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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 September 1982 to 15 October 1982
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report date:
1982

Materials and methods

Objective of study:
absorption
distribution
excretion
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3',3'',5,5',5''-hexa-tert-butyl-α,α',α''-(mesitylene-2,4,6-triyl)tri-p-cresol
EC Number:
216-971-0
EC Name:
3,3',3'',5,5',5''-hexa-tert-butyl-α,α',α''-(mesitylene-2,4,6-triyl)tri-p-cresol
Cas Number:
1709-70-2
Molecular formula:
C54H78O3
IUPAC Name:
3,3',3'',5,5',5''-hexa-tert-butyl-α,α',α''-(mesitylene-2,4,6-triyl)tri-p-cresol
Radiolabelling:
yes
Remarks:
14C-labeled on the methyl groups of the central benzene ring

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
The male Sprague-Dawley rats used in the study were purchased from Charles River Breeding Laboratories (Kingston Facility, Stoneridge, New York). On arrival, the rats were ear-tagged with unique identification numbers and placed in polycarbonate cages. Ground corn cob (Bed-0-Cob) was used as the bedding material. Commercial Purina Laboratory Chow and tap water were provided ad libitum.
The animals were housed in environmentally controlled rooms with 10 to 15 air changes per hour. The rooms were maintained at a temperature of 22 ± 2°C and humidity of 50 ± 10%, with a 12-hr light/dark cycle per day. The animals were kept in our quarantine facility for at least 7 days prior to use. They were then weighed and randomized for each test group using a computer based body weight stratification program. At the initiation of the studies, the rats were 58 to 60 days old and weighed 278 to 334 g.
Animal care and housing were in accordance with DHEW Publication No. (NIH)-78-23, 1978, "Guidelines for the Care and Use of Laboratory Animals," and the MRI Manual for Animal Care.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The dosing solution was prepared by dissolving the appropriate amounts of the nonlabeled and 14C-labeled compounds in Mazola® corn oil. Aliquots of the solution were counted to determine the amount of radioactivity. The dosing solution contained 165.7 μCi of the labelled compound per 3 ml of the vehicle (specific activity of 7,356 dpm/μg). When not in use, the dosing solution was stored frozen under nitrogen.
Duration and frequency of treatment / exposure:
Single dose, sacrificed at 24 hour
Doses / concentrations
Remarks:
Doses / Concentrations:
14C-Ethanox 330 was administered by gavage at a dose level of 50 mg/kg in a volume of 3 ml/kg.
No. of animals per sex per dose / concentration:
16 male rats
Control animals:
not specified
Positive control reference chemical:
Not specified
Details on study design:
A total of 16 male rats were used in the study. One rat was used for a preliminary experiment and was housed in a glass metabolism cage (Delmar-Roth type) in order to assess the possible respiratory elimination of radioactivity. Following dosing, expired air, urine, and faeces were collected between 0-8 and 8-24 hr intervals. The 14CO2 trapping solution was 5 M ethanolamine in 2-methoxyethanol. Other possible volatile products were trapped in methanol: water (50:50). The rat was sacrificed at 24 hr for analysis of radioactivity in selected tissues.
Following a determination of the absence of expired radioactivity, the other 15 rats were treated and housed in stainless steel metabolism cages for separate collection of urine and faeces. They were then sacrificed in groups of three each, at 0.5, 1, 2, 4, and 24 hr. For animals sacrificed at 24 hr, urine and faeces were collected between 0-8 and 8-24 hr. At sacrifice, blood was withdrawn from the abdominal aorta under light ether anesthesia and the liver, kidneys, and lungs were removed, weighed, and prepared for radioactivity analyses. For rats sacrificed at 24 hr, the GI tract plus contents and samples of retroperitoneal fat, skeletal muscle, and skin were also collected and analyzed.
Details on dosing and sampling:
Sample Preparation and Analysis
Volumes of urine, cage rinse, and the solutions used for trapping expired radioactivity were measured and samples (250-2,000 μl) were analyzed. Faeces, GI tract, liver, kidneys, lungs, and muscle samples were weighed, homogenized in five volumes of ethanol:water (10:90), and samples were measured for analysis. Aliquots (250-500 μl) of blood were measured and samples (90-110 mg) of fat and skin were weighed and assayed for 14C content. Blood, tissues, and faecal samples were combusted in a Packard Tricarb sample oxidizer (Model C306). Permafluor® V in combination with Carbo-Sorb® (Packard Instrument Company, Downers Grove, Illinois) was used as the scintillation cocktail. Urine and cage rinses were counted directly in Phase Combining Solution (PCS, Amersham).

