tert-butyl methyl ether

Transcription

1 tert-butyl methyl ether MAK value (2000) 50 ml/m 3 (ppm) 180 mg/m 3 Peak limitation (2000) Category I, excursion factor 1.5 Absorption through the skin Sensitization Carcinogenicity (2000) Prenatal toxicity (2000) Germ cell mutagenicity BAT value Synonyms Chemical name (CAS) Category 3B CAS number Pregnancy risk group C 2-methoxy-2-methylpropane methyl-tert-butyl ether tert-butyl methyl ether Structural formula (CH 3 ) 3 C O CH 3 Molecular formula C 5 H 12 O Molecular weight Melting point Boiling point 109 C 55.2 C Density at 20 C 0.74 g/cm 3 Vapour pressure at 25 C 335 hpa logp ow * ml/m 3 (ppm) 3.66 mg/m 3 1mg/m ml/m 3 (ppm) The present documentation was based on reviews of toxicological data for tert-butyl methyl ether (ECB 1995, ECETOC 1997, WHO 1998) and supplemented with more recently published data. * n-octanol/water distribution coefficient

2 120 tert-butyl methyl ether Volume 17 1 Toxic Effects and Mode of Action tert-butyl methyl ether is absorbed readily after inhalation exposure and ingestion, and is distributed rapidly in the organism. Absorbed tert-butyl methyl ether and the resulting metabolites are rapidly exhaled, and to a lesser extent also eliminated via the kidneys. The substance is transformed by cytochrome P450 to tert-butyl alcohol and formaldehyde. The formaldehyde is either rapidly detoxified or enters intermediary metabolism. tert-butyl alcohol is metabolized only slowly and excreted with the urine or exhaled. In man, single exposures to tert-butyl methyl ether concentrations above 50 ml/m 3 led to irritation of the mucous membranes of the upper respiratory tract and to adverse effects on the central nervous system. The acute toxicity of the substance was found to be low for all administration routes in experiments with various species of animal. The symptoms of intoxication are mainly irritative effects and central nervous depression. On the skin and in the eye of rabbits the substance is moderately irritative. tert-butyl methyl ether does not have sensitizing effects on guinea pig skin. For rats or mice exposed by inhalation for up to 90 days the NOEL (no observed effect level) is at least 300 ml/m 3. Higher concentrations cause irritation and central nervous effects, and increased nephropathy, changes in the blood count and increased organ weights (liver, kidneys, adrenal glands). Repeated oral administration of the substance to rats produces effects similar to those seen after inhalation exposure. Even the lowest tested dose of 90 mg/kg body weight and day had effects. tert-butyl methyl ether has no effect on the fertility or reproductive success of rats even at toxic concentrations of 8000 ml/m 3. Exposure to maternally toxic concentrations up to 2500 and 8000 ml/m 3 has no adverse effects on the offspring of rats or rabbits. In the mouse, concentrations up to 2500 ml/m 3 do not have adverse effects on reproduction; maternally toxic concentrations also have foetotoxic and teratogenic effects. tert-butyl methyl ether is not mutagenic in Salmonella typhimurium nor clastogenic in various mammalian cell lines. In mouse lymphoma cells the substance is mutagenic after metabolic activation, presumably as a result of the formaldehyde formed. All in vivo genotoxicity studies with tert-butyl methyl ether have yielded negative results even in toxic concentration and dose ranges. In the liver of the mouse, no tumour-promoting effects of tert-butyl methyl ether could be detected. In long-term inhalation studies, markedly toxic concentrations of the substance increased the incidences of kidney and Leydig cell tumours in the male rat and liver cell tumours in the female mouse. In rats given long-term oral doses of the substance, the incidence of Leydig cell tumours was increased, and in the female animals the sum of the incidences of leukaemia and lymphomas was increased but survival was low.

3 Volume 17 tert-butyl methyl ether Mechanism of Action 2.1 Nephrotoxicity tert-butyl methyl ether was nephrotoxic in rats after repeated exposure; female animals were found to be less sensitive. The effects resulted presumably from the acceleration and intensification of spontaneous age-related nephropathy observed in both sexes after long-term exposure (Bird et al. 1997, Borghoff et al. 1996), and the species and sexspecific accumulation of hyaline droplets in the epithelial cells of the proximal tubules detected in male animals after medium-term inhalation exposure and oral administration. In subsequent studies F344 rats were exposed to concentrations up to 3013 ml/m 3 (6 hours a day, for 10 days); the accumulated protein was identified as α 2 u-globulin. The accumulation was regarded as weak compared to that seen after treatment with 2,2,4-trimethylpentane (Borghoff et al. 1996, Prescott-Mathews et al. 1997). On the other hand, in male F344 rats exposed for 28 days to concentrations up to 8000 ml/m 3 (6 hours a day, on 5 days a week), protein accumulation in the epithelial cells of the proximal tubules could be detected, but the immuno-histological analysis of α 2 u-globulin in the control group and the exposed group revealed no differences in the intensity of staining (Bird et al. 1997). Evidence of the interaction of tert-butyl methyl ether and α 2 u-globulin in vivo was recently found. The study was carried out with F344 rats given oral doses of 14 C- labelled tert-butyl methyl ether of 750 mg/kg body weight and day for 4 days. The level of α 2 u-globulin in the kidneys of the treated male animals was about twice that in the controls. However, no more radioactivity accumulated in the kidneys in the male animals than in the female animals. Only in the kidney cytosol of male animals, however, could bound radioactivity be displaced by d-limonene oxide, a substance with great affinity to α 2 u-globulin (Prescott-Mathews et al. 1999). The results suggest that tert-butyl methyl ether, in addition to causing α 2 u-globulin nephropathy, which is not relevant for man, can also have toxic effects on the kidneys as a result of the acceleration or intensification of chronic age-related nephropathy, which is particularly pronounced in rats. Also in studies with one of the main metabolites of tert-butyl methyl ether, tert-butyl alcohol, evidence was found of the involvement of the two mechanisms of nephrotoxicity described above (see MAK documentation for tert-butyl alcohol, Greim 1999). 2.2 Endocrine modulation In mice exposed long-term via inhalation to tert-butyl methyl ether, a treatment-related decrease in physiologically occurring cystic hyperplasia in the endometrium was observed (Bird et al. 1997). In Sprague-Dawley rats given oral doses of the substance longterm, the incidence of mammary tumours was decreased, that of Leydig cell tumours increased (Belpoggi et al. 1995, 1997). In view of this, tert-butyl methyl ether was thought to have an anti-oestrogenic effect. This supposition was investigated in subsequent studies with female B6C3F 1 mice. In animals exposed to tert-butyl methyl

