Registration Dossier

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
basic toxicokinetics in vivo
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study is old (1970) and not conducted to regulatory guidelines or to GLP standards. Only two animals were used.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Two male Rhesus monkeys were dosed daily in the diet for 31 days with unlabelled substance. On day 32 the animals were given an oral dose by oral gavage of a mixture of AN-702 and 14C-labeled AN-702 in corn oil. Both animals were bled 2, 4, 8, 16 and 24 hours after treatment. At 24 hours one monkey was sacrificed. The remaining animal was also bled at 48 hours and then sacrificed at 66.5 hours after treatment. Tissue aliquots were prepared and the 14-C content was determined by liquid scintillation counting. All urine and feces were collected during the post-treatment period and analyzed for 14-C content.
GLP compliance:
no
Radiolabelling:
yes
Remarks:
14-C
Species:
monkey
Strain:
other: Rhesus
Sex:
male
Route of administration:
oral: gavage
Vehicle:
corn oil
Duration and frequency of treatment / exposure:
The animals were dosed daily in the diet for 31 days with unlabelled substance. On day 32 the animals were given an oral dose by oral gavage of a mixture of AN-702 and 14C-labeled AN-702 in corn oil.
Remarks:
Doses / Concentrations:
The treatment solution contained 44 mg of substance and 0.49 µC of 14-C per ml. One animal received 16.4 ml of this solution, or 722 mg of AN-702 and 3.59 x 107 dpm of 14-C. This represents a total dose of AN-702 of 134 mg/kg. The other animal received 18.3 ml of which approximately 5 percent was lost. This animal received about 765 mg AN-702 and 3.8 x 107 dpm of 14-C or about 130 mg AN-702 per kg.
No. of animals per sex per dose / concentration:
2 males
Control animals:
no
Positive control reference chemical:
None
Preliminary studies:
None
Details on absorption:
Blood and plasma levels of 14C in both test monkeys reached a peak at 8 hours post-treatment and dropped only slightly during the next 16 hours. By 48 hours, the blood and plasma level in the remaining animal fell to about 1 µg/ml and at 66.5 hours had decreased only slightly more.
Details on distribution in tissues:
After 24 hours, the 14C concentration was highest in the bile, small intestine, adrenal glands, caecum and gall bladder. All other tissues had less than 50 µg/g of sample. The small intestine, skeletal muscle and liver accounted for the largest percentage of the total 14C activity.

After 66.5 hours, the highest concentrations of 14C were observed in the bile, adrenals, fat and lungs. The muscle and liver tissues accounted for three-fourths of the total tissue 14 C activity. In four tissues - the brain, eyes, fat and lungs, the concentration of 14C were higher than in the animal sacrificed at 24 hours.
Details on excretion:
After 66.5 hours, fecal and urinary excretion accounted for nearly 67.5% of the total adminstered dose.
Metabolites identified:
no
Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study results
The test material exhibits a low bioaccumulation potential, with the total distribution of 14C material in tissues being 1.56% after 66.5 hours.
Executive summary:

The levels of 14C activity in blood, plasma, urine, feces and tissues have been determined in two monkeys intubated with approximately 130 mg/kg of a mixture of unlabelled & radiolabelled test material. One animal was sacrificed at 24 hours, and the other at 66.5 hours, after treatment.

The blood and plasma levels reached a maximum at 8 hours and showed little decline in the next 16 hours. The blood level dropped rapidly in the 24 to 48 hours interval and then fell more slowly towards 66.5 hours.

Urinary excretion accounted for not more than 0.5% by 66.5 hours. Fecal excretion during the same period was 67%.

