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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6-hr exposure, 7-day post exposure collection
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study conducted in compliance with US EPA Guideline OPPTS 870.7485

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000
Reference Type:
publication
Title:
Unnamed
Year:
2006

Materials and methods

Objective of study:
metabolism
toxicokinetics
Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Chemical name (as stated in report): tertiary-butyl acetate
- Synonym (used in test report): Acetic acid, tertiary-Butyl ester; TBAC
- Molecular formula: C6H12O2
- Molecular weight: 116.2
- Physical state: Colorless clear liquid

Radiolabelled test substance:
- Position of radiolabel: Tertiary carbon
- Source: Nycomed Amersham plc, Amersham Place, Little Chalfont, Buckinghamshire, England
- Batch number: 200799.RG1
- Specific activity: 1.41 GBq/mmol, 38 mCi/mmol
- Radiochemical purity: 99.4%
- Storage: <-15 °C in the dark

Non-radiolabelled test substance:
- Source: LyondellBasell Industries
- Batch number: 1031267
- Purity: >95%
- Storage: Ambient temperature, in the dark
Radiolabelling:
yes

Test animals

Species:
rat
Strain:
other: Sprague-Dawley (Crl:CD®(SD)IGS BR)
Sex:
male
Details on test animals and environmental conditions:
Test animals:
-Source: Charles River UK Ltd.
-Age: 6-7 weeks
-Number/sex: 12 males (6/exposure group)
-Quarantine period: at least 5 days
-Weight at study initiation: 210-220 g
-Housing: stainless steel cages with suspended mesh floor prior to dose administration; metabowls after dosing
-Diet: LAD1 supplied by Special Diet Services Ltd., Witham Essex, UK; ad libitum
-Water: Anglian Water mains supply; ad libitum
-Identification method: indelible markings on tails
-Method of animal distribution: not identified

Environmental Conditions:
-Room temperature (°C): 21 ± 2
-Humidity: 55 ± 15%
-Light: 12:12 light:dark cycle
-Ventilation: about 15 air changes/hour

In-Life Dates:
Date of Dose Administration:
Low level: 12 Oct 1999
High level: 21 Oct 1999
Termination of high level group: 28 Oct 1999

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
other: Air (clean, dried, filtered and oil-free)
Details on exposure:
For administration of doses, samples of tertiary butyl acetate were weighed into syringes. Syringes of maximum volume 5 and 50 mL were used for the low (3.68 g) and high (36.6 g) exposure levels, respectively.

The exposure system consisted of a vapor generator and exposure chamber. The vapor generator was designed to produce and maintain an atmosphere containing vapor by evaporation of the test substance from a fritted glass disc with a countercurrent of air. The vapor generator was partly immersed in a water bath (45 °C) as an aid to evaporation. The test-substance was delivered to the generator at a constant flow rate from a polypropylene syringe, with a Teflon® feed line, driven by a syringe pump. All parts of the generator in contact with the test substance, except the syringe and feed line, were made of glass. The nose-only exposure chamber (ADG Developments Ltd, Hitchin, Hertfordshire, England) used for the exposures was of cylindrical form (30 cm internal diameter, 45 cm height) and made of aluminum alloy. The internal surfaces of the chamber had a conformal chemically resistant coating. The chamber had an enclosed volume of approximately 30 liters. The rats were held for exposure in molded polycarbonate restraining tubes, which were attached at evenly spaced ports in the cylindrical section of the chamber, and were designed to allow only the nose to project into the chamber. Each rat was restrained in a forward position by an adjustable foamed plastic stopper, which also provided a seal for the tube. The conditioned test atmosphere entered through a port at the top center of the chamber and passed out through a port at the base section below the level of the rats. The chamber was positioned in a large cabinet equipped with an extract fan exhausting through an absolute filter.