4 122 tert-butyl methyl ether Volume 17 ether concentrations of 8000 ml/m 3 (6 hours per day, 5 days per week), after 3 days the relative liver weights were increased and relative uterus weights decreased, after 21 days exposure the relative uterus and ovary weights were decreased. Histological examination revealed slight swelling of the hepatocytes only after exposure for 3 days. The level of cytochrome P450 in the liver was increased to 1.4 times the control value after exposure for 3 days, and to twice the control value after exposure for 21 days. The 7-pentoxyresorufin-O-dealkylase activity, a measure of the level of cytochrome P450 2B, was increased 5-fold and 14-fold in isolated liver microsomes. The 7-ethoxyresorufin-Odeethylase activity, a measure of the levels of cytochrome P450 1A1 and 1A2, was increased 2-fold to 3-fold. Oral administration of tert-butyl methyl ether concentrations of 1800 mg/kg body weight and day for 3 days yielded similar results. In addition, the metabolic deactivation of oestradiol to water-soluble metabolites (no other details) by isolated hepatocytes from these animals was 2.1 times that in the control test run (Moser et al. 1996a), which could partially explain the anti-oestrogenic effect in vivo. In another study with female B6C3F 1 mice, anti-oestrogenic activity of tert-butyl methyl ether was detected, but only on the basis of changes in some oestrogen-dependent parameters; the oestrogen levels and oestrogen receptor density of various tissues of the genital tract were unchanged. The animals were exposed for 3 or 21 days and 4 or 8 months to tert-butyl methyl ether concentrations of 8000 ml/m 3. The uterus weights were reduced during the whole exposure period, the pituitary gland and ovary weights only after exposure for 4 or 8 months. After exposure of the animals for 8 months, the menstrual cycle was also prolonged. The number of uterine glands decreased after mediumterm exposure to tert-butyl methyl ether. DNA-synthesis in various epithelial layers of the female genital tract was also reduced. Competitive binding of tert-butyl methyl ether to the human oestrogen receptor was not observed in vitro. Also in HepG2 cells transfected with the oestrogen receptor gene, no interaction of tert-butyl methyl ether with the oestrogen receptor could be detected (Moser et al. 1998). An anti-oestrogenic effect of tert-butyl methyl ether has therefore been demonstrated for high concentrations/doses, but the underlying mechanism is still unclear. 2.3 Carcinogenicity In rats and mice of both sexes the incidence of various tumours was increased after longterm exposure of the animals to tert-butyl methyl ether (see Section 5.7). The first possibility to be discussed is a causal involvement of the genotoxic metabolite formaldehyde and thus a possible genotoxic mechanism for the carcinogenic effects of tert-butyl methyl ether. Evidence of formaldehyde-induced mutagenicity of tert-butyl methyl ether was obtained with mouse lymphoma cells in vitro under special experimental conditions (see Section 5.6.1; Mackerer et al. 1996). In cultured hepatocytes taken from female CD-1 mice, male B6C3F 1 mice or male F344 rats and incubated with tert-butyl methyl ether, only low levels of formaldehyde-induced DNA protein crosslinks or RNA formaldehyde adducts were detected. The effects were not found to be concentrationdependent for the tested range of 0.33 to 6.75 mm. If the cells, however, were incubated with formaldehyde in the same concentration range, the number of adducts increased in a

5 Volume 17 tert-butyl methyl ether 123 concentration-dependent manner; the level of DNA adducts was up to 10 times that formed with tert-butyl methyl ether. The authors concluded from their results that the metabolism of tert-butyl methyl ether to formaldehyde is already saturated at low concentrations and formaldehyde is rapidly detoxified. Accordingly, they attribute little or no importance to the genotoxic metabolite for the carcinogenic effects of tert-butyl methyl ether (Casanova and Heck 1997). The early saturation of the transformation of tert-butyl methyl ether to formaldehyde proposed by these authors is not confirmed by the available in vivo experiments. The formation of tert-butyl alcohol and thus also of formaldehyde which, however, was not determined was directly proportional to the amount of tert-butyl methyl ether administered for wide concentration and dose ranges (see Section 3). The low formaldehyde body burden is therefore more likely the result of the high detoxification capacity and the various detoxifying physiological routes of formaldehyde metabolism than of saturation of the metabolism of tert-butyl methyl ether. Quantitative data for the formaldehyde body burden in vivo after exposure to tert-butyl methyl ether are, however, not available. That the level of exposure to this genotoxic metabolite is low can also be concluded from the in vivo genotoxicity studies with tert-butyl methyl ether: up to toxic concentration and dose ranges the results were negative (see Section 5.6). The conceivable non-genotoxic mechanisms for the carcinogenicity of tert-butyl methyl ether are discussed below. 2.4 Kidney tumours (male rat) The carcinogenic effects on the kidneys of tert-butyl methyl ether may be the result of the nephrotoxicity of the substance (see above) and the resulting increase in proliferation in the target tissues. Accordingly, in male rats exposed for 28 days to concentrations of 3000 or 8000 ml/m 3 (Bird et al. 1997) or for 10 days to concentrations up to 3000 ml/m 3 (Borghoff et al. 1996, Prescott-Mathews et al. 1997), increased proliferation in the proximal tubules was detected via the incorporation of bromodeoxyuridine. Exposure to tert-butyl methyl ether without nephrotoxic effects should therefore not lead to tumours. 2.5 Leydig cell tumours (male rat) The mechanism of formation of the Leydig cell tumours, which were observed both in F344 rats after inhalation and also in Sprague-Dawley rats after oral administration of tert-butyl methyl ether, is unclear. This tumour type can occur spontaneously in F344 rats with an incidence of up to 100 % (Boorman et al. 1990, Haseman et al. 1990, 1998), while the spontaneous incidence in Sprague-Dawley rats is only about 10 % (Belpoggi et al. 1995). In rodents the Leydig cell tumours induced by non-genotoxic substances are often the result of disturbed hormonal regulation, which eventually leads to the increased release of luteinizing hormone, e.g. as a result of androgen antagonism at the androgen receptor or decreased testosterone, dihydrotestosterone and oestradiol levels. Human Leydig cells usually react much less sensitively than those of rodents to the proliferation

