Abstract  When methyl tertiary-butyl ether (MTBE) in gasoline was first introduced to reduce vehicle exhaust emissions and comply with the Clean Air Act, in the United States, a pattern of complaints emerged characterised by seven "key symptoms." Later, carefully controlled volunteer studies did not confirm the existence of the specific key symptoms, although one study of self-reported sensitive (SRS) people did suggest that a threshold at about 11-15% MTBE in gasoline may exist for SRSs in total symptom scores. Neurobehavioral and psychophysiological studies on volunteers, including SRSs, found no adverse responses associated with MTBE at likely exposure levels. MTBE is well and rapidly absorbed following oral and inhalation exposures. Cmax values for MTBE are achieved almost immediately after oral dosing and within 2 h of continuous inhalation. It is rapidly eliminated, either by exhalation as unchanged MTBE or by urinary excretion of its less volatile metabolites. Metabolism is more rapid humans than in rats, for both MTBE and tert-butyl alcohol (TBA), its more persistent primary metabolite. The other primary metabolite, formaldehyde, is detoxified at a rate very much greater than its formation from MTBE. MTBE has no specific effects on reproduction or development, or on genetic material. Neurological effects were observed only at very high concentrations. In carcinogenicity studies of MTBE, TBA, and methanol (included as an endogenous precursor of formaldehyde, without the presence of TBA), some increases in tumor incidence have been observed, but consistency of outcome was lacking and even some degree of replication was observed in only three cases, none of which had human relevance: alpha(2u)-globulin nephropathy-related renal tubule cell adenoma in male rats; Leydig-cell adenoma in male rats, but not in mice, which provide the better model of the human disease; and B-cell-derived lymphoma/leukemia of doubtful pathogenesis that arose mainly in lungs of orally dosed female rats. In addition, hepatocellular adenomas were significantly higher in female CD-1 mice and thyroid follicular-cell adenomas were increased in female B6C3F1 mice treated with TBA, but these results lack any independent confirmation, which would have been possible from a number of other studies.

Abstract  #311 chemicals were tested under code, for mutagenicity, in Salmonella typhimurium; 35 of the chemicals were tested more than once in the same or different laboratories. The tests were conducted using a preincubation protocol in the absence of exogenous metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters. Some of the volatile chemicals were also tested in desiccators. A total of 120 chemicals were mutagenic or weakly mutagenic, 3 were judged questionable, and 172 were non-mutagenic. The remaining 16 chemicals produced different responses in the two or three laboratories in which they were tested. The results and data from these tests are presented.

Abstract  Methyl-tert-butylether (MTBE) is an oxygenate widely used in the United States as a motor vehicle fuel additive to reduce emissions and as an octane booster [National Research Council, Toxicological and Performance Aspects of Oxygenated Motor Vehicle Fules, National Academy Press, Washington, DC, 1996]. But it is the potential for MTBE to enter drinking water supplies that has become an area of public concern. MTBE has been shown to induce liver and kidney tumors in rodents but the biochemical process leading to carcinogenesis is unknown. MTBE was previously shown to be non-mutagenic in the standard Ames plate incorporation test with tester strains that detect frame shift (TA98) and point mutations (TA100) and in a suspension assay using TA104, a strain that detects oxidative damage, suggesting a non-genotoxic mechanism accounts for its carcinogenic potential. These strains are deficient in excision repair due to deletion of the uvrB gene. We hypothesized that the carcinogenic activity of MTBE may be dependent upon a functional excision repair system that attempts to remove alkyl adducts and/or oxidative base damage caused by direct interaction of MTBE with DNA or by its metabolites, formaldehyde and tert-butyl alcohol (TBA), established carcinogens that are mutagenic in some Ames strains. To test our hypothesis, the genotoxicity of MTBE-induced DNA alterations was assayed using the standard Ames test with TA102, a strain similar to TA104 in the damage it detects but uvrB+ and, therefore, excision repair proficient. The assay was performed (1) with and without Aroclor-induced rat S-9, (2) with and without the addition of formaldehyde dehydrogenase (FDH), and (3) with human S-9 homogenate. MTBE was weakly mutagenic when tested directly and moderately mutagenic with S-9 activation producing between 80 and 200 TA102 revertants/mg of compound. Mutagenicity was inhibited 25%-30% by FDH. TA102 revertants were also induced by TBA and by MTBE when human S-9 was substituted for rat S-9. We conclude that MTBE and its metabolites induce a mutagenic pathway involving oxidation of DNA bases and an intact repair system. These data are significant in view of the controversy surrounding public safety and the environmental release of MTBE and similar fuel additives.