Based on the limited data from these two monkeys the largest amounts of compound during the first 24 hours were found in the small intestine, muscle and liver. In most tissues, the levels fell 50% or more during the 24 to 66.5 hour interval; in four tissues - the brain, eyes, fat and lungs, there was an apparent increases in 14C concentration.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
At the time the study was conducted there was not a recommeded guideline to be followed for the toxicokinetics studies. The study presents several deviations from the recommended guidelines. However, it is considered that the results are reliable and can be used to assess the fate of the test subtance in rats.
Objective of study:
toxicokinetics
Principles of method if other than guideline:
The objective of the assay was to study the metabolic fate of a single oral dose of the test substance in rats:
1.- The elimination of 14C in the faeces, urine and expired gases
2.- The rate of elimination of 14C
3.- The tissue radioactivity, with special reference to the liver, subcutaneous fat, body fat and skin plus hair
4.- The rate of secretion of 14C into the bile of animals equipped with biliary fistulae.
Radioactive metabolites in the faeces, urine, bile and body fat and an unlabelled metabolite in the faeces have been identified in animals dosed with [14C] test substance.
GLP compliance:
no
Remarks:
(The study was conducted before GLP were approved)
Radiolabelling:
yes
Species:
rat
Strain:
other: Porton strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 3 months old.
- Weight at study initiation: 200-225 g
- Housing: Animals were kept singly in all-glass metabolism cages.
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): ad libitum.
- Water (e.g. ad libitum): ad libitum.
Route of administration:
other: other: stomach tube
Vehicle:
olive oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
In the preliminary experiment, a solution of [14C] test substance (11 mg, 0.905 μC/mg) in 2 ml of olive oil was administered to rats.
In the main experiment, a solution of [14C] test substance (10 mg, 0.905 μC/mg) in 2 ml of olive oil was administered to rats.
Duration and frequency of treatment / exposure:
Single dose
Remarks:
Doses / Concentrations:
A solution of [14C] test substance (10 mg, 0.905 μC/mg) in 2 ml of olive oil was administered to rats.
No. of animals per sex per dose / concentration:
Main experiment: Eight animals (four animals per sex; one animal per sex per sampling time: 4, 8, 16 and 24 days)
Control animals:
no
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: In the preliminary experiment, the urine and faeces were collected and the exhaled gases were monitored for 14 CO2. The alimentary canal, feet, head, skin plus hair and tail were removed, and the carcass and remaining viscera were examined for radioactivity.
In the main experiment, the urine and faeces were collected daily. The entire alimentary canal and contents, the skin plus hair, the whole liver and a standard amount of intra-abdominal and subcutaneous fats were dissected from each cadaver and there organs and these organs and tissues together with the carcass and remaining viscera were stored at -29ºC.
- Time and frequency of sampling: In the preliminary experiment, the rats were killed 2, 4, 6, 12, 24 and 48 hours after dosage.
In the main experiment, pairs of animals (one of each sex) were killed 4, 8, 16 and 24 days after dosage.
- Other: Biliary fistulae were established in rats that had previously been intubated with a solution of 10 mg of 14C test substance in 2 ml of olive oil. Bile was collected during 2.0-3.5 hours after dosage. These animals were killed at the end of the experiment.
Preliminary studies:
Measurement of radioactivity in the urine and expired gases showed that less than 1% of the label was excreted in urine and less than 0.1% in the expired gases. In the animals killed between 6 and 48 hours after dosage, more than 5% of the label was found in the carcass and viscera remaining after removal of the entire gut. The conclusion was drawn that at least partial absorption of orally administered test substance occurred, that the rate of its metabolism was slow, and that the elimination of test substance and its metabolic products was mainly by the faecal route.
Details on absorption:
Considerable alimentary absorption occurred in rats dosed orally with [14C] test substance which is shown by the retention of radioactivity in the body tissues ant by the secretion of 14C into the bile.
Details on distribution in tissues:
The carcass and viscera remaining after removal of the gut of the male rat, killed 4 days after dosage, contained 9.5% of 14C and the corresponding value for the female rat was 6.42% of 14C. These values fell to 3.46 (male) and 3.25% (female) in animals killed 24 days after dosage. After an oral dose of [14C] test substance more 14C is stored in the fatty tissues, including the subcutaneous fats, body fats and pelts, than in organs. After 24 days, the concentrations of 14C in the liver of male and female rats had fallen to < 0.1 and 0.1 ppm, respectively, whereas the concentrations of 14C in the subcutaneous fat, body fat and skin plus hair of male rats after 8 days had fallen to one-half or less of the values at 4 days and they remained stationary during 8-24 days. There is greater retention of 14C in the fatty tissues of female rats than of males.

