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Basic toxicokinetics

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basic toxicokinetics in vivo
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Investigators report, produced as a part of Health Effects Institue Research Report 102; well-conducted and documented study, adequate for assessment.

Data source

Reference Type:

Materials and methods

Objective of study:
Principles of method if other than guideline:
Exposure of male and female rats to two target concentrations of ETBE for 4 hours, wtih subsequent quantification of ETBE and its metabolites in urine and blood.
GLP compliance:
not specified

Test material

Constituent 1
Reference substance name:
EC Number:
EC Name:
Cas Number:
tert-butyl ethyl ether
Details on test material:
- Name of test material (as cited in study report): ETBE
- Analytical purity: > 99%
- Supplier: Tokyo Kasei Kogyo (Tokyo, Japan)

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 12 weeks
- Weight at study initiation: 210-240 g (males); 190-220 g (females)
- Fasting period before study:
- Housing: during the exposure: separate Macrolon cages; after the exposure metabolic cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 3 days

- Temperature (°C): 21
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Details on exposure:

- Exposure apparatus: dynamic exposure chamber, total volume 8 m3
- Method of holding animals in test chamber: caged
- Rate of air: 28 m3/hour
- Temperature: 22 °C
- humidity: 50-60%
Duration and frequency of treatment / exposure:
4 hours
Doses / concentrations
Doses / Concentrations:
4 and 40 ppm
No. of animals per sex per dose / concentration:
Control animals:
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled : urine, blood
- Time and frequency of sampling: urine was collected at 4 °C for 72 h at 6-h intervals. Blood samples were taken at the end of the exposure.
- Other:

- Tissues and body fluids sampled: urine, blood
- Method type(s) for identification: GC-MS
Statistical analysis were performed using a Student t test. To determine possible sex differences, all data from the male and female animals were compared using a t test. P values of < 0.05 were considered significant. Half-times were calculated using exponential regression in Excel. The curve-fitting function of the program was used, and curves were stripped based on correlation coefficients. All correlation values (r2) > 0.95 were considered for separation.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The concentrations of ETBE in rat blood immediately after the end of exposure to 4 and 40 ppm ETBE were 1.0 ± 0.7 and 5.3 ± 1.2 μM, respectively.
Besides ETBE, tert-butyl alcohol (TBA) was also detected in blood of the rats after the end of exposure at concentrations 5.7 ± 0.8 and 21.7 ± 4.9 μM, respectively.
TBA was also detected in low concentrations in blood samples taken from control rats; however, blood samples taken from rats after exposure to 4 ppm showed statistically significant increases in TBA concentrations at the end of exposure.
Details on distribution in tissues:
No data.
Details on excretion:
After exposure to 4 ppm ETBE, the concentrations of 2-hydroxyisobutyrate and 2-methyl-1,2-propanediol in urine samples from exposed rats were significantly increased between 6 and 18 hours after exposure. The concentrations of TBA were not significantly increased. In the urine samples from exposed rats, the concentrations of 2-hydroxyisobutyrate and 2-methyl-1,2-propanediol were significantly increased (as compared to control levels) in urine samples collected within 24 hours after the end of 40-ppm ETBE inhalation. TBA concentrations were significantly above background only between 6 and 18 hours after the end of the exposure. Based on the amount of 2-hydroxyisobutyrate recovered, this compound also represents the major urinary metabolite of ETBE; in rats; TBA and 2-methyl-1,2-propanediol were minor urinary metabolites of ETBE.
Toxicokinetic parametersopen allclose all
Test no.:
Toxicokinetic parameters:
half-life 1st: 0.4 ± 0.0 (4 ppm)
Test no.:
Toxicokinetic parameters:
half-life 1st: 0.8 ± 0.2 (40 ppm)

Metabolite characterisation studies

Metabolites identified:
Details on metabolites:
TBA, 2-methyl-1,2-propanediol, and 2-hydroxyisobutyrate are ETBE metabolites excreted in urine. This observation suggests that TBA is also a major intermediate metabolite of ETBE formed by CYP-mediated oxidation reaction. With ETBE, oxidation by CYP seems to occur exclusively at the alpha-carbon atom of the ethyl ether moiety since metabolites whose formation could be explained by oxidation of the beta-carbon (such as t-butyl glycol) were not observed. The observation of a preferred oxidation on the alpha-carbon is in line with observations on the biotransformation of ethyl ether where ether cleavage by alpha-carbon oxidation is the major pathway of biotransformation (Axelrod 1956). In ETBE biotransformation, the intermediate hemiacetal also decomposes to give TBA and acetaldehyde. The further fate of the aldehydes formed in ETBE biotransformation has not been investigated; but they are expected to be rapidly metabolized to formate and acetate, respectively.

Any other information on results incl. tables

Formation of the other ETBE metabolites involves further biotransformation of TBA formed in the first step of metabolic pathway. Conjugation of TBA with activated glucuronic acid results in excretion of the glucuronide conjugate. The two other metabolites (2-methyl-1,2-propanediol and 2-hydroxyisobutyrate) present in urine of TBA-treated animals and also in urine of rats exposed to ETBE suggest further oxidative metabolism of the intermediate metabolite TBA. The likely pathway for formation of these metabolites involves oxidation of TBA by CYP to give 2-methyl-1,2-propanediol. TBA is not a substrate for alcohol dehydrogenase, but it is oxidized by rat liver microsomes to formaldehyde and acetone under conditions consistent with an involvement of CYP (Cederbaum and Cohen 1980, Cederbaum et al 1983). CYPmediated oxidation of a C–H bond in one of the methyl groups of TBA results in excretion of the diol metabolite. Further oxidation of 2-methyl-1,2-propanediol results in 2-hydroxyisobutyrate, which is excreted as a major metabolite of TBA, as well as of ETBE.

Applicant's summary and conclusion