Abstract  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.

Abstract  T-Butyl alcohol (TBA) was administered in drinking water to F344/N rats and B6C3F1 mice for two years using 60 animals/dose/sex/species. Male rats received doses of 0. 1.25. 2.5. or 5 mg/ml and females received 0, 2.5, 5, or 10 mg/ml. resulting in average daily doses of approximately 85, 195, or 420 mg TBA/kg body weight for males and 175, 330, or 650 mg/kg for females. Ten rats per group were evaluated after 15 months. Male and female mice received doses of 0, 5, 10, or 20 mg/m, resulting in average daily doses of approximately 535, l,035, or 2.065 mg TBA/kg body weight for males and 510, 1,015, or 2,105 mg/kg for females. Survival was significantly reduced in male rats receiving 5mg/ml, female rats receiving 10 mg/ml, and male mice receiving 20 mg/ml. Long-term exposure to TBA produced increased incidences of renal tubule adenoma and carcinoma in male rats; transitional epithelial hyperplasia of the kidney in male and female rats; follicular cell adenoma of the thyroid in female mice: and follicular cell hvperplasia of the thyroid and inflammation and hyperplasia of the urinary bladder in male and female mice. In addition, a slight increase in follicular cell adenoma or carcinoma of the thyroid (combined) in male mice may have been related to the administration of TBA.

Abstract  Male and female Sprague-Dawley rats of different ages at the start of the experiments (12 day embryos, and 7 and 25 weeks old) were administered fromaldehyde in drinking water at different doses (2,500 or 1,500, 1,000, 500, 100, 50, 10, 0 ppm). An increased incidence of leukemias and of gastro-intestinal tumors was observed in formaldehyde treated rats. Gastro-intestinal tumors are exceptionally rare in the rats of the colony used. These results, together with the ones obtained by other Authors on rats exposed by inhalation to formaldehyde, indicate that this compound is an experimental multipotential carcinogen. The experimental results presented in this report give scientific support to the epidemiological observation of a higher incidence of leukemias and of gastro-intestinal cancers among the people occupationally exposed.

Abstract  Unleaded gasoline causes acute and chronic nephrotoxicity and renal tumors in male rats, but not female rats or mice of either sex. An active nephrotoxic component of unleaded gasoline has been identified as 2,2,4-trimethylpentane (TMP). The first objective of this study was to characterize light microscopic renal lesions induced in male F344 rats by a 21-day gavage regimen of 50 to 500 mg/kg TMP. The second objective was to localize and quantitate sites of renal cell proliferation induced by the same TMP dose regimens using histoautoradiographic analysis after [3H]thymidine incorporation. Light microscopic lesions in the proximal convoluted tubule consisted of protein droplet and crystalloid body accumulation, degeneration, and necrosis, and were similar to lesions noted in previous inhalation and gavage studies with other hydrocarbon compounds. The above renal lesions were not dose-related, although tubular dilation of thin limb segments with granular cell debris was dose-related. In cell proliferation studies TMP induced a non-dose-related five- to sixfold increase in the labelling index of the same proximal convoluted tubule portions (P2 segment) that contained severe crystalloid body accumulation, degeneration, and necrosis. Less pronounced, but statistically significant (p less than or equal to 0.05), increases in cell proliferation were also observed in other nephron segments, indicating a generalized regenerative response of the kidney to TMP. The cytotoxic and regenerative renal effects of TMP administered by gavage suggest that similar mechanisms may be involved in the induction of kidney tumors in male rats following chronic inhalation exposure to unleaded gasoline.