Maximum concentration of 14C in the bile of two animals occurred respectively during 4-8 hours and 6-12 hours after dosage. After 18 hours, the concentration of 14C in the bile of each animal fell very slowly. These observations suggest that a low degree of alimentary absorption had taken place and that diffusion of 14C products from plasma to bile was slow. At least 14-16% of the 14C excreted in the faeces during 24 days originated in the bile, since 10% (male)-11% (female) of 14C was secreted in the 30 hour bile and 6.0 (males) - 3.2% (females) of 14C was eliminated from the organs and tissues during 4-24 days. When allowance is made for retention of 14C 24 days after dosage, it follows that approximately 20% of a single dose of [14C] test substance was absorbed in rats.
Details on excretion:
Less than 1% of the original label was excreted in the urine, 89.4-97.5% in the faeces during 24 days, where these figures include the contents of the gut on the 24th day.

Initial elimination in the faeces was comparatively rapid, 86.58-94.62 in 4 days. Rats do not show a sex difference in the pattern of elimination of test substance and its metabolites.
Metabolites identified:
yes
Details on metabolites:
The test substance was not present amongst the metabolites in the urine. The single peak of 14C in the extracts prepared at pH 6.0 correspond to [14C]3,5-di-tert.-butyl-4-hydroxybenzoic acid, which was identified by isotope-dilution methods and the single peak of 14C in extracts prepared at pH 1.5-2.0 corresponded to 3,5-di-tert.-butyl-4-hydroxybenzoyl-β-D-glucopyranosiduronic acid.

Unchanged [14C] test substance which accounts for 87% of the 14C in solvent extracts of the faeces of treated rats was separated from the total lipid fraction of rat faeces. A small proportion of contaminating [14C] quinone methide was separated from the fraction containing [14C] test substance and estimated. A middle fraction from the column contained a small amount of [14C]3,5-di-tert.-butyl-4-hydroxy-benzoic acid. When the late fractions, which contained a small amount of a slow-moving radioactive component, were chromatographed on paper, radioautography revealed traces of 3,5-di-tert.-butyl-4-hydroxy-benzoic acid and of an unidentified component with Rf 0.1. 2,6-di-tert.-butyl-pbenzoquinone was also isolated from a late fraction of faecal lipids.

A scheme is suggested for the metabolism of the test substance. The test substance is initially oxidized into the quinone methide, which was detected and isolated in radioactive form, and which was shown to be identical with 3,5 -di-tert.-butyl-4 -hydroxyphenyl-(2,6 -di-tert.-butyl-p-benzoquinone) methide. Further oxidation of the [14C]quinone methide to [14C]3,5 -di-tert.-butyl-4 -hydroxybenzoic acid and the unlabelled 2,6 -di-tert.-butyl-p-benzoquinone would be expected to proceed via the stable phenoxy radical.

Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study results
A big proportion of a single oral dose of 14C test substance to rats is eliminated in 24 days: 89.4-97.5% of the label is excreted in the faeces (much of this is eliminated in the first 4 days after dosage), 1% in the urine and < 0.1% in the expired gases. 3.46 and 3.42% of 14C respectively are present in the carcass and viscera of male and female rats after removal of the gut. Rats do not show a sex difference in the pattern of elimination of the test substance and its metabolites.

After ingestion of [14C] test substance the fatty tissues account for the retention of 14C.

