Abstract  Ethyl tertiary-butyl ether (ETBE) is commonly used as an oxygenated gasoline additive. In this study, the prenatal developmental toxicity of ETBE was determined in rabbits. New Zealand white rabbits were given ETBE by gavage at 100, 300, or 1,000 mg/kg/day on gestational days (GDs) 6-27, and the pregnancy outcome was determined on GD 28. Neither death nor abortion occurred in the pregnant rabbits at any dose. Slightly and significantly suppressed maternal body-weight gain and transiently decreased maternal food consumption were found at 1,000 mg/kg/day during the administration period. At this dose, no changes in clinical or macroscopic finding were noted in dams. No treatment-related changes were observed in any dam treated at 300 mg/kg/day or less. There was no significant effect of ETBE on the numbers of corpora lutea, implantations, live fetuses, resorptions and dead fetuses, incidences of pre- and postimplantation loss, viability of fetuses, fetal body weight, sex ratio of fetuses, or weights of gravid uteri. No significant difference was detected in the incidences of fetuses with malformations or variations between the ETBE-treated and control groups. Also, no adverse effects on the progress of ossification were noted in fetuses of dams given ETBE. Based on these findings, it is concluded that the no observed adverse effect levels of ETBE were 300 mg/kg/day for dams and 1,000 mg/kg/day for fetuses in rabbits.

Abstract  Carcinogenicity of ethyl tertiary-butyl ether (ETBE) was examined with inhalation exposure using F344/DuCrlCrlj rats. Groups of 50 male and 50 female rats, 6 week old at commencement, were exposed to ETBE at 0, 500, 1,500 or 5,000 ppm (v/v) in whole-body inhalation chambers for 6 h/day, 5 days/week for 104 weeks. A significant increase in the incidence of hepatocellular adenomas was indicated in males exposed at 5,000 ppm, but not in females at any concentration. In addition, significantly increased incidences of eosinophilic and basophilic cell foci were observed in male rats at 5,000 ppm. Regarding non-neoplastic lesions, rat-specific changes were observed in kidney, with an increase in the severity of chronic progressive nephropathy in both sexes at 5,000 ppm. Increased incidences of urothelial hyperplasia of the pelvis were observed at 1,500 ppm and above, and mineral deposition was apparent in the renal papilla at 5,000 ppm in males. There were no treatment-related histopathological changes observed in any other organs or tissues in either sex. The present 2-year inhalation study demonstrated hepatotumorigenicity of ETBE in male, but not in female rats.

Abstract  The effects of ethyl tertiary-butyl ether (ETBE) on two-stage urinary bladder carcinogenesis in male F344 rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were investigated at various dose levels with regard to possible promoting activity. Groups of 30 rats were given drinking water containing 500 ppm BBN, as an initiator, for 4 weeks and starting one week thereafter received ETBE by gavage (daily, 7 days/week) at dose levels of 0 (control), 100, 300, 500 or 1000 mg/kg/day until experimental week 36. No statistically significant differences in incidences of preneoplastic lesions, papillomas, and carcinomas of the urinary bladder were evident in rats treated with 100-1000 mg/kg/day ETBE as compared with control values. Furthermore, the average numbers of preneoplastic or neoplastic lesions per unit length of basement membrane in rats given 100-1000 mg/kg/day ETBE were also comparable to control values. However, papillomatosis of the urinary bladder was found in 4 out of 30 rats (13%) in the group given 1000 mg/kg/day ETBE, and soft stones in the urinary bladder were found in 3 out of these 4 rats. The results thus demonstrated that ETBE did not exert promotional activity on urinary bladder carcinogenesis. However, papillomatosis of the urinary bladder developed in small numbers of the rats given ETBE at 1000 mg/kg/day but not in rats given 500 mg/kg/day or lower doses.