Measurement of Radioactivity
Vials were cooled for a minimum of 24 hr before counting in a liquid scintillation counter (Packard Tricarb Model 3255). Correction for background was carried out automatically on the counter. Background determinations were obtained from the average of natural counts of several tissue homogenates from non treated animals. The sample oxidizer recovery was monitored using 14C Spec-Chec® supplied by Packard. The counting efficiency was determined using the automatic external standard (AES) method. An AES versus efficiency curve was prepared by processing a quench curve set through the counter under the conditions used throughout the experiment. Assays not within ± 10% of the mean of the duplicates were re-assayed in duplicate except when the sample was no longer available or when radioactivity counts were low and non significant, i.e., less than two times the background, 36.5 cpm (counts per minute).
Statistics:
Carbon-14 contents in blood, tissues, and excreta were presented in terms of microgram equivalents per millilitre (blood) or gram (tissues), and/or percentage of the dose administered to each animal. Individual calculations for each sample were performed with an Apple II computer as follows:

1. Cpm (counts per minute) for each sample was converted to dpm (disintegration per minute).
Cpm ÷ efficiency = dpm/sample

2. Dpm per g or ml was calculated.
Dpm/sample x 1,000 ÷ sample weight or volume (mg or μl) = dpm/g or ml

3. Dpm per g or ml was divided by the specific activity of the compound (dpm/μg) to obtain the μg equivalents/g or ml.
Dpm/g or ml ÷ specific gravity = μg/g or ml

4. Total residues in the organ or excreta were obtained by multiplying the μg/g or ml by the total weight of volume of the organ or excreta.
μg/g or ml x total weight or volume = μg/organ or excreta

5. The μg/organ or excreta was divided by the total dose administered in order to obtain the percentage of the administered dose.
μg/organ or excreta x 100 ÷ total dose (in μg) = % of administered dose

Significant counts were considered to be those containing more than two times the average background activity (36.5 cpm). Depending on the sample size, the limit of accurate detection in this study was determined to be 0.01 to 0.1 μg/g or ml of tissue or blood. However, all the data generated including those resulting from counts lower than 36.5 cpm were included in this report.

Results and discussion

Preliminary studies:
A preliminary experiment using one rat indicated that practically no radioactivity (~ 0.001% of the dose) was eliminated in the expired air. In this experiment the administered dose was recovered in faeces (~87%) and in the GI tract (~ 12%) at the time of sacrifice (24 hr). Less than 0.01% of the dose was found in urine and only 0.06% was recovered in blood and tissue.
Main ADME resultsopen allclose all
Type:
absorption
Results:
less than 0.2%
Type:
distribution
Results:
trace amounts were found in urine and in the tissues examined
Type:
excretion
Results:
All the administered radioactivity was essentially eliminated in faeces

Toxicokinetic / pharmacokinetic studies

Details on absorption:
The primary objective of this study was to assess the absorption of Ethanox® 330 following oral administration to rats. To achieve this objective, 15 rats were treated with 14C-labeled Ethanox® 330 and sacrificed in groups of three each at early (0-5, 1, 2, and 4 hr) and late (24 hr) time periods for determination of radioactivity in blood and selected tissues. For the rats sacrificed at 24 hr, elimination of 14C in urine and faeces was also determined. A preliminary experiment using one rat indicated that practically no radioactivity (~ 0.001% of the dose) was eliminated in the expired air. In this experiment the administered dose was recovered in faeces (~ 87%) and in the GI tract (~ 12%) at the time of sacrifice (24 hr). Less than 0.01% of the dose was found in urine and only 0.06% was recovered in blood and tissue.
Details on distribution in tissues:
The counts in most samples were low and were considered insignificant (less than two times the background). The low levels in livers increased at later times and were highest at the 24-hr sampling period. Radioactivity in lungs of one rat sacrificed at 1 hr and two rats sacrificed at 2 hr was higher than in other tissues. Although all tissues were rinsed thoroughly following necropsy, these lung samples may have been contaminated. Alternatively, the trace impurities in the labelled compound may have been absorbed and preferentially concentrated in these lungs.
Details on excretion:
An average of ~ 71% of the administered doses was eliminated in faeces and ~ 28% was recovered in the GI tract.

