Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
publication
Title:
Metabolic studies in the rat with 2,4,6-tri-t-butylphenol: a haemorrhagic antioxidant structurally related to butylated hydroxytoluene
Author:
Takahashi O & Hiraga K
Year:
1983
Bibliographic source:
Xenobiotica. 13: 319-326

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Butylated hydroxytoluene (BHT), a sterically hindered phenolic antioxidant, can cause a dose-dependent decrease in the prothrombin index, and haemorrhagic death at high dosage, when administered in the diet to male rats. The nature of the haemorrhagic effect has not yet been determined, but may be attributable to the structural characteristics of BHT rather than to its antioxidant properties.
It is considered that the quinone methide of BHT may take part in the haemorrhagic effect. The test material, which is structurally related to BHT and has a stronger haemorrhagic effect than BHT, cannot be oxidised to the corresponding quinone methide. If the haemorrhagic effects of these two chemicals involve the same mechanism, a common active metabolite other than the quinone methide must be involved.
This study was conducted to investigate the metabolism and clearance of the test material in the rat. Clearance studies (dosed by oral gavage and in the diet) and the analysis of urinary and faecal metabolites (dosed via the diet) took place. The toxicological implications of the metabolism are also considered.
GLP compliance:
not specified

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): 2,4,6-Tri-tert-butylphenol
- Supplier: Aldrich Chemical Co. (Milwaukee, WI, USA)
- Purity: >97 %
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 190 g
- Fasting period before study: For clearance studies, some rats were fasted overnight prior to dosing
- Individual metabolism cages: No data
- Diet: Rats used for the clearance studies were given ad libitum access to the diet
- Other: In the animals used for the analysis of biliary excreted metabolites, the bile duct was cannulated with polyethylene tubing of internal diameter 0.20 mm, exterior diameter 0.50 mm (Natsume Seisakusho, Tokyo, Japan), for the collection of bile.

Administration / exposure

Route of administration:
other: Gavage and in the diet
Vehicle:
soya oil
Remarks:
for the gavage doses
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Details on diet: No information relating to the diet was reported other than the test material concentration.

VEHICLE
- Details on vehicle: The doses administered by gavage were prepared in soy bean oil (Wako Pure Chemical Co., Osaka, Japan).
Duration and frequency of treatment / exposure:
For clearance studies, rats received oral doses of the test material. Rats given diet ad libitum were also used for clearance studies.
For the analysis of urinary and faecal metabolites, rats were fed a diet containing the test material for two days.
Doses / concentrations
Remarks:
Doses / Concentrations:
Rats dosed by oral gavage received doses of 260 mg/kg.
For the analysis of urinary and faecal metabolites, rats were fed a diet containing 0.2 % test material.
Control animals:
no
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: rats were killed by a blow to the head and blood, liver, spleen, kidneys, testes and samples of epididymal adipose tissue were collected for analysis.
- Time and frequency of sampling: At various times

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: For the analysis of biliary excreted metabolites, rats were anaesthetised intraperitoneally with Nembutal® sodium solution (Abbott Laboratories, North Chicago, IL, USA). For the analysis of urinary and faecal metabolites, urine and faeces were collected.
- Time and frequency of sampling: At various times

ANALYTICAL PROCEDURES
Each tissue was mixed with anhydrous Na₂SO₄, and homogenised with acetone (1 g tissue in l0 mL of acetone) using a Polytron homogeniser (Kinematica GmbH, Lucerne, Switzerland). The acetone solution was concentrated under N₂ and the residual solution was extracted with n-hexane. The solvent was evaporated under N₂ and the residue dissolved in n-hexane for analysis by GLC.
Urine was extracted with n-hexane-acetone (95:5, v/v). The hexane layer was washed with 5 % aqueous Na₂CO₃, evaporated under N₂ and the residue dissolved in n-hexane for analysis by GLC. The aqueous layer with an equal volume of 2 M NaOH was heated at 90 to 100 °C for 3 hours and acidified with concentrated HCl. This solution was extracted with diethyl ether, the ether extract dried under N₂ and the residue dissolved in n-hexane for analysis by GLC.
Faeces, softened by the addition of water, were homogenised with acetone using a Polytron homogeniser. The homogenate was extracted with n-hexane-water (1:1) and the hexane layer analysed by GLC.
GLC was performed on a Shimadzu GC-9A gas chromatograph equipped with a flame-ionisation detector and with a Shimadzu Chromatopack C-R1B data managing system (Shimadzu Corporation, Kyoto, Japan). A glass column (2 m x 3 mm internal diameter) packed with Gaschrom Q containing 2 % Silicone OV-17 was used. The operating temperatures were 170 °C (column), 250 °C (detector) and 250 °C (injection port) and the carrier gas (N₂) flow rate was 50mL/minute.
Mass spectra were recorded on a JEOL JMS D-300 gas chromatograph-mass spectrometer at 70 eV.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on absorption:
BLOOD CONCENTRATIONS OF THE TEST MATERIAL IN STARVED RATS
Absorption after oral administration was rapid.
Peak concentration was attained 15 to 60 minutes after dosing. The blood half-lives of the test material were 18.2 minutes and 11.8 hours for the rapid α- and the slower β-phase, respectively.
Details on distribution in tissues:
CONCENTRATIONS OF THE TEST MATERIAL IN TISSUES IN STARVED RATS
The peak concentration in the liver was reached at 2 to 6 hours after dosing, and it could not be detected after 24 hours. In the spleen and kidneys, the test material appeared in a biphasic manner and did not decrease much until after 24 hours. The testicular concentration of the test material could not be determined, but trace amounts were detected at 15 minutes and at 2.5 to 3 hours. The concentrations in epididymal fat increased from 3 to 24 hours. Maximum concentrations were 100, 10, 5, 2 and 200 µg/g in blood, liver, spleen, kidneys and fat, respectively.