Abstract  No data are available regarding aldehyde dehydrogenase 2 (ALDH2) polymorphisms related to the reproductive toxicity possibly caused by ethyl tertiary butyl ether (ETBE). In this study, two inhalation experiments were performed in Aldh2 knockout (KO), heterogeneous (HT) and wild type (WT) C57BL/6 male mice exposed to ETBE, and the data about general toxicity, testicular histopathology, sperm head numbers, sperm motility and sperm DNA damage were collected. The results showed that the 13-week exposure to 0, 500, 1,750 and 5,000 ppm ETBE significantly decreased sperm motility and increased levels of sperm DNA strand breaks and 8-hydroxy-deoxyguanosine in both WT and KO mice, the effects were found in 1,750 and 5,000 ppm groups of WT mice, and all of the three exposed groups of KO mice compared to the corresponding control; furthermore, ETBE also caused decrease in the relative weights of testes and epididymides, the slight atrophy of seminiferous tubules of testis and reduction in sperm numbers of KO mice exposed to ≥500 ppm. In the experiment of exposure to lower concentrations of ETBE (0, 50, 200 and 500 ppm) for 9 weeks, the remarkable effects of ETBE on sperm head numbers, sperm motility and sperm DNA damage were further observed in KO and HT mice exposed to 200 ppm ETBE, but not in WT mice. Our findings suggested that only exposure to high concentrations of ETBE might result in reproductive toxicity in mice with normal active ALDH2, while low active and inactive ALDH2 enzyme significantly enhanced the ETBE-induced reproductive toxicity in mice, even exposed to low concentrations of ETBE, mainly due to the accumulation of acetaldehyde as a primary metabolite of ETBE.

Abstract  Ethyl tertiary-butyl ether (ETBE) is commonly used as an oxygenated gasoline additive. In this study, we evaluated its developmental toxicity in rats. ETBE was administered by gavage to 21 or 22 pregnant female Sprague-Dawley rats per group at dose levels of 0, 100, 300 and 1000 mg/kg/day from days 5 through 19 postcoitum to assess its effects on pregnant animals and their embryos and fetuses applied to the OECD testing guideline (no. 414) correspondingly. There were no toxicological effects attributable to ETBE regarding clinical signs, body weight, food intake, necropsy or examination at caesarean section in pregnant animals. There were also no toxicological effects on external, visceral and skeletal examinations of embryos and fetuses. These results indicate that, under the conditions of this study, ETBE had no toxicological effects on pregnant rats or their embryos and fetuses and that the no observed adverse effect level was 1000 mg/kg/day both for pregnant rats and their embryos and fetuses.

Abstract  The purpose of this study was to evaluate whether repeated 6-h exposure (65' exposures over a 14-week period) of male and female Fischer-344 rats (n = 12 rats/sex/concentration) to ethyl tertiary-butyl ether (ETBE) atmospheres at 500, 1750, or 5000 ppm would result in neurotoxicity. Neurotoxicity was assessed by a blinded functional observational battery (FOB), motor activity, and terminal neuropathology. Motor activity was assessed 4 days prior to ETBE exposure and following 20, 42, and 65 days of exposure. The FOB was assessed 4 days prior to ETBE exposure and following 1, 6, 10, 20, 42, and65 days of exposure. Transient ataxia, a sign of narcosis, was noted in male rats immediately following the 6-h exposure to 5000 ppm ETBE. Statistically significant treatment effects on motor activity were not observed. Minor changes in grip strength and hind limb splay were observed; however, none demonstrated a dose-response relationship or a consistent pattern of neurological dysfunction. No gross or microscopic abnormalities were observed in the central, peripheral, or autonomic nervous systems of rats exposed to 5000 ppm ETBE. No statistically significant differences in brain weight or size were observed in ETBE-exposed rats. A statistically significant increase in body weight was observed in female rats exposed to 5000 ppm following 42 and 65 exposure days. Although ataxia was a common feature of acute ETBE neurotoxicity in rats following high-level exposure, adverse neurological effects are not expected in the general public at the anticipated exposure levels associated with automotive refueling.

