.7 microM in rats and 1.3+/-0.7 microM in humans. ETBE was rapidly cleared from blood. After the end of the 40-ppm ETBE exposure period, the blood concentrations of t-butanol were 13.9+/-2.2 microM in humans and 21.7+/-4.9 microM in rats. After 4-ppm ETBE exposure, blood concentrations of t-butanol were 1.8+/-0.2 microM in humans and 5.7+/-0.8 microM in rats. t-Butanol was cleared from human blood with a half-life of 9.8+/-1.4 h in humans after 40-ppm ETBE exposure. In urine samples from controls and in samples collected from the volunteers and rats before the exposure, low concentrations of t-butanol, 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were present. In the urine of both humans and rats exposed to ETBE, the concentrations of these compounds were significantly increased. 2-Hydroxy-isobutyrate was recovered in urine as the major excretory product formed from ETBE; t-butanol and 2-methyl-1,2-propane diol were minor metabolites. All metabolites of ETBE excreted with urine were rapidly eliminated in both species after the end of the ETBE exposure. Excretion half-lives for the different urinary metabolites of ETBE were between 10.2 and 28.3 h in humans and 2.6 and 4.7 h in rats. The obtained data indicate that ETBE biotransformation and excretion are similar for rats and humans, and that ETBE and its metabolites are rapidly excreted by both species. Between 41 and 53% of the ETBE retained after the end of the exposure was recovered as metabolites in the urine of both humans and rats.

Biotransformation of [(12)C]- and [(13)C]-tert-amyl methyl ether and tert-amyl alcohol.

tert-Amyl methyl ether (TAME) is intended for use as a gasoline additive to increase oxygen content. Increased oxygen content in gasoline reduces tailpipe emissions of hydrocarbons and carbon monoxide from cars. Due to possible widespread use of TAME, the toxicity of TAME is under investigation. We studied the biotransformation of TAME in rats and one human volunteer after inhalation of (12)C- or (13)C-labeled TAME. In addition, the biotransformation of [(13)C]-tert-amyl alcohol was studied in rats after gavage. Urinary metabolites were identified by GC/MS and (13)C NMR. Rats (two males and two females) were individually exposed to 2000 ppm [(12)C]- or [(13)C]TAME for 6 h, and urine was collected for 48 h. Free and glucuronidated 2-methyl-2,3-butanediol and a glucuronide of tert-amyl alcohol were identified by (13)C NMR, GC/MS, and LC/MS/MS as major urinary metabolites on the basis of the relative intensities of the (13)C NMR signals. The presence of several minor metabolites was also indicated by (13)C NMR; they were identified as tert-amyl alcohol, 2-hydroxy-2-methylbutyric acid, and 3-hydroxy-3-methylbutyric acid. One human volunteer was exposed to an initial concentration of 27 000 ppm [(13)C]TAME by inhalation for 4 min from a 2 L gas sampling bag, and metabolites of TAME excreted in urine were analyzed by (13)C NMR. All TAME metabolites identified in rats were also present in the human urine samples. To study tert-amyl alcohol biotransformation, male rats (n = 3) were treated with 250 mg/kg [(13)C]-tert-amyl alcohol dissolved in corn oil by gavage, and urine was collected for 48 h. (13)C NMR of the urine samples showed the presence of metabolites identical to those in the urine of [(13)C]TAME-treated rats. Our results suggest that TAME is extensively metabolized by rats and humans to tert-amyl alcohol which may be further oxidized to diols and carboxylic acids. These reactions are likely mediated by cytochrome P450-dependent oxidations.

Biotransformation and kinetics of excretion of methyl-tert-butyl ether in rats and humans.

Methyl-tert-butyl ether (MTBE) is widely used as an additive to gasoline to increase oxygen content and reduce tail pipe emission of pollutants. Therefore, widespread human exposure may occur. To contribute to the characterization of potential adverse effects of MTBE, its biotransformation was compared in humans and rats after inhalation exposure. Human volunteers (3 males and 3 females) and rats (5 each, males and females) were exposed to 4 (4.5 +/- 0.4) and 40 (38.7 +/- 3.2) ppm MTBE for 4 h in a dynamic exposure system. Urine samples from rats and humans were collected for 72 h in 6-h intervals, and blood samples were taken in regular intervals for 48 h. In urine, MTBE and the MTBE metabolites tertiary-butanol (t-butanol), 2-methyl-1,2-propane diol, and 2-hydroxyisobutyrate were quantified; MTBE and t-butanol were determined in blood samples. After the end of the exposure period, inhalation of 40 ppm MTBE resulted in blood concentrations of MTBE 5.9 +/- 1.8 microM in rats and 6.7 +/- 1.6 microM in humans. The MTBE blood concentrations after inhalation of 4 ppm MTBE were 2.3 +/- 1.0 in rats and 1.9 +/- 0.4 microM in humans. MTBE was rapidly cleared from blood with a half-life of 2.6 +/- 0.9 h in humans and 0.5 +/- 0.2 h in rats. The blood concentrations of t-butanol were 21.8 +/- 3.7 microM in humans and 36.7 +/- 10.8 microM in rats after 40 ppm MTBE, and 2.6 +/- 0.3 in humans and 2.9 +/- 0.5 in rats after 4 ppm MTBE. In humans, t-butanol was cleared from blood with a half-life of 5.3 +/- 2.1 h. In urine samples from controls and in samples collected from the volunteers and rats before the exposure, low concentrations of t-butanol, 2-methyl-1,2-propane diol and 2-hydroxyisobutyrate were present. In urine of both humans and rats exposed to MTBE, the concentrations of these compounds were significantly increased. 2-Hydroxyisobutyrate was recovered as a major excretory product in urine; t-butanol and 2-methyl-1,2-propane diol were minor metabolites. All metabolites of MTBE excreted with urine were rapidly eliminated in both species after the end of the MTBE exposure. Elimination half-lives for the different urinary metabolites of MTBE were between 7.8 and 17.0 h in humans and 2.9 to 5.0 h in rats. The obtained data indicate that MTBE biotransformation and excretion are similar in rats and humans, and MTBE and its metabolites are rapidly excreted in both species. Between 35 and 69% of the MTBE retained after the end of the exposure was recovered as metabolites in urine of both humans and rats.