Measurement of 14C in the bile of animals with biliary fistulae in conjunction with othe rdata implies that one-fifth of a single dose of [14C] test substance is absorbed. 87% of 14C in the faeces is due to unchanged test substance, 5% to the quinone methide, 5% to the free acid and 3% to an unidentified polar constituent. Three-fifths of 14C in the urine is due to 3,5-di-tert.-butyl-4-hydroxybenzoid acid and the remainder to the ester glucuronide. In several animals, one-half of 14C in the bile is due to the free acid, onequarter to the ester glucuronide and the remainder to unchanged antioxidant, whereas, in others, all of 14C in the bile is due to test substance. 97% of 14C in the body fat is due to unchanged antioxidant and the remainder to the free acid.

Up to 20% of a single oral dose of the test substance is absorbed in rats: 13-14% is metabolized. 3,5-di-tert.-butyl-4-hydroxybenzoic acid accounts for > 5% of a dose of the test substance, 3,5-di-tert.-butyl-4-hydroxybenzoyl-β-Dglucopyranosiduronic acid for < 0.4%, the quinone methide for > 5% and an unidentified compound for < 3%.
Executive summary:

The objective of the assay was to study the metabolic fate of a single oral dose of the test substance in rats:

1.- The elimination of 14C in the faeces, urine and expired gases

2.- The rate of elimination of 14C

3.- The tissue radioactivity, with special reference to the liver, subcutaneous fat, body fat and skin plus hair

4.- The rate of secretion of 14C into the bile of animals equipped with biliary fistulae.

Radioactive metabolites in the faeces, urine, bile and body fat and an unlabelled metabolite in the faeces have been identified in animals dosed with [14C] test substance.

A big proportion of a single oral dose of 14C test substance to rats is eliminated in 24 days: 89.4-97.5% of the label is excreted in the faeces (much of this is eliminated in teh first 4 days after dosage), 1% in the urine and < 0.1% in the expired gases. 3.46 and 3.42% of 14C respectively are present in the carcass and viscera of male and female rats after removal of the gut. Rats do not show a sex difference in the pattern of elimination of the test substance and its metabolites.

After ingestion of [14C] test substance the fatty tissues account for the retention of 14C.

Measurement of 14C in the bile of animals with biliary fistulae in conjunction with other data implies that one-fifth of a single dose of [14C] test substance is absorbed. 87% of 14C in the faeces is due to unchanged test substance, 5% to the quinone methide, 5% to the free acid and 3% to an unidentified polar constituent. Three-fifths of 14C in the urine is due to 3,5-di-tert.-butyl-4 -hydroxybenzoid acid and the remainder to the ester glucuronide. In several animals, one-half of 14C in the bile is due to the free acid, one-quarter to the ester glucuronide and the remainder to unchanged antioxidant, whereas, in others, all of 14C in the bile is due to test substance. 97% of 14C in the body fat is due to unchanged antioxidant and the remainder to the free acid.

Up to 20% of a single oral dose of the test substance is absorbed in rats: 13-14% is metabolized. 3,5-di-tert.-butyl-4 -hydroxybenzoic acid accounts for > 5% of a dose of the test substance, 3,5-di-tert.-butyl-4-hydroxybenzoyl-βDglucopyranosiduronic

acid for < 0.4%, the quinone methide for > 5% and an unidentified compound for < 3%.