Abstract  The 1990 Clean Air Act Amendments require that oxygenates be added to automotive fuels to reduce emissions of carbon monoxide and hydrocarbons. One potential oxygenate is the aliphatic ether ethyl tertiary butyl ether (ETBE). Our objective was to provide data on the potential toxic effects of ETBE. Male and female Fisher 344 rats and CD-1 mice were exposed to 0 (control), 500, 1750, or 5000 ppm of ETBE for 6 h/day and 5 days/wk over a 13-week period. ETBE exposure had no effect on mortality and body weight with the exception of an increase in body weights of the female rats in the 5000-ppm group. No major changes in clinical pathology parameters were noted for either rats or mice exposed to ETBE for 6 (rats only) or 13 weeks. Liver weights increased with increasing ETBE-exposure concentration for both sexes of rats and mice. Increases in kidney, adrenal, and heart (females only) weights were noted in rats. Degenerative changes intesticular seminiferous tubules were observed in male rats exposed to 1750 and 5000 ppm but were not seen in mice. This testicular lesion has not been reported previously for aliphatic ethers. Increases in the incidence of regenerative foci, rates of renal cell proliferation, and "alpha"2u-globulin containing protein droplets were noted in the kidneys of all treated male rats. These lesions are associated with the male rat-specific syndrome of "alpha"2u-globulin nephropathy. Increases in the incidence of centrilobular hepatocyte hypertrophy and rates of hepatocyte cell proliferation were seen in the livers of male and female mice in the 5000-ppm group, consistent with a mitogenic response to ETBE. These two target organs for ETBE toxicity, mouse liver and male rat kidney, have also been reported for methyl tertiary butyl ether and unleaded gasoline.

Abstract  Ethyl tertiary-butyl ether (ETBE) is a motor fuel oxygenate used in reformulated gasoline. The current use of ETBE in gasoline or petrol is modest but increasing. To investigate the effects of ETBE on splenocytes, mice were exposed to 0 (control), 500 ppm, 1750 ppm, or 5000 ppm of ETBE by inhalation for 6 h/day for 5 days/wk over a 6- or 13-week period. Splenocytes were harvested from the control and exposed mice, and the following cell phenotypes were quantified by flow cytometry: (1) B cells (PerCP-Cy5.5-CD45R/B220), (2) T cells (PerCP-Cy5-CD3e), (3) T cell subsets (FITC-CD4 and PE-CD8a), (4) natural killer (NK) cells (PE-NK1.1), and (5) macrophages (FITC-CD11b). Body weight and the weight of the spleen were also examined. ETBE-exposure did not affect the weight of the spleen or body weight, while it transiently increased the number of RBC and the Hb concentration. The numbers of splenic CD3+, CD4+, and CD8+ T cells, the percentage of CD4+ T cells and the CD4+/CD8+ T cell ratio in the ETBE-exposed groups were significantly decreased in a dose-dependent manner. However, ETBE exposure did not affect the numbers of splenic NK cells, B cells, or macrophages or the total number of splenocytes. The above findings indicate that ETBE selectively affects the number of splenic T cells in mice.

Abstract  The modifying potential of ethyl tertiary-butyl ether (ETBE) on tumor development was investigated in a medium-term multi-organ carcinogenesis bioassay using male F344 rats. Animals were sequentially given 5 carcinogens with different target sites in the first 4 weeks for multi-organ initiation. After one week they received ETBE by gavage at dose levels of 0 (control), 300 or 1000mg/kg/day until experimental week 28. Further groups were also given ETBE at doses of 0 or 1000mg/kg/day without prior carcinogen application. Incidences and multiplicities of follicular cell hyperplasias and neoplasms in the thyroid were significantly increased at dose levels of more than 300mg/kg/day. Combined incidences of squamous cell hyperplasias and papillomas of the forestomach were also significantly increased at 300 and 1000mg/kg/day. Incidences and multiplicities of adenocarcinomas in the colon were increased at 1000mg/kg/day. The numbers and areas of glutathione S-transferase placental form (GST-P) positive foci per unit area of the liver sections, and the incidence of hepatocellular adenomas were also significantly increased at 1000mg/kg/day, along with multiplicities of atypical hyperplasias of renal tubules of the kidney and the incidence of papillomatosis of the urinary bladder. This latter lesion was also seen at low incidence at 1000mg/kg/day without initiation. Thus, the current results indicate that ETBE has tumor promoting potential for the thyroid and forestomach at dose levels of 300mg/kg/day and more, and for the colon, liver, kidney and urinary bladder at 1000mg/kg/day, under the present experimental conditions.