6 124 tert-butyl methyl ether Volume 17 stimulus caused by luteinizing hormone. Also direct stimulation of the Leydig cells by oestrogen agonists, without the involvement of luteinizing hormone, has been observed (Cook et al. 1999). No evidence of this could be found, however, in 9-week-old male Sprague-Dawley rats given gavage doses of tert-butyl methyl ether of up to 1500 mg/kg body weight and day for 15 or 28 days. Only in the high dose groups and after exposure for 15 days were the levels of testosterone in serum and in the interstitial fluid of the testes, and the level of prolactin in serum slightly decreased. After treatment for 28 days the levels of luteinizing hormone and dihydrotestosterone in serum were decreased. Triiodothyronine levels were significantly decreased in the two high dose groups. It is questionable whether these marginal changes, which are only induced by high doses, are causally involved in the formation of Leydig cell tumours (Williams et al. 2000). 2.6 Hepatocellular tumours (female mouse) The mechanism of formation of the hepatocellular adenomas which were found in female CD-1 mice after exposure to tert-butyl methyl ether concentrations of 8000 ml/m 3 is unclear. Studies of the mechanism of action revealed a substance-related stimulation of the proliferation of liver cells by high concentrations. In a 28-day study, the increase in the incidence of hepatocellular proliferation in female mice after exposure to 8000 ml/m 3 for 5 days was statistically significant. A slight, but not significant increase was found also in the female animals of the 3000 ml/m 3 group and in the male animals of the 8000 ml/m 3 group. The increase in proliferation was transient and no longer detectable on the last day of exposure (Bird et al. 1997). Other authors observed a slight, but not significant increase in proliferation in the livers of B6C3F 1 mice after exposure to tertbutyl methyl ether concentrations of 8000 ml/m 3 for 3 days; it was not accompanied by cytotoxic processes. After exposure of the animals for 21 days, the incidence of proliferation was, however, significantly decreased (Moser et al. 1996a). It may be assumed that tert-butyl methyl ether concentrations which do not increase the proliferation of hepatocytes, also cannot have tumorigenic effects. First effects on the liver were observed in the mouse after exposure to concentrations of 3000 ml/m 3. Tumour-promoting activity of tert-butyl methyl ether in the liver of B6C3F 1 mice was not detectable, however, even after concentrations of 8000 ml/m 3 (see Section 5.7.1; Moser et al. 1996b). To what extent also hormonal processes (see above) are involved in the carcinogenic effects on the liver of tert-butyl methyl ether is unclear. 2.7 Lymphomas and leukaemia (female rat) In female Sprague-Dawley rats given long-term oral doses of tert-butyl methyl ether, a statistically significant, dose-dependent increase in the incidence of lymphomas and leukaemia was found (Belpoggi et al. 1995, 1997; see also Section 5.7 and Table 4). The pathogenesis is unclear. Also the incidence of dysplastic proliferation in lymphoreticular tissue, which can be regarded as a preneoplastic change, was significantly increased, although not in a dose-dependent manner. On the other hand, in the study of the carcino-

7 Volume 17 tert-butyl methyl ether 125 genicity of inhaled tert-butyl methyl ether in F344 rats no effects were observed in the lymphoreticular system, and there were no such tumours (Bird et al. 1997). The authors attributed the lymphomas/leukaemia to the metabolite formaldehyde, as in a 2-year study in the same laboratory in which formaldehyde was administered to male and female Sprague-Dawley rats in the drinking-water the increase in the incidence of leukaemia was dose-dependent and statistically significant. Survival and body weight gains were not impaired in any group, and no other non-neoplastic adverse effects were reported (Soffritti et al. 1989). From the formaldehyde concentrations in drinking-water, an assumed average water consumption of 15 ml/day and an average body weight of 300 g, formaldehyde doses of 0.5 to 75 mg/kg body weight and day can be calculated. The increase in the incidence of leukaemia was statistically significant from doses of 5 mg/kg body weight and day. In a 2-year drinking-water study with paraformaldehyde administered in similar doses to Wistar rats, however, no evidence of carcinogenic effects of formaldehyde was found (Til et al. 1989); a causal involvement of formaldehyde in the development of tert-butyl methyl ether-related leukaemia and lymphomas therefore seems questionable. 3 Toxicokinetics and Metabolism The toxicokinetics of tert-butyl methyl ether has been investigated in extensive studies with rats, in vitro studies and a few studies with volunteers, monkeys and mice. To date, no important differences in the way various species react to the substance have become known; the toxicokinetic properties of tert-butyl methyl ether can therefore be adequately described for man on the basis of the extensive database for rats. A summary is given below based on the data of Clary (1997), ECETOC (1997), Hutcheon et al. (1996) and WHO (1998). 3.1 Absorption and distribution The pulmonary retention of tert-butyl methyl ether in man during exposure to concentrations of 5, 25 or 50 ml/m 3 with light physical activity (2 hours, 50 watts) was about 40 %. Absorption from the respiratory tract was rapid; increased tert-butyl methyl ether concentrations were detected in blood only a few minutes after the beginning of exposure. These concentrations in blood correlated linearly with the exposure concentration, and reached a maximum of 110, 550 and 1100 µg/l blood for the 3 exposure concentrations. Equilibrium was reached within the 2-hour exposure period only at the low concentration of 5 ml/m 3 (Johanson et al. 1995, Nihlén et al. 1998a). Another study with volunteers, which is described in the above reviews, yielded similar results. The volunteers were exposed at rest to 25 or 75 ml/m 3 for 4 hours. The maximum tert-butyl methyl ether concentrations in blood were found to be 970 and 2556 µg/l. The plasma elimination