Any other information on results incl. tables

TABLE 1 – RADIOACTIVITY IN BLOOD, TISSUE AND EXCRETA AT 24 HR FOLLOWING ORAL ADMINISTRATION OF14C-ETHANOX® 330 (50 mg/kg) TO A MALE SPRAGUE-DAWLEY RAT (PRELIMINARY STUDY)

 

Concentration

(μg equivalent/g or ml)

Percent

of Administered Dose

 

Rat No. 1

Rat No. 1

Blooda

0.038d

0.006d

Liver

0.098d

0.009d

Kidneys

0.056

0.001

Lungs

0.031

0.000

Muscleb

0.0421

0.034

Fat

0.041

ND

Skinc

0.030

0.010

GI tract

-

11.941d

Expired air

-

0.001

Urine

-

0.007d

Faeces

-

86.876d

Recovery

-

98.885

aBased on 7% of body weight.

bBased on 40% of body weight.

cBased on 16% of body weight.

dSignificant counts, i.e., more than two times the background (36.5 cpm). All others are below the limit of accurate detection.

ND = not determined.

 

TABLE 2 – RADIOACTIVITY IN BLOOD AND TISSUES OF MALE SPRAGUE-DAWLEY RATS TREATED WITH14C-ETHANOX® 330 (50 mg/kg)

 

Concentration (μg equivalent/g or ml)

Percent of Administered Dose

Time After Dosing (hr)

Rat No.

Blood

Liver

Kidneys

Lungs

Blooda

Liver

Kidneys

Lungs

0.5

2

0.001

0.000

0.000

0.003

0.000

0.000

0.000

0.000

 

3

0.002

0.000

0.001

0.000

0.000

0.000

0.000

0.000

 

4

0.000

0.000

0.006

0.000

0.000

0.000

0.000

0.000

 

Average

0.001

0.000

0.002

0.001

0.000

0.000

0.000

0.000

1

5

0.004

0.010

0.015

0.307b

0.001

0.001

0.000

0.002b

 

6

0.003

0.000

0.011

0.000

0.000

0.000

0.000

0.000

 

7

0.002

0.000

0.000

0.001

0.000

0.000

0.000

0.000

 

Average

0.003

0.003

0.009

0.103

0.000

0.000

0.000

0.001

2

8

0.012

0.010

0.004

0.004

0.002

0.002

0.000

0.000

 

9

0.011

0.021

0.006

1.190b

0.002

0.002

0.000

0.013b

 

10

0.016b

0.017

0.013

0.097b

0.002b

0.002

0.000

0.001b

 

Average

0.013

0.016

0.008

0.430

0.002

0.002

0.000

0.005

4

11

0.005

0.040

0.000

0.000

0.001

0.004

0.000

0.000

 

12

0.002

0.037

0.000

0.000

0.000

0.004

0.000

0.000

 

13

0.005

0.046

0.005

0.005

0.001

0.004

0.000

0.000

 

Average

0.004

0.041

0.002

0.002

0.001

0.004

0.000

0.000

24

14

0.002

0.044

0.000

0.000

0.000

0.005

0.000

0.000

 

15

0.004

0.058

0.010

0.000

0.001

0.007

0.000

0.000

 

16

0.002

0.058

0.001

0.000

0.000

0.006

0.000

0.000

 

Average

0.003

0.053

0.004

0.000

0.000

0.006

0.000

0.000

aBased on 7% of body weight.

bSignificant counts, i.e., more than two times the background (36.5 cpm). All others are below the limit of accurate detection.