Abstract  A one-generation reproductive toxicity study was conducted to evaluate the effects of ethyl tertiary butyl ether (ETBE), a bio-fuel, on reproduction of parental rats, as well as development and growth of their offspring at dose levels of 0, 100, 300 and 1000 mg/kg-d by gavage. No treatment-related changes were observed in either F0 parents or their F1 offspring in the 100 and 300 mg/kg groups in any parameters examined. Some parental animals in the 1000 mg/kg group exhibited transient salivation, possibly a reflex to a bitter taste of ETBE, immediately after dosing, although their body weights, food consumption, reproductive parameters, and gross pathological findings were not affected. Their absolute and relative liver weights increased significantly in the 1000 mg/kg group, suggesting enhanced activities of metabolic enzymes. Pup viability was slightly reduced during the early lactation period in the 1000 mg/kg group. These results lead to the conclusion that the no-observed-adverse-effect-level (NOAEL) of ETBE on both parental rats and their offspring is 300 mg/kg-d under the current study condition.

Abstract  These experiments were conducted to follow up on a report of testis seminiferous tubular degeneration in Fischer 344 rats treated with high doses of ethyl t-butyl ether (ETBE). Also, high doses of a related compound, methyl t-butyl ether (MTBE), had been shown to reduce circulating testosterone (T) in rats. Isolated rat Leydig cells were used to compare hCG-stimulated T production following exposure to ETBE, MTBE, and their common main metabolite, TBA. In addition, male Fischer 344 rats were gavaged daily with 600 mg/kg, 1200 mg/kg or 1800 mg/kg ETBE in corn oil (n=12) for 14 days, the 1200 mg/kg dose chosen for comparison with a prior 14-day MTBE gavage experiment. In cell culture experiments, TBA was more potent than either ETBE or MTBE, both of which caused similar inhibition of T production at equimolar concentrations. In the in vivo study, no significant plasma T reduction was seen 1h after the final 1200 mg/kg ETBE dose, whereas 1200 mg/kg MTBE had significantly lowered T when administered similarly to Sprague-Dawley rats. Some rats treated with 1800 mg/kg ETBE had noticeably lower T levels, and the group average T level was 66% of corn oil vehicle control (p>0.05) with high variability also evident in ETBE-treated rats. 17beta-Estradiol had been increased by 1200 mg/kg MTBE, and was elevated in the 1200 and 1800 mg/kg ETBE dose groups (p<0.05), both groups also experiencing significantly reduced body weight gain. None of these effects were seen with 600 mg/kg/day ETBE. No definitive evidence of androgen insufficiency was seen in accessory organ weights, and no testicular pathology was observed after 14 days in a small subset of 1800 mg/kg ETBE-treated animals. Like MTBE, ETBE appears to be capable of altering reproductive steroid levels in peripheral blood sampled 1h after treatment, but only with extremely high doses that inhibit body weight gain and may produce mortality.

Abstract  The carcinogenicity of ethyl tertiary-butyl ether (ETBE) was examined by oral administration using F344/DuCrlCrlj rats. Groups of 50 male and 50 female rats were given drinking water containing ETBE at doses of 0, 625, 2,500 or 10,000 ppm (w/w) for 104 weeks. No significant increase in the incidence of tumors was detected in any organ of either sex. Rat-specific non-neoplastic lesions were observed in the kidney: An increase in the severity of chronic progressive nephropathy was observed in the male and female 10,000 ppm groups, and increased incidences of urothelial hyperplasia of the pelvis and mineral deposition in the renal papilla were observed in the male 2,500 and 10,000 ppm groups. Besides these lesions, no treatment-related histopathological changes were observed in any organ or tissue in either sex. Thus, the present study demonstrated that a two year administration ETBE in the drinking water did not exert any carcinogenic effects in either male or female rats.