8 126 tert-butyl methyl ether Volume 17 half-time for the slow phase was 5 hours. The elimination of tert-butyl alcohol from the blood was found to involve a single phase with a half-time of 11.9 hours. In Finnish tanker drivers exposed for about 30 minutes to tert-butyl methyl ether while filling tanks with petrol, a good correlation (r = ) was found between the individual levels of exposure ( mg/m 3 ) and the levels of tert-butyl methyl ether in the blood ( nmol/l corresponding to µg/l), which were determined about 20 minutes after the end of exposure. Correlations with the tert-butyl alcohol levels in blood or urine were not found (Vainiotalo et al. 1998). The pulmonary retention in rats has, to date, not been determined experimentally, but can be estimated on the basis of physico-chemical data and data for structurally related compounds. For concentrations up to 100 ml/m 3 it has been estimated to be 60 %, up to 1000 ml/m 3 to be 30 % and above 1000 ml/m 3 to be 15 %. Equilibrium was reached after exposure for about 3 hours. Absorption of the substance from the gastrointestinal tract takes place rapidly and completely. Peak concentrations in plasma were reached about one hour after ingestion. When tert-butyl methyl ether doses of 40 or 400 mg/kg body weight were applied to the skin of rats in a closed chamber for 6 hours, 20 % and 39 % of the amount absorbed from the same oral doses was taken up. The peak concentrations in plasma were about 20 times lower than after oral intake of the same doses, and were reached about 2 hours after the beginning of exposure. After non-occlusive application of the substance, lower absorption is to be expected because of the high vapour pressure of tert-butyl methyl ether. From experiments with animals and from the distribution coefficients between various tissues and blood or air determined in vitro for tert-butyl methyl ether, it was deduced that the substance is distributed evenly in the body. The highest concentrations were determined in adipose tissue, as expected for this highly lipophilic substance; at equilibrium they were about 10 times the concentrations in blood. Because of the rapid elimination and metabolism of the substance, accumulation is not to be expected after repeated exposure. 3.2 Metabolism and excretion tert-butyl methyl ether is transformed by cytochrome P450-dependent monooxygenases by means of O-demethylation to tert-butyl alcohol and formaldehyde. Direct evidence of formaldehyde formation has to date been found only in vitro. In vivo, methanol, which is probably formed from formaldehyde via alcohol dehydrogenase, was detected. The main P450 isoforms involved were identified in earlier studies as cytochrome P450 2E1 and P450 2B1. According to more recent in vitro studies with liver microsomes from rats treated with various cytochrome P450 inducers, isoform 2B1 plays the main role, and isoform 2E1 only a minor one (Turini et al. 1998). Other authors were able to confirm that cytochrome P450 2E1 plays only a limited role, if any at all, in the metabolism of tert-butyl methyl ether and demonstrate the involvement of cytochrome P450 2A6. Microsomes of Sf9 cells which expressed human cytochrome P450 2A6 transformed about 20 times more tert-butyl methyl ether than did microsomes of the same cells which expressed human cytochrome P450 2E1 (Hong et al. 1997b). Studies with microsomes

9 Volume 17 tert-butyl methyl ether 127 from human liver biopsies also yielded good evidence that cytochrome P450 2A6 is the main enzyme involved in the metabolism of tert-butyl methyl ether (Hong et al. 1999a). Liver microsomes of cytochrome P450 2E1 knock-out mice metabolized tert-butyl methyl ether to the same extent as did the corresponding microsomes from wild-type mice (Hong et al. 1999b). The activity of microsomes from cells of the olfactory nasal epithelium of rats for metabolism of tert-butyl methyl ether was found to be 46 times that of hepatic microsomes. Corresponding preparations from the kidney and lung were inactive (Hong et al. 1997a). Evidence was found that repeated administration of tert-butyl methyl ether causes the induction of cytochrome P450 enzymes and thus promotes its own metabolism. The extent of induction was, however, judged to be too small to affect the toxicological profile of the substance. In inhalation studies, the metabolite tert-butyl alcohol was detected in blood 10 minutes after the beginning of exposure, and equilibrium was reached within the 6-hour exposure period. The amount of tert-butyl alcohol formed was directly proportional to the amount of administered tert-butyl methyl ether over a wide concentration and dose range. Saturation of metabolism occurred in rats at concentrations of about 8000 ml/m 3 or oral doses of 400 mg/kg body weight. tert-butyl alcohol is distributed rapidly in the organism, mainly in aqueous compartments. Accumulation in adipose tissue does not occur. In rodents, the elimination half-time of tert-butyl alcohol from the plasma is at least 4 hours. tert-butyl alcohol is not transformed by alcohol dehydrogenase. Conjugation with sulfuric acid or glucuronic acid, oxidative demethylation by cytochrome P450 enzymes to form acetone and formaldehyde, or hydroxylation of a methyl group to form 2-methyl-1,2-propanediol with further oxidation to 2-hydroxyisobutyric acid, for example, take place. The metabolites and initial substance are eliminated via the kidneys or the lungs; quantitative data are not available (see MAK documentation for tert-butyl alcohol, Greim 1999). The formaldehyde formed is metabolized rapidly to methanol, formic acid or CO 2. Formaldehyde and formic acid can also enter the physiological C 1 pool. Comparison of the rate of transformation of tert-butyl methyl ether to formaldehyde with the rate of detoxification of formaldehyde showed that an increase in the intracellular formaldehyde concentration is not to be expected even when large amounts of tert-butyl methyl ether are taken up (ECETOC 1997). The excretion of tert-butyl methyl ether and its metabolites after single exposures is more or less complete within 24 hours. In volunteers exposed for 2 hours to 5, 25 or 50 ml/m 3, three-phase (Johanson et al. 1995) or four-phase elimination of tert-butyl methyl ether from the blood (Nihlén et al. 1998a) with half-times of about 10 and 90 minutes and 20 hours, or 1, 10 and 90 minutes and 19 hours was observed. In volunteers exposed for 4 hours to tert-butyl methyl ether concentrations of 4 or 40 ml/m 3,other authors determined total elimination half-times from plasma of about 1.8 and 2.6 hours (Amberg et al. 1999). Elimination with the urine was found to involve two phases with half-times of 0.3 and 3 hours. The elimination half-lives of tert-butyl alcohol in urine and blood were given as 8.2 and 10 hours, respectively (Johanson et al. 1995, Nihlén et al. 1998a) and 11 and 6 hours (Amberg et al. 1999). Less than 1 % of the absorbed amount of tert-butyl methyl ether was eliminated unchanged or in the form of tert-butyl alcohol