Biotransformation of 12C- and 2-13C-labeled methyl tert-butyl ether, ethyl tert-butyl ether, and tert-butyl alcohol in rats: identification of metabolites in urine by 13C nuclear magnetic resonance and gas chromatography/mass spectrometry.

The biotransformation of the fuel oxygenates methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) was studied in rats after inhalation exposure; the biotransformation of the initial metabolite of these ethers, tert-butyl alcohol, was studied after oral gavage. To study ether metabolism, rats were exposed for 6 h to initial concentrations of 2000 ppm of MTBE or ETBE, respectively [2-13C]MTBE and [2-13C]ETBE. Urine was collected for 48 h after the end of the exposure, and urinary metabolites were identified by 13C NMR (13C-labeled ethers) and gas chromatography/mass spectrometry (GC/MS) (12C- and 13C-labeled ethers). To study tert-butyl alcohol metabolism, rats were dosed either with tert-butyl alcohol at natural carbon isotope ratio or with 13C-enriched tert-butyl alcohol (250 mg/kg of body weight), urine was collected, and metabolites were identified by NMR and GC/MS. tert-Butyl alcohol was identified as a minor product of the biotransformation of MTBE and ETBE. In addition, small amounts of a tert-butyl alcohol conjugate, likely a glucuronide, were present in the urine of the treated animals. Moreover, the mass spectra obtained indicate the presence of small amounts of [13C]acetone in the urine of [13C]MTBE and [13C]ETBE-treated rats. 2-Methyl-1,2-propanediol, 2-hydroxyisobutyrate, and another unidentified conjugate of tert-butyl alcohol, most probably a sulfate, were major urinary metabolites of MTBE and ETBE as judged by the intensities of the NMR signals. In [13C]-tert-butyl alcohol-dosed rats, [13C]acetone, tert-butyl alcohol, and its glucuronide represented minor metabolites; as with the ethers, 2-methyl-1,2-propanediol, 2-hydroxyisobutyrate, and the presumed tert-butyl alcohol sulfate were the major metabolites present. In one human individual given 5 mg/kg [13C]-tert-butyl alcohol orally, 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate were major metabolites in urine detected by 13C NMR analysis. Unconjugated tert-butyl alcohol and tert-butyl alcohol glucuronide were present as minor metabolites, and traces of the presumed tert-butyl alcohol sulfate were also present. Our results suggest that tert-butyl alcohol formed from MTBE and ETBE is intensively metabolized by further oxidation reactions. Studies to elucidate mechanisms of toxicity for these ethers to the kidney need to consider potential toxicities induced by these metabolites.

Pseudoexfoliation syndrome and secondary cataract.

AIM/BACKGROUND: The pseudoexfoliation (PEX) syndrome is frequently associated with impairment of the blood-aqueous barrier. This study analysed if this might stimulate secondary cataract following cataract extraction. METHODS: This historical cohort study included 197 eyes of 197 patients (99 with and 98 without PEX) that underwent extracapsular cataract extraction with posterior chamber lens implantation (PMMA optic) between 1985 and 1991. Secondary cataract was defined as opacification of the axial posterior capsule and decrease of visual acuity by two or more lines. Mean follow up was 23.8 months. For statistical analysis, the Kaplan-Meier method and multivariate Cox regression analysis were used. RESULTS: Secondary cataract was observed within 24 months in 35% (SD 7%) of all eyes, and was significantly more frequent in eyes with PEX (45 (11)%) than in eyes without PEX (24 (9)%, p < 0.03). Eyes with diabetes mellitus (n = 32) showed a significantly lower frequency of secondary cataract (11 (11)%) than eyes without diabetes mellitus (39 (8)%, p < 0.01). The influences of sex, open angle glaucoma, type of cataract, surgeon, positioning of IOL, and phacoemulsification versus nuclear expression on secondary cataract did not reach statistical significance. CONCLUSION: The higher frequency of secondary cataract could be considered as another potential complication of cataract surgery in eyes with PEX.