Abstract  Physiologically based pharmacokinetic models for volatile organic chemicals typically describe the respiratory tract as a single compartment in which chemicals in the alveolar air space and the arterial blood are in instantaneous equilibrium. These models also assume that the distribution of chemical in the airstream is uniform throughout the respiratory tract and that uptake is significant only in the alveolar region. A functional role for the upper respiratory tract in the uptake of volatile chemicals has been largely ignored. While these models have worked well for chemicals with low aqueous solubility in biological fluids, systemic uptake of highly soluble volatiles is overestimated. Thus, there is a significant effort to describe the critical determinants for uptake of soluble chemicals and to formulate models with more biologically relevant descriptions of respiratory tract structure and function. Investigators have addressed this problem from several viewpoints. Airflow patterns in the respiratory tract, regional metabolism, diffusion-dependent uptake, and the cyclic nature of respiration are now being incorporated into current models. Use of dosimetric models that incorporate relevant biology for inhaled chemicals will ultimately result in more meaningful human risk assessments.

Abstract  In Catalonia (northeast Spain), a monitoring program was carried out to determine methyl tert.-butyl ether (MTBE), its main degradation products, tert.-butyl alcohol (TBA), tert.-butyl formate (TBF), and other gasoline additives, the oxygenate dialkyl ethers ethyl tert.-butyl ether, tert.-amyl methyl ether and diisopropyl ether and the aromatic compounds benzene, toluene, ethylbenzene and xylene (BTEX) in 21 groundwater wells that were located near different gasoline point sources (a gasoline spill and underground storage tank leakage). Purge-and-trap coupled to gas chromatographyûmass spectrometry was optimised for the simultaneous determination of the above mentioned compounds and enabled to detect concentrations at ng/l or sub-Ág/l concentrations. Special attention was given to the determination of polar MTBE degradation products, TBA and TBF, since not much data on method performance and environmental levels are given on these compounds in groundwater. All samples analysed contained MTBE at levels between 0.3 and 70 Ág/l. Seven contaminated hot spots were identified with levels up to US Environmental Protection Agency drinking water advisory (20û40 Ág/l) and a maximum concentration of 670 Ág/l (doubling the Danish suggested toxicity level of 350 Ág/l). Samples with high levels of MTBE contained 0.1û60 Ág/l of TBA, indicating (but not proving) in situ degradation of parent compound. In all cases, BTEX was at low concentrations or not detected showing less solubility and persistence than MTBE. This fact confirms the suitability of MTBE as a tracer or indicator of long-term gasoline contamination than the historically used BTEX.

Abstract  Methyl tertiary butyl ether (MTBE) is widely used in gasoline as an oxygenator and octane enhancer. There is also an interest in using the ethyl tertiary butyl (ETBE) and methyl tertiary amyl (TAME) ethers. We measured the blood, water, and olive oil/air partition coefficients in vitro of MTBE, ETBE, TAME and tertiary butyl alcohol (TBA), a metabolite of MTBE and ETBE. The results indicate similar uptake and distribution behavior for the three ethers and a slight affinity for fatty tissues. The partition coefficients of TBA indicate that this metabolite is not excreted via the lungs to any great extent and that it is preferentially distributed in body water. Further, we exposed 10 healthy male volunteers to MTBE vapor at 5, 25 and 50 ppm for 2 h during light physical exercise. Uptake and disposition were studied by measuring MTBE and TBA in inhaled and exhaled air, blood and urine. Low uptake, high post-exposure exhalation, and low blood clearance indicate slow metabolism of MTBE relative to many other solvents. A low recovery of TBA in urine (below 1% of uptake) indicates further metabolism of TBA. The concentration of MTBE and TBA in blood was proportional to exposure level suggesting linear kinetics up to 50 ppm. The half life of 7-10 h in blood and urine indicates that TBA would be more suitable than the parent compound as a biomarker for MTBE exposure. Subjective ratings (discomfort, irritative symptoms, CNS effects) and eye (redness, tear film break-up time, conjunctival damage, blinking frequency) and nose (peak expiratory flow, acoustic rhinometry, inflammatory markers in nasal lavage) measurements indicated no or minimal effects of MTBE.