10 128 tert-butyl methyl ether Volume 17 with the urine (Amberg et al. 1999, Johanson et al. 1995, Nihlén et al. 1998a). About 20 % was exhaled unchanged (Johanson et al. 1995, Nihlén et al. 1998a). In a later study from this working group, the known tert-butyl alcohol metabolites α-hydroxyisobutyric acid and 2-methyl-1,2-propanediol (see MAK documentation for tert-butyl alcohol, Greim 1999) could also be identified as additional elimination products (3 11 % and 1 % of the amount of tert-butyl methyl ether absorbed) after exposure of 4 volunteers to 50 ml/m 3 for 2 hours. All other toxicokinetic parameters were in agreement with the earlier results (Nihlén et al. 1999). In another study with volunteers which is described in the above-mentioned reviews, persons were exposed to 25 or 75 ml/m 3 for 4 hours at rest; a half-time of 5 hours was found for the slow elimination phase of tert-butyl methyl ether from the plasma. The elimination of tert-butyl alcohol from the blood was found to involve a single phase with a half-time of 11.9 hours. 58 % of the amount of tert-butyl methyl ether taken up was exhaled unchanged. 1.4 % was excreted unchanged with the urine and 1.2 % as tert-butyl alcohol. In rats, an elimination half-time from plasma of less than one hour was determined for tert-butyl methyl ether (Amberg et al. 1999). After administration of the radioactively labelled substance, most of the absorbed radioactivity (70 90 %) was exhaled, mainly as tert-butyl methyl ether, and in small amounts also as tert-butyl alcohol and CO 2. About 5 % was found in the urine (mainly tert-butyl alcohol metabolites) and about 1 % in the faeces. In the 48-hour urine of F344 rats exposed via inhalation to tert-butyl methyl ether concentrations of 2000 ml/m 3 for 6 hours the main metabolites found were α-hydroxyisobutyric acid and 2-methyl-1,2-propanediol and an incompletely identified tert-butyl alcohol conjugate (probably a sulfate conjugate). In addition, tert-butyl alcohol and another conjugate (probably a glucuronide conjugate) and small amounts of acetone were identified (Bernauer et al. 1998). A comparison of the biotransformation and excretion of inhaled tert-butyl methyl ether by man and the rat did not reveal any great differences. tert-butyl methyl ether and its metabolites are rapidly metabolized and excreted by both species. Between 35 % and 69 % of the absorbed amount of tert-butyl methyl ether was found after the end of exposure in the 48-hour urine (Amberg et al. 1999). 4 Effects in Man In 2 studies with volunteers exposed for one hour to tert-butyl methyl ether concentrations of 5 and 6 mg/m 3 (1.4 and 1.7 ml/m 3 ), with the exception of detection of the odour no exposure-related symptoms were reported. The volunteers answered questions about their subjective symptoms in a questionnaire, and objective parameters for irritation of the eyes (tear film, reddening, inflammation parameters in tear fluid) and nose (inflammation parameters in nasal lavage fluid) were investigated. One of these studies also investigated adverse effects on behaviour; there were no abnormal findings (ECETOC 1997, WHO 1998).

