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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 TAME for 4 hours, with subsequent quantification of TAME and its metabolites in urine and blood.
GLP compliance:
not specified

Test material

Details on test material:
- Name of test material (as cited in study report): TAME
- Analytical purity: > 97%
- Supplier: Aldrich Chemical Company (Deisenhofen, Germany)

Test animals

Fischer 344
Details on test animals 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:
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 TAME in rat blood immediately after the end of exposure to 4 and 40 ppmTAME were 1.4 ± 0.8 and 9.6 ± 1.4 μM, respectively.
Besides TAME, tert-amyl alcohol (TAA) was also detected in blood of the rats after the end of exposure at concentrations 1.8 ± 0.2 and 8.1 ± 1.5 μM, respectively.
Details on distribution in tissues:
No data.
Details on excretion:
In urine samples of rats collected before TAME exposure and in samples collected from control rats, low concentrations of 2-hydroxy-2-methyl butyrate, 3-hydroxy-3-methyl butyrate, 2-methyl-2,3-butanediol, and TAA were present. No significant increases in concentrations of 2-hydroxy-2-methyl butyrate were observed in rat urine samples collected after the 4-ppm exposure, but concentrations were significantly increased in a few urine samples collected within 18 hours after the end of the 40-ppm exposure. Also, no significant increases above background excretion rates were observed in the concentration of 3-hydroxy-3-methyl butyrate in any urine samples collected from TAME-exposed rats. Due to much lower background levels, the concentrations of 2-methyl-2,3- butanediol were significantly higher than controls in all samples collected between 0 and 42 hours after both 4-ppm and 40-ppm TAME exposures. The glucuronide of 2-methyl-2,3-butanediol was not detectable in urine samples after 4-ppm exposure, but it was detected in all urine samples collected up to 48 hours after the end of the 40-ppm exposure. The glucuronide of TAA was detected in all urine samples collected up to 24 hours after the end of 4-ppm and 40-ppm exposures. The other metabolites quantified after the 40-ppm exposure were rapidly excreted, and their concentrations in urine samples were below the limit of detection after 24 hours; the presence of these minor metabolites was not detected in urine samples collected from rats after exposure to 4 ppm TAME.
Toxicokinetic parametersopen allclose all
Test no.:
Toxicokinetic parameters:
half-life 1st: 1.1 ± 0.5 (4 ppm)
Test no.:
Toxicokinetic parameters:
half-life 1st: 0.6 ± 0.1 (40 ppm)

Metabolite characterisation studies

Metabolites identified:
Details on metabolites:
Based on the amounts of 2-methyl-2,3-butanediol and its glucuronide, these compounds and the TAA glucuronide were the major urinary metabolites of TAME in rat urine.

Any other information on results incl. tables

The structures of TAME metabolites delineated from experiments in rats suggest a complex biotransformation of TAME. The first step in the biotransformation of TAME is oxidation of the methyl group to give an unstable hemiacetal that decomposes to TAA. The low concentrations of TAA recovered in urine of rats exposed to both TAME and to TAA suggest intensive further metabolism of this alcohol by conjugation and by further oxidation, similar as observed for TBA (Bernauer et al 1998, Cederbaum and Cohen 1980, Johanson et al 1995a). Glucuronidation of TAA seems to be a major step in its biotransformation resulting in excretion of a glucuronide. In addition, TAA is oxidized to several diols in reactions that are likely to involve CYP–catalyzed oxidation of a C–H bond. The major pathway of TAA oxidation occurs at the C3 atom to give 2-methyl-2,3-butanediol, which, including the glucuronide formed, is the major product of TAME biotransformation excreted in urine of TAME-exposed rats. Oxidation of the carbon atom in the 3-position to the alcohol moiety seems to be a minor process resulting in 2-methyl-2,4-butanediol as an intermediate, which is further oxidized to 3-hydroxy-3-methyl butyrate. Oxidation of the methyl group next to the alcohol group in TAA also represents a minor pathway that results in intermediate formation of 2-methyl-1,2-butanediol, which may be further oxidized to give 2-hydroxy-2-methyl butyrate.

Applicant's summary and conclusion