A supply of clean, dried, filtered and oil-free air was connected to the vapor generator and the supply pressure was adjusted to give a flow rate of 15 liters per minute measured at the generator outlet tube. The chamber exhaust was calibrated at the point of attachment to the exposure chamber and was adjusted to produce a slightly negative chamber pressure. During preliminary generation trials, samples were taken from the chamber in order to determine appropriate liquid feed rates to the vaporizer. The concentration of tertiary butyl acetate in the chamber was determined by chemical analysis. Samples were obtained following equilibration and then approximately at hourly intervals. Air samples were withdrawn at 2 liters/minute through an ethyl acetate gas absorption trap (bubbler). The contents of the bubbler were retained for chemical analysis. The volume of the air sample was measured with a wet-type gas meter. No sampling or analysis was carried out during exposure with radiolabelled tertiary butyl acetate.
Duration and frequency of treatment / exposure:
6 hours/1 day
Doses / concentrations
Remarks:
Doses / Concentrations:
100 and 1000 ppm
No. of animals per sex per dose:
6 males/group
Control animals:
no
Details on study design:
The absorption, distribution, metabolism and excretion of 14C-tertiary butyl acetate in the rat was studied after a single 6-hr exposure by the inhalation route at exposure concentrations of 100 ppm (low level) and 1000 ppm (high level). These levels are equivalent to 2% and 20% of the LC50 of this compound in the test species. Each of two groups of 6 animals was exposed to the 14C-tertiary butyl acetate at one of the exposure levels. To determine total radioactivity inhaled, two rats from each group were sacrificed immediately at the end of the exposure period, and their carcasses were analyzed for radioactivity. The remaining four animals were individually placed in metabolism cages designed for the separate collection of urine, feces, and expired air. A series of four expired air traps was attached to each metabolism cage. Expired air traps (in the following sequence) consisted of CO2-cooled cold trap (for volatiles), ethyl digol (to trap volatile radioactivity), 1M KOH (to trap expired carbon dioxide), and 2-ethoxyethanol/ethanolamine (to trap residual volatile radioactivity and carbon dioxide). Expired air, urine and feces were collected for 7 days after the termination of the exposure period. Cage washings were also done at each sample collection period. Expired air traps, excreta and cage washings were analyzed for radioactivity.

Animals were sacrificed at 7 days after the termination of the exposure period. The nasal tissues, trachea, larynx, liver, kidneys, lungs, spleen, remaining carcass and samples of whole blood and fat were analyzed for radioactivity by liquid scintillation counting.

Expired air traps and excreta samples containing significant quantities of radioactivity were examined by chromatographic procedures (HPLC with radiodetection). GC-MS with radiodetection was also used for test material purity analysis and identification of some urinary metabolites.
Details on dosing and sampling:
The radiolabelled tertiary butyl acetate was removed from its container and diluted with non-radiolabelled tertiary butyl acetate for administration at the appropriate concentrations. Two samples of each batch of radiolabelled tertiary butyl acetate were weighed and made up to a volume (100 mL) in volumetric flasks with methanol. Aliquots (0.1 mL) were analyzed by liquid scintillation counting. Specific activities were 3098 dpm/µg, 0.052 MBq/mg (low dose) and 315.5 dpm/µg, 0.0053 MBq/mg (high dose). The lower specific activity for the higher dose means that the detection limits for radioactivity (as µg/g) in radioactive biological samples will be correspondingly (ca 10-fold) higher.

During preliminary generation trials, the nominal concentration of the test substance was calculated from the amount of tertiary butyl acetate delivered to the vaporizer and the total volume of air flowing through the exposure system during the period of generation. No sampling or analysis was carried out during exposure with radiolabelled tertiary butyl acetate.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
After inhalation exposure of male rats to 14C-tertiary butyl acetate at a mean vapor concentration of 100 ppm for 6 hours, a mean dose level of 50.7 mg/kg was absorbed.