11 Volume 17 tert-butyl methyl ether 129 There are also 2 studies with volunteers exposed to workplace-relevant concentrations (5, 25 and 50 ml/m 3 for 2 hours (Johanson et al. 1995, Nihlén et al. 1998b) and 25 or 75 ml/m 3 for one hour or 3 hours (ECETOC 1997, WHO 1998)). In the study in which 10 male volunteers were exposed to concentrations up to 50 ml/m 3 during light physical exercise, there was no increase in the prevalence of reported symptoms. Also objective parameters for eye irritation (blinking frequency, state of the conjunctival epithelium, reddening of the eyes, tear film) and the inflammation parameters in the nasal lavage fluid were unchanged. The nasal peak expiratory flow was significantly decreased, but not concentration-dependent. All the volunteers registered the smell of tert-butyl methyl ether when they entered the exposure chamber, but during the course of exposure perception of the odour decreased. The 13 male volunteers in the other study reported only very slight subjective symptoms: mainly a congested feeling in the head and slight irritation of the mucosa. The index of symptoms differed significantly from the control index only for persons exposed for 3 hours to a concentration of 75 ml/m 3. Reaction time and body sway while standing with open eyes and closed eyes were not affected by the treatment. The controlled studies described were carried out in accordance with modern methodological requirements and the results are highly reliable. Therefore it may be concluded that the irritation threshold for tert-butyl methyl ether lies between 50 ml/m 3 and 75 ml/m 3 (ECETOC 1997, WHO 1998). Since the prescribed introduction in 1992 in most parts of the USA of oxygenated motor fuel with a tert-butyl methyl ether content of up to 15 %, several populationrelated studies of potentially adverse effects on health as a result of exposure to the new type of petrol have been carried out. The data for subjective symptoms from persons frequently exposed to petrol at work or privately were compared with data from persons exposed only rarely or not at all, or such data from areas with petrol containing tert-butyl methyl ether were compared with that from areas where the petrol did not contain this substance. No tangible relationship could be found between exposure to tert-butyl methyl ether and the symptoms reported. The level of exposure was evaluated either by determining the concentration in the air or in blood, and in all studies was found to be low (a maximum of 22 mg/m 3 and of 37 µg/l blood). This information is not of use, however, for evaluating a MAK value; for more details reference is made to Balter (1997), ECETOC (1997) and WHO (1998). tert-butyl methyl ether is also used therapeutically to break down gall stones. With a catheter the substance is repeatedly instilled directly into the gall bladder and removed by suction. In about 30 % of the patients treated in this way the side-effects were nausea, vomiting and slight dizziness. All symptoms regressed rapidly. After the treatment tertbutyl methyl ether was detectable in the blood and exhaled air. The accidental injection of tert-butyl methyl ether into a blood vessel led to haemolysis, a leakage of 21 ml into the small intestine to kidney failure after the substance had been absorbed into the circulatory system (ECETOC 1997). The incidence of apoptosis was determined in the isolated peripheral lymphocytes of 60 persons who had consumed for 5 8 years tap water which was contaminated with tertbutyl methyl ether concentrations up to 76 µg/l and benzene concentrations up to 14 µg/l. The incidence was about twice that in the controls. The effect was attributed to a cell

12 130 tert-butyl methyl ether Volume 17 cycle block at the S-G 2 /M boundaries by tert-butyl methyl ether and benzene (Vojdani et al. 1997). A causal relationship with the exposure to tert-butyl methyl ether cannot be deduced from these data because of the simultaneous exposure to benzene and the possibility of other interfering factors. There are no reports of other effects in man. 5 Animal Experiments and in vitro Studies 5.1 Acute toxicity The acute toxicity of tert-butyl methyl ether is low for all routes of absorption (see Table 1). The symptoms after inhalation exposure were typical of volatile, low-molecular organic solvents: mainly irritation of the mucous membranes, irregular breathing, ataxia, and central nervous symptoms. The surviving animals recovered within a few hours of the end of exposure. After oral administration, diarrhoea, slight central nervous depression, laboured breathing and tremor were observed, while after dermal application only local irritation was seen. Detailed reviews have been published (ECB 1995, ECETOC 1997, WHO 1998). Table 1. Acute toxicity of tert-butyl methyl ether according to ECB (1995), ECETOC (1997) and WHO (1998) Species LC 50 [mg/m 3 ] (exposure duration) oral LD 50 [mg/kg body weight] dermal LD 50 [mg/kg body weight] rat (4 hours) > 6800 mouse (5 minutes) (10 minutes) (15 minutes) rabbit > Exposure of F344 rats to tert-butyl methyl ether concentrations of 800, 4000 or 8000 ml/m 3 (2880, or mg/m 3 ) for 6 hours led in the two high concentration groups to transient symptoms of central nervous depression (laboured breathing, ataxia, unsteady gait, decreased muscle tone, decreased gripping strength in the hind paws, decreased performance in the treadmill); 6 and 24 hours after the end of exposure the results of the various tests no longer differed from those found for the control group. Concentrations of 800 ml/m 3 did not produce neurotoxic effects (Daughtrey et al. 1997).

13 Volume 17 tert-butyl methyl ether Subacute, subchronic and chronic toxicity Inhalation The subacute toxicity of tert-butyl methyl ether after daily exposure for several hours (for 5 to 28 days) was found to be low in numerous studies with rats and mice and in one study with monkeys. In the rat the lowest NOEL was 300 ml/m 3, in the mouse 400 ml/m 3. At the next higher concentrations of at least 1000 ml/m 3, symptoms of central nervous depression, irritation of the eyes or increased liver and kidney weights were found. In rhesus monkeys exposed to 5000 ml/m 3 for 6 hours a day on 5 days, central nervous effects were not observed (ECETOC 1997, WHO 1998). Because of the lesser importance of inhalation studies with only short-term exposure for the evaluation of a MAK value, the studies are not discussed here in any more detail. In an unpublished 13-week study from 1980, groups of 10 male and 10 female Sprague-Dawley rats were exposed to tert-butyl methyl ether concentrations of 250, 500 or 1000 ml/m 3 for 6 hours a day, on 5 days a week. The narcotic effects of the substance increased with the concentration. Clinico-chemical parameters in blood and urine were unchanged. In the male animals of the high concentration group, the haemoglobin level in the erythrocytes was increased. With the exception of slightly reduced relative and absolute lung weights in the female animals of the high concentration group, gross pathological and histological examination of the organs did not reveal any pathological findings (ECETOC 1997, WHO 1998). As a result of the central nervous depression detected even at the lowest concentration, 250 ml/m 3 is to be seen as the LOEL (lowest observed effect level), but adverse effects on organs or tissue did not occur at concentrations below 500 ml/m 3. In another 13-week study with F344 rats which was carried out in 1989 but published much more recently (Lington et al. 1997), central nervous effects could not be confirmed in this low concentration range. The animals (15 of each sex per group) were exposed to tert-butyl methyl ether concentrations of 800, 4000 or 8000 ml/m 3 for 6 hours a day on 5 days a week. In the high concentration group, body weights were significantly decreased from the first week of the experiment to the end of the study, in the 4000 ml/m 3 group only at some of the observation times during the study. In the high concentration group, during the first 4 weeks ataxia was observed after the end of the daily exposure. Later ataxia and hypoactivity were observed also during exposure. At 4000 ml/m 3 the animals were hypoactive during exposure. Some haematological parameters (see below) were changed slightly in the two high concentration groups (deviations from the control value by about 5 %); the male animals were found to be more sensitive than the females. In male rats the haemoglobin levels were decreased, and the haematocrit and number of reticulocytes and leukocytes increased. In the female animals exposed to concentrations of 8000 ml/m 3, the haematocrit and the polymorphonuclear neutrophil count were increased at the end of the study. In serum of animals exposed to 8000 ml/m 3, some parameters were slightly changed, but this was not considered to be of toxicological relevance. The significant increase in the level of cortisone was interpreted as a stress reaction. The levels of aldosterone and adrenocorticotrophic hormone were, however, unchanged.