Abstract  This report illustrates the project of long-term experimental studies on the gasoline oxygenated additives and of gasoline containing several of the same oxygenates, performed by the Cancer Research Centre (CRC) of the European Ramazzini Foundation of Oncology and Environmental Sciences (RF). The compounds and mixtures studied by this project are: methyl alcohol, ethyl alcohol, methyl-tertiary-butyl ether (MTBE), ethyl-tertiary-butyl ether (ETBE), tert-amyl-methyl ether (TAME) and di-isopropyl ether (DIPE), as well as gasoline containing methyl alcohol, ethyl alcohol, MTBE and ETBE. All experiments were performed on Sprague-Dawley rats from the CRC/RF colony, exposed to test materials by ingestion (stomach intubation in extra virgin olive oil solution, or in drinking water) and kept under control until spontaneous death. The report also presents the first results of the study on ETBE. The compound was administered to groups of 120 rats (60 males and 60 females) at a daily dose of 1000, 250, and 0 (in olive oil) mg/kg b.w., for 4 days weekly, over 104 weeks. In the tested experimental conditions ETBE causes an increase in: 1) total malignant tumours (more evident in females); 2) total oncological lesions of the mouth (more evident among males); 3) total oncological lesions of the forestomach in males and, more specifically, squamocellular carcinomas in both males and females, exposed to the lower concentration of ETBE; 4) malignant uterine tumours (in particular sarcomas) in the females exposed to the lower concentration; and 5) haemolymphoreticular neoplasias (lymphomas and leukaemias), in particular lymphoimmunoblastic lymphomas. No dose-response relationship between neoplastic effects and ETBE concentrations was found: these results may be explained (at least partly) by the higher mortality in the group treated with the higher dose.

Abstract  The objective of this prenatal developmenjtal toxicity study was to evaluate the potential effects of the test item, ETHYL TERTIARY BUTYL ETHER (ETBE; CASE No. 637-92-3), on pregnancy and embryo-fetal development in Sprague-Dawley rats folllowing daily oral (gavage) adminstration at doses up to 1000 mg/kg/day from implantation to one day prior to scheduled hysterectomy (that is, day 5 to day 19 post-coitum, inclusive).

Abstract  Ethyl tertiary butyl ether (ETBE) is used as an additive to gasoline to reduce carbon monoxide emissions in some developed countries. So far, ETBE was not found with positive results in many genotoxic assays. This study is undertaken to investigate the modifying effects of deficiency of aldehyde dehydrogenase 2 (ALDH2) on the toxicity of ETBE in the livers of mice. Eight-week-old wild-type (WT) and Aldh2 knockout (KO) C57BL/6 mice of both sexes were exposed to 0, 500, 1,750, and 5,000 ppm ETBE for 6 h/day with 5 days per weeks for 13 weeks. Histopathology assessments and measurements of genetic effects in the livers were performed. Significantly increased accidences of centrilobular hypertrophy were observed in the livers of WT and KO mice of both sexes in 5,000 ppm group; there was a sex difference in centrilobular hypertrophy between male and female KO mice, with more severe damage in the males. In addition, DNA strand breaks, 8-hydroxyguanine DNA-glycosylase (hOGG1)-modified oxidative base modification, and 8-hydroxydeoxyguanosine as genetic damage endpoints were significantly increased in three exposure groups in KO male mice, while these genotoxic effects were only found in 5,000 ppm group of KO female mice. In WT mice, significant DNA damage was seen in 5,000 ppm group of male mice, but not in females. Thus, sex differences in DNA damage were found not only in KO mice, but also in WT mice. These results suggest that ALDH2 polymorphisms and sex should be taken into considerations in predicting human health effects of ETBE exposure.