Description of key information

In a study on rats, a big proportion of a single oral dose of 14C test substance to rats is eliminated in 24 days: 89.4-97.5% of the label is excreted in the faeces (much of this is eliminated in the first 4 days after dosage), 1% in the urine and < 0.1% in the expired gases. 3.46 and 3.42% of 14C respectively are present in the carcass and viscera of male and female rats after removal of the gut. Rats do not show a sex difference in the pattern of elimination of the test substance and its metabolites.
After ingestion of [14C] test substance the fatty tissues account for the retention of 14C.
Measurement of 14C in the bile of animals with biliary fistulae in conjunction with other data implies that one-fifth of a single dose of [14C] test substance is absorbed. 87% of 14C in the faeces is due to unchanged test substance, 5% to the quinone methide, 5% to the free acid and 3% to an unidentified polar constituent. Three-fifths of 14C in the urine is due to 3,5-di-tert.-butyl-4-hydroxybenzoid acid and the remainder to the ester glucuronide. In several animals, one-half of 14C in the bile is due to the free acid, one quarter to the ester glucuronide and the remainder to unchanged antioxidant, whereas, in others, all of 14C in the bile is due to test substance. 97% of 14C in the body fat is due to unchanged antioxidant and the remainder to the free acid.
Up to 20% of a single oral dose of the test substance is absorbed in rats: 13-14% is metabolized. 3,5-di-tert.-butyl-4-hydroxybenzoic acid accounts for > 5% of a dose of the test substance, 3,5-di-tert.-butyl-4-hydroxybenzoyl-β-Dglucopyranosiduronic acid for < 0.4%, the quinone methide for > 5% and an unidentified compound for < 3%.

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential

Additional information

A reliable study in rats (Hathaway and Wright, 1965), a big proportion of a single oral dose of 14C test substance to rats is eliminated in 24 days: 89.4-97.5% of the label is excreted in the faeces (much of this is eliminated in the first 4 days after dosage), 1% in the urine and < 0.1% in the expired gases. 3.46 and 3.42% of 14C respectively are present in the carcass and viscera of male and female rats after removal of the gut. Rats do not show a sex difference in the pattern of elimination of the test substance and its metabolites.

After ingestion of [14C] test substance the fatty tissues account for the retention of 14C.

Measurement of 14C in the bile of animals with biliary fistulae in conjunction with other data implies that one-fifth of a single dose of [14C] test substance is absorbed. 87% of 14C in the faeces is due to unchanged test substance, 5% to the quinone methide, 5% to the free acid and 3% to an unidentified polar constituent. Three-fifths of 14C in the urine is due to 3,5-di-tert.-butyl-4 -hydroxybenzoid acid and the remainder to the ester glucuronide. In several animals, one-half of 14C in the bile is due to the free acid, one-quarter to the ester glucuronide and the remainder to unchanged antioxidant, whereas, in others, all of 14C in the bile is due to test substance. Only a low percentage of the test substance was retained in body fat. 97% of 14C in the body fat is due to unchanged antioxidant and the remainder to the free acid.

Up to 20% of a single oral dose of the test substance is absorbed in rats: 13-14% is metabolized. 3,5-di-tert.-butyl-4 -hydroxybenzoic acid accounts for > 5% of a dose of the test substance, 3,5-di-tert.-butyl-4-hydroxybenzoyl-βDglucopyranosiduronic

acid for < 0.4%, the quinone methide for > 5% and an unidentified compound for < 3%.

In an additional unreliable study on monkeys (Smith, 1970) the levels of14C activity in blood, plasma, urine, faeces and tissues have been determined in two monkeys which were dosed daily in the diet for 31 days with unlabelled substance then on day 32 intubated with approximately 130 mg/kg of a mixture of unlabelled & radiolabelled test material in corn oil. One animal was sacrificed at 24 hours, and the other at 66.5 hours, after treatment.The blood and plasma levels reached a maximum at 8 hours and showed little decline in the next 16 hours. The blood level dropped rapidly in the 24 to 48 hours interval and then fell more slowly towards 66.5 hours.The test material exhibits a low bioaccumulation potential, with the total distribution of 14C material in tissues being 1.56% after 66.5 hours.

Results from old unreliable in vivo pharmacology studies indicate that the substance undergoes much better absorption from non-aqueous media.

It is anticipated that absorption via the oral route is higher than absorption after dermal or inhalation exposure due to the emulsifying function of bile acids and longer contact time and surface area in the GI tract.