14 132 tert-butyl methyl ether Volume 17 Autopsy did not reveal pathological lesions. The relative liver and kidney weights were significantly increased in a concentration-dependent manner in the male animals from 800 ml/m 3, and in the females from 4000 ml/m 3. The relative weights of the adrenal glands were significantly increased in both sexes from 4000 ml/m 3. The histological examination did not reveal any pathological findings in the female animals. In the lymph nodes of the male animals of the 8000 ml/m 3 group, lymphoid hyperplasia was found significantly more often (in 11/15 animals) than in the control group (0/14), and moderate haemosiderosis was found in the spleen of all animals. In the 15 control animals, haemosiderosis was found only 11 times and was less pronounced. In the cells of the proximal renal tubules, the number of hyaline droplets was increased; there were signs of this also in the middle concentration group (Lington et al. 1997). In the study described above, an additional 10 animals were included per group to test for neurotoxic effects (a series of behavioural tests were carried out, and motor activity and the histology of the nervous system were investigated). From concentrations of 4000 ml/m 3 tert-butyl methyl ether induced acute but reversible central nervous depression. Permanent effects or effects of increasing intensity did not occur after repeated exposure. Likewise, histological examination of the brain and peripheral nervous system did not reveal any pathological changes (Daughtrey et al. 1997). From these studies a NOAEL (no observed adverse effect level) of 800 ml/m 3 was deduced for rats exposed repeatedly to tert-butyl methyl ether. The increased liver and kidney weights at this concentration were not regarded as adverse, as the histological examination and clinico-chemical parameters yielded no evidence of organ damage. The doses taken up under the above-mentioned exposure conditions and with a concentration of 800 ml/m 3 were estimated to be 230 and 245 mg/kg body weight and day for male and female animals, respectively (ECETOC 1997) Ingestion The toxicity of tert-butyl methyl ether after repeated oral administration has been investigated only in Sprague-Dawley rats exposed for 14, 28 and 90 days. Adverse effects on the central nervous system were observed directly after administration of the substance after as little as 90 mg/kg body weight; these effects, however, regressed rapidly. They may have been the result of bolus administration. With higher doses, the observed effects included irritation of the gastrointestinal tract and changes in the liver, kidney and lung weights. Histological examination revealed changes only in the kidneys of the male animals with the accumulation of hyaline droplets in the epithelial cells of the proximal tubules and an increase in age-related chronic nephropathy. The results are shown in Table Local effects on skin and mucous membranes Occlusive application of undiluted tert-butyl methyl ether (0.5 ml) to the intact or scarified dorsal skin of rabbits for 24 hours led to moderate erythema and oedema; the

15 Volume 17 tert-butyl methyl ether 133 substance was therefore regarded as slightly irritative. The effects were somewhat more pronounced on the scarified skin. In another experiment with rabbits using the same methods, a tert-butyl methyl ether product of 96.2 % purity was found to be not irritative, while another product of 99.1 % was also barely irritative. In a skin irritation test carried out according to OECD guideline 404, tert-butyl methyl ether was described as moderately irritative (ECB 1995, ECETOC 1997, WHO 1998). In the rabbit eye, undiluted tert-butyl methyl ether causes moderate irritation. In various studies, some carried out according to the OECD guideline, after instillation of undiluted tert-butyl methyl ether into the conjunctival sac, erythema, thickening, chemosis and hypersecretion were observed. All symptoms regressed within 7 days (ECB 1995, ECETOC 1997, WHO 1998). Irritation of the eye was observed also after short-term inhalation exposure of the rat and mouse to concentrations of 1000 ml/m 3 andabove(seesection5.2.1). Table 2. Toxicity of tert-butyl methyl ether after repeated oral administration to Sprague-Dawley rats (according to ECETOC 1997, WHO 1998) Number of animals; dose; duration Effects 10 per dose and sex; 357, 714, 1071, 1428 mg/kg body weight and day in corn oil; 14 days 10 per dose and sex; 90, 440, 1750 mg/kg body weight and day in water; 28 days 10 per dose and sex; 100, 300, 900, 1200 mg/kg body weight and day (undiluted); 90 day from 357 mg/kg: diarrhoea, body weights decreased, irritation of the upper gastrointestinal tract, occasional clinical symptoms (no other details), only SS: absolute and relative lung weights decreased from 1071 mg/kg: transient narcosis after administration, cholesterol level in serum increased, only á: relative kidney weights increased, haematocrit and haemoglobin increased, monocytes decreased, aspartate aminotransferase and lactate dehydrogenase decreased 1428 mg/kg: blood urea nitrogen decreased, only SS: creatinine decreased, only á: hyaline droplets in epithelial cells of the proximal renal tubules increased (autopsy did not yield pathological findings; histological examination only carried out for the kidneys) from 90 mg/kg: salivation, hypoactivity and ataxia immediately after administration (rapidly reversible), only SS: relative kidney weights increased (not at 440 mg/kg body weight) from 440 mg/kg: only á: relative kidney weights increased, hyaline droplets in epithelial cells of the proximal renal tubules 1750 mg/kg: cholesterol increased, relative liver weights increased, histological examination revealed irritation of the forestomach, only á: relative adrenal gland weights increased from 100 mg/kg: salivation, hypoactivity and ataxia immediately after administration (rapidly reversible), diarrhoea, cholesterol increased, blood urea nitrogen decreased, only á: number of hyaline droplets in the kidney increased from 300 mg/kg: slight variation in haematological parameters, only SS: relative kidney weights increased (not dose-dependent) from 900 mg/kg: only á: absolute and relative kidney weights and relative liver weights increased, only SS: relative thymus and heart weights increased 1200 mg/kg: transient narcosis, only SS: body weights decreased, adrenal gland weights increased, only á: absolute and relative lung weights increased, agerelated chronic nephropathy more severe