Abstract  Pregnant CD-1 mice (30 per group) and female New Zealand White rabbits (15 per group) were exposed by inhalation to 0, 1000, 4000 and 8000 ppm methyl tertiary-butyl ether (MTBE) vapor for 6 h a day during gestational days (GD) 6-15 and 6-18, respectively. Maternal body weights, clinical observations and food consumption were recorded throughout gestation for both species. At scheduled euthanization (GD 18 for mice and GD 29 for rabbits), fetuses were weighed, sexed and examined for external, visceral (including craniofacial) and skeletal alterations. For both species, the pregnancy rate was high and equivalent across all groups; no pregnant animals died or aborted. There were no does that delivered early, but there were three mouse dams in the control group and two dams in the 4000 ppm group that delivered early and were removed from the study. In mice, maternal body weights, body weight gain, corrected maternal gestational weight change and food consumption were significantly reduced in mice at 8000 ppm. Hypoactivity and ataxia were observed in dams exposed to 4000 and 8000 ppm. Gestational parameters affected at 8000 ppm included post-implantation loss (due to increased late resorptions and dead fetuses) and altered sex ratio (decreased males); fetal body weights per litter were reduced at 4000 and 8000 ppm. There was a significantly increased incidence of cleft palate at 8000 ppm; this resulted in increased incidences of pooled external and visceral malformations and of total malformations at this exposure concentration. There were also treatment-related increases in the incidence of individual skeletal variations at 4000 and 8000 ppm. In rabbits, maternal weight gain and food consumption were significantly reduced at 4000 and 8000 ppm. Relative liver weights were also reduced at 8000 ppm. All gestational parameters were equivalent across all groups, including pre- and post-implantation loss, fetal sex ratios, litter size and fetal weights/litter. There was no evidence of treatment-related teratogenicity observed at any dose tested in rabbits. The no-observed-effect levels (NOELs) for maternal and developmental toxicity were both 1000 ppm in mice and 1000 ppm and at least 8000 ppm, respectively, in rabbits.

Abstract  This report has been prepared by TRC Environmental Corporation (TRC) for ARCO Chemical Company. It presents the results and conclusions from an odor and taste thresholdevaluation project conducted at TRC's Odor Laboratory in February, 1993.The gasoline oxygenates Methyl Tertiary Butyl Ether (MTBE) and Ethyl Tertiary Butyl Ether (ETBE) were evaluated for their odor detectibility and recognition thresholds in air and water as well as their taste threshold in water. MTBE (99.9% purity) and ETBE (99.0-99.5% purity) were obtained from ARCO Chemical Company.

Abstract  In a continuing review of long-term toxicology and carcinogenesis studies in rats and mice, the National Toxicology Program (NTP) is confronted with many problems concerning the interpretation of tumor data. A frequently raised question is: "Should certain neoplasms be combined for overall assessment of rodent carcinogenesis data?" NTP policy is that certain neoplasms may be combined for statistical assessment of tumor data and that hyperplastic responses may be used as supportive evidence. The primary reason for combining neoplastic lesions is to gain more insight into the evidence of the carcinogenicity of a given chemical in that species of animal. This report gives the rationale, criteria, and guidelines used by the NTP for combining neoplasms for the evaluation of long-term rodent toxicology and carcinogenesis studies. The guidelines are based mainly on lesions occurring in the F344/N inbred rat and (C57BL/6 X C3H)F1 mouse and may or may not be appropriate for other strains or species. The concepts of combining neoplasms and sites should be viewed in terms of the study as a whole, since tumor formation is only one of many responses caused by chemicals in mammals. The resulting information becomes part of the "weight of the evidence" for estimating the potential hazard of a given chemical.

Abstract  SUMMARY Ethyl-tert-Butyl Ether (ETBE) was applied at a dose of 2 g/kg of body weight to thes haved backs of five male and five female rabbits. The test article was left in contact with the skin for 24 hours and then removed. The rabbits were observed during this time and for 14 days thereafter. No deaths occurred during the study. Therefore, the acute median lethal dermal dose (LD50) was estimated to be greater than 2 g/kg of body weight. Dermal irritation (i.e., edema, erythenia and/or eschar formation) was observed within the application sites of all rabbits following unwrapping. Mean body weight increased during the study. No gross pathological lesions attributable to treatment were evident in any of the rabbits at necropsy.