 

TABLE 3 – RADIOACTIVITY IN BLOOD, TISSUE AND EXCTRETA AT 24 HR FOLLOWING ORAL ADMINISTRATION OF14C-ETHANOX® 330 (50 mg/kg) TO MALE SPRAGUE-DAWLEY RATS

 

Concentration (μg equivalent/g or ml)

Percent of Administered Dose

 

Rat No. 14

Rat No. 15

Rat No. 16

Mean±S.E.

Rat No. 14

Rat No. 15

Rat No. 16

Mean±S.E.

Blooda

0.002

0.004

0.002

0.003±0.001

0.000

0.001

0.000

0.000±0.000

Liver

0.044

0.058

0.058

0.053±0.005

0.005

0.007

0.006

0.006±0.001

Kidneys

0.000

0.010

0.001

0.004±0.003

0.000

0.000

0.000

0.000

Lungs

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

Muscleb

0.000

0.000

0.000

0.000

0.000

0.000

0.000

0.000

Fat

0.003

0.008

0.011

0.007±0.002

ND

ND

ND

ND

Skinc

0.003

0.002

0.003

0.003±0.000

0.001

0.001

0.001

0.001±0.000

GI tract

-

-

-

-

54.442d

9.609d

20.528d

27.526±12.850

Urine

-

-

-

-

0.012d

0.129d

0.202d

0.114±0.055

Faeces

-

-

-

-

45.585d

88.898d

77.922d

70.802±13.000

Recovery

-

-

-

-

98.045

98.645

98.659

98.450±0.202

aBased on 7% of body weight.

bBased on 40% of body weight.

cBased on 16% of body weight.

dSignificant counts, i.e., more than two times the background (36.5 cpm). All others are below the limit of accurate detection.

ND = not determined.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
The data generated from this study indicate that Ethanox® 330 orally administered to rats was not absorbed to any appreciable extent (less than 0.2% of the administered doses).
Executive summary:

The primary objective of this study was to assess the absorption of Ethanox® 330 following oral administration to rats. To achieve this objective, 15 rats were treated with 14C-labeled Ethanox® 330 and sacrificed in groups of three each at early (0-5,1, 2, and 4 hr) and late (24 hr) time periods for determination of radioactivity in blood and selected tissues. For the rats sacrificed at 24 hr, elimination of 14C in urine and faeces was also determined. A preliminary experiment using one rat indicated thatprac­tically no radioactivity (~0.001% of the dose) was eliminated in the expired air. In this experiment the administered dose was recovered in faeces(~87%) and in the GI tract(~12%) atthe time of sacrifice (24 hr). Less than 0.01% of the dose was found in urine and only 0.06% was recovered in blood and tissue.

The counts in most samples were low and were considered insignificant (less than two times the background). The low levels in livers increased at later times and were highestat the 24-hr sampling period. Radioactivity in lungs of one rat sacrificed at 1 hr and two rats sacrificed at 2 hr washigher than in other tissues. Although all tissues were rinsed thoroughly following necropsy, these lung samples may have been contaminated. Alternatively, the trace impurities in the labelled compound may have been absorbed and preferentially concentrated in these lungs.

An average of ~71% of the administered doses was eliminated in faeces and ~28%was recovered in the GI tract.Only trace amounts were excreted in urine (~0.1%) or recovered in the tissues examined (< 0.01%). Very low concentrations of radioactivity were found in blood, liver, kidneys, fat, and skin, while no radioactivity was found in lungs or muscle.

 

The data generated from this study indicate that Ethanox® 330 orally administered to rats was not absorbed to any appreciable extent (less than 0.2% of the administered doses). All the administered radioactivity was essentially eliminated in faeces or recovered in the GI tract; only trace amounts were found in urine and in the tissues examined. The lack of appre­ciable radioactivity in liver at early or late time periods following dosing suggests that Ethanox® 330 was not absorbed from the GI tract and then excreted by the biliary route before elimination in faeces. Although the presence of small amounts of radioactivity in urine and tissue indicates that minor absorption of orally administered Ethanox® 330 occurred, this may have resulted from the absorption of trace impurities present in the 14C-labeled Ethanox® 330.