After inhalation exposure of male rats to 14C-tertiary butyl acetate at a mean vapor concentration of 1000 ppm for 6 hours, a mean dose level of 272 mg/kg was absorbed.
Details on distribution in tissues:
100 ppm:
Mean concentrations were calculated from samples, of which, some contained radioactivity concentrations below their relative limits of accurate determination. Such concentrations were considered as equal to the limit of accurate determination. Mean concentrations of 0.925, 0.328 and 0.891 µg/g were detected in the nasal tissues, larynx, and trachea, respectively, and 0.131 µg/g was detected in the lungs. Mean concentrations of 0.112 and 0.170 µg/g were detected in the liver and kidneys, respectively, and 0.084 µg/g in the spleen. Fat contained a relatively high mean concentration of 0.246 µg/g compared to other tissues not associated with the respiratory system, and a mean of 0.126 µg/g was found in the blood.

1000 ppm:
Mean concentrations in the nasal tissues, larynx, and trachea, respectively, were all below their limits of accurate determinations (<7.33, <1.81, and <3.85 µg/g, respectively) as were concentrations in the spleen (<0.61 µg/g) and whole-blood (<0.44 µg/g). A mean concentration of 0.50 µg/g was detected in the lungs. Mean concentrations of 0.40 and 0.55 µg/g were detected in the liver and kidneys, respectively. Fat contained a relatively high mean concentration of 1.17 µg/g.

Where tissue concentrations are measurable at both exposure levels (liver, kidneys, lungs and fat), there is an increase of approximately 3- to 5-fold in concentrations, for a 10-fold increase in exposure rate, which is in agreement with the excretion data. This complements the 5-fold increase in total absorption following low and high level exposure levels and indicates that differences in tissue concentration are related to total absorption and not metabolic differences.

Where tissue concentrations of radioactivity were below their respective limits of accurate determination, at the high dose level but not the low, the concentrations at low dose levels were close to their limits of accurate determination. The lower specific activity of 14C-tertiary butyl acetate at the high dose level means that the limits of accurate determination are higher. This coupled to the lesser quantity of radioactivity absorbed will mean that the concentrations in these tissues are more likely to fall below their respective limits of accurate determination. Such data do not therefore indicate any difference in tissue concentration that cannot be related to the total dose absorbed.
Details on excretion:
100 ppm Group:
During 7 days after exposure, a mean total of 4.76% normalized dose was excreted in expired air, mostly during the first 12 hours (4.45% normalized dose). Most expired radioactivity (3.69% normalized dose) was associated with volatiles recovered in traps 1 and 2. Only 0.63% normalized dose was absorbed by the expired CO2 trap (trap 3; 1 M potassium hydroxide). Means of 89.15 and 2.69% normalized dose were excreted in urine and feces, respectively. Of the urinary radioactivity 72.3, 9.4 and 4.5% normalized dose were excreted during 0-24, 24-48 and 48-72 hours, respectively. Very little of the recovered radioactivity remained in the carcass and tissues (0.69% normalized dose). These results indicate the absorbed radioactivity was rapidly excreted after termination of exposure.

1000 ppm Group:
The excretion pattern differed from that after the lower dose, in that excretion in expired air was significantly greater. During 7 days after exposure, a mean total of 26.74% normalized dose was excreted in expired air, mostly during the first 12 hours (26.51% normalized dose). Most expired radioactivity was associated with volatile compounds recovered in trap 2 (18.33% normalized dose) and trap 4 (5.66% normalized dose). Only 2.75% normalized dose was absorbed by the expired CO2 trap (trap 3; 1M potassium hydroxide). Means of 68.99 and 0.97% normalized dose were excreted in urine and feces, respectively. Of the urinary radioactivity, 61.79, 5.33, and 1.29% of the normalized dose were excreted during 0-24, 24-48 and 48-72 hours, respectively. These results show a ca 5.5-fold increase in mean dose level for a 10-fold increase in the exposure rate when compared to the low dose data. As in the low dose experiment, the absorbed radioactivity was rapidly excreted after the termination of the exposure period. A much greater proportion of the absorbed radioactivity was recovered from the expired air, indicating some saturation of the metabolic processes. Very little of the recovered radioactivity remained in the carcass and tissues (0.22% normalized dose).

