1,3,5-tris(3,5-di-tert-butyl-4-hydroxybenzyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

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

Test guideline

Qualifier:

according to guideline

Guideline:

other: Not specified

GLP compliance:

yes

Test material

Test material information

Constituent 1

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

IUPAC Name:

4,4',4''-[(2,4,6-trimethylbenzene-1,3,5-triyl)tris(methylene)]tris(2,6-di-tert-butylphenol)

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 results

Main ADME results 1

Type:

absorption

Results:

less than 0.2%

Main ADME results 2

Type:

distribution

Results:

trace amounts were found in urine and in the tissues examined

Main ADME results 3

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
Blood^a	0.038^d	0.006^d
Liver	0.098^d	0.009^d
Kidneys	0.056	0.001
Lungs	0.031	0.000
Muscle^b	0.0421	0.034
Fat	0.041	ND
Skin^c	0.030	0.010
GI tract	-	11.941^d
Expired air	-	0.001
Urine	-	0.007^d
Faeces	-	86.876^d
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	Blood^a	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	0.307^b	0.001	0.001	0.000	0.002^b
	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	1.190^b	0.002	0.002	0.000	0.013^b
	10	0.016^b	0.017	0.013	0.097^b	0.002^b	0.002	0.000	0.001^b
	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.
Blood^a	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
Muscle^b	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
Skin^c	0.003	0.002	0.003	0.003±0.000	0.001	0.001	0.001	0.001±0.000
GI tract	-	-	-	-	54.442^d	9.609^d	20.528^d	27.526±12.850
Urine	-	-	-	-	0.012^d	0.129^d	0.202^d	0.114±0.055
Faeces	-	-	-	-	45.585^d	88.898^d	77.922^d	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 te test substance orally administered to rats was not absorbed to any appreciable extent (less than 0.2% of the administered doses) with some 87% excreted in faecal matter.

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.