16 134 tert-butyl methyl ether Volume Allergenic effects In a Magnusson and Kligman sensitization test with guinea pigs, tert-butyl methyl ether did not produce skin reactions. Epicutaneous induction and provocation treatment were carried out, however, with only 1 % tert-butyl methyl ether (no other details) (ECETOC 1997, WHO 1998). Also an experiment with intradermal induction only (one injection of 0.5 ml, followed by 9 injections of 0.1 ml of a 1 % aqueous solution over 3 weeks) did not produce sensitization after intradermal provocation 2 weeks later with 0.05 ml of a 0.01 % aqueous tert-butyl methyl ether solution (ECB 1995, ECETOC 1997, WHO 1998). In view of the only moderately irritative effects of tert-butyl methyl ether, the concentrations used in both experiments seem to be too low to be able to evaluate the sensitizing potential of the substance. 5.5 Reproductive and developmental toxicity Fertility In a 1-generation study with Sprague-Dawley rats and exposure concentrations of 250, 1000 or 2500 ml/m 3 (6 hours a day, 5 days a week), up to the highest concentration no effects were seen on the reproductive parameters when the same parent animals were mated twice in succession. Clinical symptoms occurred neither in the parent animals nor the offspring, and gross pathological examination of the organs and histological examination of the gonads at the end of the study revealed no abnormalities (ECETOC 1997, WHO 1998). Also in a 2-generation study with Sprague-Dawley rats and exposure concentrations of 400, 3000 or 8000 ml/m 3 (6 hours a day, 5 days a week), the reproductive success of the two generations was not affected. The two high concentrations led in the parent animals to hypoactivity, loss of the startle reflex and ataxia. 10 weeks exposure before mating at the highest concentration led to persistent reductions in body weight gains, at 3000 ml/m 3 to only transient reductions. In the adult animals of the F 1 generation from the middle and high concentration groups, the relative liver weights were increased; the histological examination, however, did not reveal any pathological findings. In the offspring of both parent generations of the two higher concentration groups, body weights were reduced from day 14 of life, in some cases also from day 7. In the offspring of the 8000 ml/m 3 group F 1 generation, postnatal survival was reduced on day 4. According to this study, 400 ml/m 3 was the NOEL for parental and postnatal toxicity. The NOEL for adverse effects on reproduction was over 8000 ml/m 3 (Bevan et al. 1997a) Developmental toxicity Potential adverse effects of tert-butyl methyl ether on reproduction were investigated in the species mouse, rat and rabbit (Bevan et al. 1997b, ECETOC 1997, WHO 1998). Foetotoxic and teratogenic effects were observed only in CD-1 mice from concentrations

17 Volume 17 tert-butyl methyl ether 135 of 4000 ml/m 3, which also caused marked maternal toxicity. The lowest concentration of 1000 ml/m 3 had no effects on the dams or offspring in this study (Bevan et al. 1997b), in another study 2500 ml/m 3 had no effects (ECETOC 1997, WHO 1998). In Sprague- Dawley rats, the highest tested concentration of 2500 ml/m 3 was neither maternally toxic nor toxic for the offspring, with the exception of irritative effects (ECETOC 1997, WHO 1998). For New Zealand White rabbits a NOEL for maternal toxicity of 1000 ml/m 3 was found; adverse effects on development did not occur even at the highest concentration of 8000 ml/m 3. Other data from these studies can be found in Table 3. Table 3. Studies of the reproductive toxicity of tert-butyl methyl ether Species; number/group; concentration (ml/m 3 ); exposure conditions Effects References mouse (CD-1); 25/group; 250, 1000, 2500; days 6 15 of gestation, 6 hours/day mouse (CD-1); 30/group; 1000, 4000, 8000; days 6 15 of gestation, 6 hours/day rat (Sprague-Dawley); 25/group; 250, 1000, 2500; days 6 15 of gestation, 6 hours/day rabbit (New Zealand White); 15/group; 1000, 4000, 8000; days 6 18 of gestation, 6 hours/day from 250 ml/m 3 : slight lacrimation in the dams, no adverse effects on reproduction acc. ECETOC 1997, WHO 1998 from 4000 ml/m 3 : irritation and central Bevan et al. nervous depression in the dams, 1997b foetal body weight decreased, skeletal variations (in particular delayed ossification) increased in a concentration-dependent manner 8000 ml/m 3 : maternal body weight gains and food consumption decreased number of resorptions and dead foetuses increased, ratio á/ss decreased, incidence of cleft palate and therefore the total incidences for external and visceral malformations increased from 250 ml/m 3 : slight lacrimation in the dams, no adverse effects on reproduction from 4000 ml/m 3 : maternal body weight gains and food consumption decreased 8000 ml/m 3 : relative liver weights decreased, irritation and central nervous depression in the dams, no adverse effects on reproduction acc. ECETOC 1997, WHO 1998 Bevan et al. 1997b