BLOOD AND ADIPOSE CONCENTRATIONS OF THE TEST MATERIAL IN NON-STARVED RATS
Absorption of the test material after oral administration to unstarved was slower than in starved rats. The peak concentration in adipose tissue was attained two days later and the concentration declined until day 8. The maximum concentrations were 1.50 and 200 µg/g in blood and fat, respectively.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
CHARACTERISATION OF METABOLITES
Neither the test material nor its metabolites were detected in extracts of urine.
Only a single peak was detected in the extract of faeces. The mass spectrum of this compound resembles that of the test material), but its base peak (m/z 246) and molecular ion (m/z 261) were smaller by 1 unit than the test material. This compound was also detected in bile. Unchanged test material was found in some tissues.

Any other information on results incl. tables

Discussion

The test material is well absorbed and the blood clearance is rapid. Most of this compound after disappearing from the blood moves into adipose tissue. It is not metabolised to less lipophilic compounds (as in BHT metabolism), but can be oxidised to the 2,4,6-tri-tert-butylphenoxy radical which is excreted in the faeces via the bile. This phenoxy radical can be detected as it is relatively stable due to the delocalisation of electrons.

This finding indicates that the biological oxidation of BHT may also yield the corresponding phenoxy radical as an intermediate to the corresponding quinone methide.

 

The more severe bleeding induced by the test material than by BHT may be attributed to the following reasons:

- The test material is not metabolised to less lipophilic compounds as occurs in the metabolism of BHT, so concentration of an active metabolite (the radical) may be higher.

- If the phenoxy radicals are active metabolites involved in the mechanism of haemorrhage, the life of the test material radical is longer.

 

It is inferred that these radical species may play an important role in the haemorrhagic effects of some hindered phenols.

The oxidation of the test material to its radical species can probably also be effected in systems such as hepatic microsomal suspensions and/or in incubation with gut microflora.

Applicant's summary and conclusion

Conclusions:
Single oral doses were well absorbed in the rat. Peak blood levels of the test material were reached in 15 to 60 minutes. The blood elimination half-lives were 18.2 minutes for the α-phase and 11.8 hours for the slower β-phase.
Executive summary:

This study was conducted to investigate the metabolism and clearance of the test material in the rat. Clearance studies (dosed by oral gavage and in the diet) and the analysis of urinary and faecal metabolites (dosed via the diet) took place. The toxicological implications of the metabolism are also considered.

For clearance studies, male Sprague-Dawley rats received oral doses (260 mg/kg) of the test material by stomach tube in soy bean oil following overnight starvation. Rats given diet ad libitum were also used for clearance studies. At various times rats were killed by a blow to the head and blood, liver, spleen, kidneys, testes and samples of epididymal adipose tissue were collected for analysis. For the analysis of biliary excreted metabolites, the bile duct was cannulated with polyethylene tubing for the collection of bile. For the analysis of urinary and faecal metabolites, rats were fed a diet containing 0.2 % test material for two days, and urine and faeces were collected.

Single oral doses were well absorbed in the rat. Peak blood levels of the test material were reached in 15 to 60 minutes. The blood elimination half-lives were 18.2 minutes for the α-phase and 11.8 hours for the slower β-phase.

Maximum tissue concentrations were reached after 2 to 3 hours in the liver, 2 to 6 hours in the kidneys, 1.5 to 2.5 hours in the spleen and >24 hours in epididymal adipose tissues.

The test material and its metabolites were not excreted in the urine; a metabolite but not the parent compound was detected in the faeces. The faecal metabolite had a molecular weight of 261 and was considered to be 2,4,6-tri-tbutylphenoxy radical. The phenoxy radical was also detected in the bile of rats.

The test material is well absorbed and the blood clearance is rapid. Most of this compound after disappearing from the blood moves into adipose tissue. It is not metabolised to less lipophilic compounds. It is inferred that the radical species may play an important role in the haemorrhagic effects of some hindered phenols.