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
Metabolite characterization studies
Details on metabolites in urine and expired air:

Proportions of radioactive components in excreta:

Expired air (1000 ppm group)- In expired air trap 2 (ethyl digol: 16.25% dose) 3 compounds were detected. Metabolite A2 (8.5% dose) was chromatographically identical to parent tertiary butyl acetate. Metabolite A1 accounted for 6.7% dose. Co-chromatography of urine with expired air showed that detected radioactive metabolites in these matrices were not the same compounds.

Urine- Up to 7 metabolites (identified as U1, U2, U4, U6, U7 and U8) were detected in urine of which 4 (U1, U2, U4, and U6) were major. In 0-6 hour β-glucuronidase-treated urine of animals receiving the low (100 ppm) exposure, the proportion of metabolite U6 declined from 25 to 0%, with an increase in the proportion of metabolite U1 from 20 to 45%. Metabolite U6 was not hydrolyzed either by incubation at pH 5 or incubation with sulphatase. This indicated that U6 was a glucuronide conjugate of U1.

Following the low dose (100 ppm) exposure, proportions of metabolites U1, U2, U4, and U6 were 9.3, 45.5, 11.0, and 10.0% dose respectively before incubation with β-glucuronidase, and were 19.8, 47.6, 11.1, and 0.0% dose respectively after incubation.

Following the high dose (1000 ppm) exposure, proportions of metabolites U1, U2, U4, U6 were 7.9, 38.9, 15.2, and 2.6% dose respectively before incubation with β-glucuronidase, and were 9.8, 39.0, 15.3, and 0.7% dose respectively after incubation.

These results show a lower proportion of the urinary glucuronide U6 in animals receiving the high dose compared to the low dose. In the high dose group, there was also a lesser proportion of U2 and a 35% increase (approximately) in the quantity of U4.


Metabolite U1
No structure could be assigned to this compound from LC-MS analysis data. However metabolite U6 was shown to be the glucuronide of this compound by deconjugation experiments. U6 was subsequently identified as the glucuronide conjugate of 2-hydroxymethylisopropyl acetate.

Metabolite U4
The spectrum produced by MS/MS is consistent with the compound being the glucuronide conjugate of t-butanol. The fragment ions at m/z 75, 85, and 113 are characteristic fragments of glucuronic acid under negative ion conditions. The glucuronide bond was not hydrolyzed under the conditions used.

Metabolite U6
MS/MS product ion spectra were recorded for the m/z 326 positive ion and m/z 307 negative ion. The negative ion spectrum produced is consistent with the compound being the glucuronide conjugate of 2-hydroxymethylisopropylacetate. The fragment ions at m/z 75, 85 and 113 are characteristic fragments of glucuronic acid under negative ion conditions. The compound loses 60 amu from the parent ion to form the fragment at m/z 247, i.e., loss of CH3CO2H. This is an indication that the hydroxyl addition is on the t-butyl group.

Metabolite U7
No structure has been assigned to this compound.

Metabolite U8
MS/MS product ion spectra were recorded for the m/z 326 positive ion and m/z 307 negative ions. The negative ion spectrum produced is consistent with the compound being the glucuronide conjugate of t-butyl-2-hydroxyacetate. The fragment ions at m/z 75, 85, and 113 are characteristic fragments of glucuronic acid under negative ion conditions. A loss of 74 amu (t-butanol) to produce the fragment ion at m/z 233 is observed. This is an indication that the hydroxyl addition is on the acetyl group.

Metabolite U2
Metabolite U2 could not be identified by LC-MS analysis of high dose 0-6 hour urine. The urinary metabolite was partially purified from high dose 6-12 hour urine by preparative HPLC. Total Ion Chromatograms (TIC) were obtained by GC-MS analyses of BSTFA-derivatized solutions of 2-hydroxyisobutyric acid and metabolite U2. Only one peak (ca 2.95 minutes) was detected for both the U2 metabolite and 2-hydroxyisobutyric acid. The mass spectra obtained for 2-hydroxyisobutyric acid and metabolite U2 were virtually identical. No molecular ion is formed. The fragmentation ions of EI mass spectrum are consistent with the structure of 2-hydroxyisobutyric acid.