Abstract  The objective of this study was to evaluate the n-octanol/water partitioning coefficient (Kow) for ethyl tertiary butyl ether (ETBE). The purpose of this report is to summarize the data from two determinations of Kow when both concentrations of the analyte are below the limit of solubility, and differ by a factor of 10. The test substance, ETBE, was partitioned between n-octanol saturated MilIiQ purified water and MilliQ purified water saturated n-octanol at two concentrations (1 x 10-2 and 1 x l0-3M). One time zero sampling point, and two post equilibrium sampling points were used to determine partitioning. Aliquots were taken from each of the aqueous and organic phases, from each sample, and analyzed for ETBE using gas chromatography with FID detection. Equilibrium time was determined from a non-GLP prestudy screen to be <0.5 hours. The results of the study indicate that the analytical method was sensitive enough to detect the concentration of the analyte in both the water and n-octanol phases at both concentrations used. The study shows that the ETBE preferentially partitions to the n-octanol phase, and the average values for Kow and log (Kow) at 25░C +/- 1░C are as follows: High Concentration (1 x 10-2M): Kow = 28.3 log(Kow) = 1.45 Low Concentration (1 x 10-3M): Kow = 31.4 log(Kow) = 1.50 Average of all Kow values for Both Concentrations: Kow = 30.1 ± 3.02 SD log(Kow) = 1.48 ± 0.0434 SD

Abstract  A strain that efficiently degraded methyl tert-butyl ether (MTBE) was obtained by initial selection on the recalcitrant compound tert-butyl alcohol (TBA). This strain, a gram-positive methylotrophic bacterium identified as Mycobacterium austroafricanum IFP 2012, was also able to degrade tert-amyl methyl ether and tert-amyl alcohol. Ethyl tert-butyl ether was weakly degraded. tert-Butyl formate and 2-hydroxy isobutyrate (HIBA), two intermediates in the MTBE catabolism pathway, were detected during growth on MTBE. A positive effect of Co2+ during growth of M. austroafricanum IFP 2012 on HIBA was demonstrated. The specific rate of MTBE degradation was 0.6 mmol/h/g (dry weight) of cells, and the biomass yield on MTBE was 0.44 g (dry weight) per g of MTBE. MTBE, TBA, and HIBA degradation activities were induced by MTBE and TBA, and TBA was a good inducer. Involvement of at least one monooxygenase during degradation of MTBE and TBA was shown by (i) the requirement for oxygen, (ii) the production of propylene epoxide from propylene by MTBE- or TBA- grown cells, and (iii) the inhibition of MTBE or TBA degradation and of propylene epoxide production by acetylene. No cytochrome P-450 was detected in MTBE- or TBA-grown cells. Similar protein profiles were obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts from MTBE- and TBA-grown cells. Among the polypeptides induced by these substrates, two polypeptides (66 and 27 kDa) exhibited strong similarities with known oxidoreductases.

Abstract  #The ambient air concentrations of 88 volatile organic compounds were determined in samples taken at 13 semi-rural to urban locations in Maine, Massachusetts, New Jersey, Pennsylvania, Ohio, Illinois, Louisiana, and California. The sampling periods ranged from 7 to 29 months, yielding a large data set with a total of 23,191 individual air concentration values, some of which were designated "ND" (not detected). For each compound at each sampling site, the air concentrations (ca, ppbV) are reported in terms of means, medians, and means of the detected values. The analytical method utilized adsorption/thermal desorption with air-sampling cartridges. The analytes included numerous halogenated alkanes, halogenated alkenes, ethers, alcohols, nitriles, esters, ketones, aromatics, a disulfide, and a furan. At some sites, the air concentrations of the gasoline-related aromatic compounds and the gasoline additive methyl tert-butyl ether were seasonally dependent, with concentrations that maximized in the winter. For each site studied here, the concentrations of some compounds were highly correlated one with another (e.g., the BTEX group (benzene, toluene, ethylbenzene, and the xylenes). Other aromatic compounds were also all generally correlated with one another, while the concentrations of other compound pairs were not correlated (e.g., benzene was not correlated with CFC-12). The concentrations found for the BTEX group were generally lower than the values that have been previously reported for urbanized and industrialized areas of other nations.

Abstract  This publication originates from a workshop held in 1997 to consider how rodent tumours of urinary bladder, renal cortex, and thyroid gland should be treated within the IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. The workshop also addressed the predictive value of these tumours for the identification of carcinogenic hazards to humans when they occur alone and when they occur along with tumours in other organs. In the following sections, etiological risk factors in humans and experimental animals as well as the hypotheses underlying the proposed species-specific mechanisms for each of the above-mentioned tumour types are summarized. Finally, for each tumour type, recommendations are presented on how the mechanistic data could be used in the overall evaluation of carcinogenicity to humans.

Abstract  Tert-Butanol is an important intermediate in industrial chemical synthesis, particularly of fuel oxygenates. Human exposure to tert-butanol may occur following fuel oxygenate metabolism or biodegradation. It is poorly absorbed through skin, but is rapidly absorbed upon inhalation or ingestion and distributed to tissues throughout the body. Elimination from blood is slower and the half-life increases with dose. It is largely metabolised by oxidation via 2-methyl-1,2-propanediol to 2-hydroxyisobutyrate, the dominant urinary metabolites. Conjugations also occur and acetone may be found in urine at high doses. The single-dose systemic toxicity of tert-butanol is low, but it is irritant to skin and eyes; high oral doses produce ataxia and hypoactivity and repeated exposure can induce dependence. Tert-Butanol is not definable as a genotoxin and has no effects specific for reproduction or development; developmental delay occurred only with marked maternal toxicity. Target organs for toxicity clearly identified are kidney in male rats and urinary bladder, particularly in males, of both rats and mice. Increased tumour incidences observed were renal tubule cell adenomas in male rats and thyroid follicular cell adenomas in female mice and, non-significantly, at an intermediate dose in male mice. The renal adenomas were associated with α2u-globulin nephropathy and, to a lesser extent, exacerbation of chronic progressive nephropathy. Neither of these modes of action can function in humans. The thyroid tumour response could be strain-specific. No thyroid toxicity was observed and a study of hepatic gene expression and enzyme induction and thyroid hormone status has suggested a possible mode of action.

Abstract  For many years, toxicological investigations have shown that the sensitivity of kidney to xenobiotics evolves depending on the stage of life. The increasing requirement for information on the potential nephrotoxic effect of drugs during human embryonic development, childhood, adulthood and senescence has potentiated toxicological studies in vivo. Rodents, specifically rats, are the primary animal models used in toxicology testing. Despite the popularity of this approach, there are a number of doubts about the appropriateness of rats for the examination of changes in toxicological responses during different stages of life. This perspective tackles the issue of evaluating whether rats fail to adequately mimic the human kidney response to xenobiotic agents through a critical analysis of the literature. We conclude that rats constitute a good model for toxicological investigations during embryonic development, youth and adulthood. However, senescent rats frequently undergo spontaneous kidney degeneration caused by chronic progressive nephropathy, making them a poor model for the study of kidney responses to xenobiotics.

Abstract  Current suggestions towards amending the OECD two-generation protocol include omission of the second generation and inclusion of additional parameters. This study analysed the relative parameter sensitivity in 18 individually published multi-generation studies with substances toxic to fertility. Among parameters that most often determined the reproductive LOAEL were weight of testis, dam and pup as well as litter size. Several other parameters were found to be unaffected in all studies evaluated. Some substances affected a specific set of parameters, indicating that rarely affected parameters may prove crucial in individual situations. This argues for the inclusion of a wide spectrum of parameters to cover all possible effects. Less sensitive parameters, mechanistically related to more sensitive ones, may be omitted as they will unlikely contribute to the overall LOAEL. This study gives first insights and needs follow-up by more extensive analyses before firm conclusions on the design of the two-generation study protocol can be drawn.