Any other information on results incl. tables

A proposed metabolic pathway of tertiary butyl acetate is located in the "Overall remarks, attachments" section below.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study results
During exposure to 100 or 1000 ppm 14C-tertiary butyl acetate for a period of 6 hours, male rats absorbed around 0.30 and 0.16%, respectively, of the total available radioactivity. A lower proportion of available radioactivity was absorbed by animals at the high concentration level. The reduction in proportion absorbed and the increased proportion in expired air suggest some saturation of absorption and metabolism. At the lower exposure level, the absorbed radioactivity was rapidly eliminated, mainly in urine (89% of dose, 72% of dose during the first 24 hours) and in expired air (4.8% of dose, 4.5% of dose during the first 12 hours) with less than 3% in feces. At the higher exposure level, the excretion in expired air was considerably greater (27% of dose) with 69% of the absorbed dose being excreted in urine. Less than 1% of the absorbed dose was detected in feces. Very little residual radioactivity remained in the carcass and tissues at either exposure level (< 1% of the normalized dose). At both exposure levels, most of the excreted radioactivity was recovered during the first 24 hours and greater than 99% of the dose was eliminated in excreta over the 7 days after exposure.

Four major metabolites and several minor ones were detected in urine. Of the urinary metabolites, 2 of the major metabolites were glucuronides of tertiary butyl alcohol and 2-hydroxymethylisopropyl acetate. A third major metabolite in urine was the aglycone of 2-hydroxymethylisopropylacetate glucuronide. The fourth major metabolite was identified as 2-hydroxyisobutyric acid. One of the minor urinary metabolites was identified as the glucuronide of t-butyl-2-hydroxyacetate. Two major radioactive components were detected in expired air. A large proportion of radioactivity in expired air of animals exposed to the higher concentration of 14C-tertiary butyl acetate was chromatographically identical to the parent tertiary butyl acetate.

Metabolism appears to follow 2 major routes, hydroxylation of the t-butanol moiety or cleavage of the ester linkage to produce tertiary butyl alcohol. In both cases, the available hydroxyl groups were conjugated with glucuronic acid. Free tertiary butyl alcohol was not detected in urine. A major metabolite of tertiary butyl alcohol in rats, 2-hydroxyisobutyric acid, was detected. A minor route of metabolism was the hydroxylation of the acetate moiety in the parent molecule (t-butyl-2-hydroxyacetate), followed by glucuronide conjugation.
Executive summary:

The absorption, distribution, metabolism, and excretion of 14C-tertiary butyl acetate was studied in rats exposed by inhalation for 6 hours to concentrations of 100 or 1000 ppm which are approximately 2% and 20%, respectively, of the LC50 of this compound. Rats inhaled five times more tertiary butyl acetate during a 6 hour exposure to 1000 ppm 14C-tertiary butyl acetate than from exposure to100 ppm and excretion in air during the first 12 hours following exposure was five times higher at 1000 ppm. There was some evidence of partial saturation of tertiary butyl acetate absorption and metabolism at some concentration below 1000 ppm. Approximately 5% of the lower dose and 26% of the higher dose was expired within 12 hours, while the retained material was rapidly metabolized and excreted, mostly in the urine, within 24 hours. Very little radioactivity remained in the tissues after Day 7. Tertiary butyl acetate metabolites were identified by molecular weight and fragment ions from MS/MS. The metabolism of tertiary butyl acetate appears to follow two major routes: hydroxylation of the tertiary-butyl moiety to form 2-hydroxymethylisopropyl acetate and ester hydrolysis to form tertiary butyl alcohol. A minor route involves oxidation of the acetate moiety followed by glucuronide conjugation. Based on the proportion of metabolites that can clearly be assigned to one or the other major pathways, hydroxylation of the tertiary-butyl moiety prevails at 100 ppm, while hydrolysis of the ester bond predominates at 1000 ppm.