Abstract  Three major enzyme systems have been shown to metabolize epoxidized xenobiotics in vertebrate tissues, and this study demonstrates that these enzyme systems can be differentially induced. The cytosolic epoxide hydrolase activity was routinely monitored with trans-beta-ethylstyrene oxide, the microsomal epoxide hydrolase activity with benzo(a)pyrene, 4,5-oxide, and the glutathione S-transferase activity with 2,4-dichloro-4-nitrobenzene. Commonly used inducers of microsomal mixed-function oxidase, microsomal epoxide hydrolase, and cytosolic glutathione S-transferase activities failed to cause significant induction of the cytosolic epoxide hydrolase while leading to the expected induction of the other epoxide metabolizing enzymes. The compounds tested by ip injection into male Swiss-Webster mice included phenobarbital, 3-methylcholanthrene, Aroclor 1254, trans- and cis-stilbene oxides, pregnenolone-16 alpha-carbonitrile, chalcone, and 4-bromochalcone. To determine if there were strain, sex, or species differences, the enzymes were monitored in male C57BL/6 mice, female Swiss-Webster mice, and male Sprague-Dawley rats following ip injection of phenobarbital, 3-methylcholanthrene, and/or pregnenolone-16 alpha-carbonitrile. The time dependence of enzyme induction was followed in Sprague-Dawley rats following trans-stilbene oxide administration. Male Swiss-Webster mice were additionally exposed to dietary alpha-naphthoflavone and 2(3)-tert-butyl-4-hydroxyanisole while male Sprague-Dawley rats were fed 2,6-di-tert-butyl-4-methylphenol. In no case was significant induction of cytosolic epoxide hydrolase activity observed. Dietary di-(2-ethylhexyl)phthalate, 2-ethyl-l-hexanol, and clofibrate proved to be potent inducers of the cytosolic epoxide hydrolase in male Swiss-Webster mice while probucol (a nonperoxisome proliferating hypolipidemic drug) failed to cause significant induction. Data from isoelectric focusing experiments and other data are consistent with the epoxide hydrolase activities induced by 2-ethyl-l-hexanol and clofibrate being due to the same protein that is present in control animals. The lack of induction of the cytosolic epoxide hydrolase by a variety of compounds which were selected to demonstrate induction of other xenobiotic metabolizing enzymes, may indicate that the cytosolic epoxide hydrolase has a constitutive role whereas its induction by clofibrate could be related to some of the pharmacological and/or carcinogenic actions of this drug

Abstract  Increasing pressure is exerted by some stakeholders to reduce the two-generation study to a one-generation study, a measure that would considerably reduce the number of animals and other costs involved in these lengthy studies. The present study retrospectively evaluates 176 multi-generation studies to assess potential differences between the first and the second generation, both in terms of the types of effects observed and in terms of the effective doses. All substances classified as reproductive toxicants by the Directive 92/32/EEC or considered as toxic to fertility by the California EPA for which we found a multi-generation study were included (n = 58 studies). The second generation in the two-generation studies considered affected neither the overall NOAEL nor the critical effect. Therefore, it had no impact on the ensuing risk assessment, nor on classification and labeling. However, several substances did show an increased sensitivity of the F1 adults in comparison to the P0. These results support the proposal of replacing the current two-generation study by a one-generation study with a more extensive assessment of parameters at F1 adulthood.

Abstract  Tertiary butyl alcohol (TBA) was administered to groups of 15 female B6C3F1 mice in drinking water at concentrations of 0, 2.0 or 20 mg TBA ml(-1), for 14 days, for assessment of gross and histological changes in the liver and thyroid, thyroid hormones (T3, T4, and TSH), total hepatic cytochrome P450 (Cyp) content, specific Cyp activities and quantitative PCR analysis of specific Cyp enzymes (Cyp1a1, Cyp2b9, Cyp2b10, Cyp3a11), sulfuryltransferases (ST1a1, ST2a2, and STn) and glucuronyltransferases (UGT1a1, UGT2b1, and UGT2b5). Phenobarbital (PB) was administered to a positive control group by oral gavage at a daily dose of 80 mg kg(-1). TBA caused, on day 14, a reduction in circulating T3 (12-15% decrease) and a dose-dependent reduction in T4 (13-22% decrease), with no evidence of thyroid pathology. Two of five livers examined in the 20 mg TBA ml(-1) dose group showed mild, diffuse centrilobular hypertrophy. On day 14, Cyp 7-benzoxyresorufin-O-debenzylase activity was significantly induced 12-fold by TBA at 20 mg ml(-1), and 1.8-fold at the 2.0 mg TBA ml(-1) concentration. Cyp 7-pentoxyresorufin-O-dealkylase activity was slightly induced (2.1-fold) by 20 mg TBA ml(-1) on day 14. Quantitative PCR analysis of gene transcripts showed a significant induction of Cyp2b10 and ST1a1 with both TBA concentrations, and a slight induction of Cyp2b9 at 20 mg TBA ml(-1) only. PB induced all phase I and phase II gene transcripts except for Cyp1a1 and Cyp2b9. These findings suggest that TBA, at and below doses used in chronic studies, is an inducer of phase I and phase II liver enzymes, with resulting decreases in circulating thyroid hormones in B6C3F1 mice.