Trinonyl benzene-1,2,4-tricarboxylate

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 1981 to 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Well conducted study in accordance with standards applicable at the time

Data source

Reference

Reference Type:

secondary source

Title:

Unnamed

Year:

1984

Materials and methods

Objective of study:

absorption

metabolism

toxicokinetics

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Deviations:

yes

Remarks:

only single dose applied

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

EC Number:

222-020-0

EC Name:

Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

Cas Number:

3319-31-1

Molecular formula:

C33H54O6

IUPAC Name:

tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate

Constituent 2

Reference substance name:

Tri-(2-ethylhexyl)trimellitate

IUPAC Name:

Tri-(2-ethylhexyl)trimellitate

Details on test material:

- Name of test material (as cited in study report): [Hexyl-2-14C] tri-(2-ethylhexyl)trimellitate (TEHT, TOTM)
- Molecular formula (if other than submission substance): C33-H54-O6
- Molecular weight (if other than submission substance): 546.79
- Smiles notation (if other than submission substance): CCCCC(CC)COC(=O)c1ccc(c(c1)C(=O)OCC(CC)CCCC)C(=O)OCC(CC)CCCC
- InChl (if other than submission substance): 1S/C33H54O6/c1-7-13-16-25(10-4)22-37-31(34)28-19-20-29(32(35)38-23-26(11-5)17-14-8-2)30(21-28)33(36)39-24-27(12-6)18-15-9-3/h19-21,25-27H,7-18,22-24H2,1-6H3

- Analytical purity: 91.7%
- Impurities (identity and concentrations):di-(2-ethylhexyl)terephthalate
- Radiochemical purity (if radiolabelling): >99%
- Specific activity (if radiolabelling): 4.82 mCi/mmole
- Locations of the label (if radiolabelling): [Hexyl-2-14C] tri-(2-ethylhexyl)trimellitate
- Stability under test conditions: stable

Radiolabelling:

yes

Remarks:

[Hexyl-2-14C] tri-(2-ethylhexyl)trimellitate

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Wilmington, MA, USA
- Weight at study initiation: 200 to 300 g
- Fasting period before study: 16 h
- Individual metabolism cages: yes (during study each rat was placed in a separate glass metabolism chamber)
- Diet: Purina Rodent Laboratory Chow #5001 (provided from St. Louis, MO, USA), available immediately after dosing, ad libitum
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- no data

IN-LIFE DATES: From: 3 NOV 1981 To: 9 NOV 1981

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Radiolabelled TOTM was mixed with non-labelled TOTM and dissolved in corn oil

VEHICLE
- Justification for use and choice of vehicle (if other than water): commonly used vehicle for poorly water soluble substances
- Concentration in vehicle: no data
- Amount of vehicle (if gavage): no data
- Lot/batch no. (if required): no data

HOMOGENEITY AND STABILITY OF TEST MATERIAL: no data

Duration and frequency of treatment / exposure:

single dose

Doses / concentrations

Remarks:

Doses / Concentrations:
100 mg/kg body weight (16-18 microCi/animal)

No. of animals per sex per dose / concentration:

4

Control animals:

yes

Details on study design:

Immediately after dosing sampling of expired air, urine and faeces from treated animals began.
Dehumified air was drawn through the metabolism chamber (rate about 500 ml/min). The expired air was passed through two silica gel traps to remove volatile organic compounds and then through two NaOH traps to remove CO2. The silica gel traps were changed daily and the NaOH traps were changed at frequent intervals up to 144 hours post dose.
Urine and faeces were collected at various times up to 144 hours post dose and radioactivity was measured. At each collection period the metabolism cages were rinsed with water. On termination following 144 hours cages were also washed with methanol followed by methylene chloride. Rats were killed 144 hours post dose.

Radioactivity Measurements - A Liquid Scintillation Spectrometer (Tri-Carb Model 2660, from Packard Instrument Co., Downers Grove, IL, USA) was used for measurement of radioactivity. Efficiency corrections were made by external standardisation. Most samples were counted in a naphthalene-dioxane based scintillator fluid ()(Eastmen-Ready to use I or II).

Processing of Tissues and Excreta - Extrationi from tissues were made twice using 95% aqueous acetone. Combined extracts were counted in scintillation fluid. Residues were dried, powdered, combusted and counted. Carcasses were shredded in a blender with 300 mL acetone. The mixture was refluxed overnight and then filtered. The residue was refluxed with n-hexane and filtered. The acetone and n-hexane filtrates were then counted in scintillation fluid (II and I, respectively) . Solid residues were pulverised and portions were measured for radioactivity. Faeces were homogenised in acetone. Solids were separated by centrifugation (10000 rpm, 2 minutes) and residues extracted twice with acetone. Combined extracts were counted in scintillation fluid II. Residues were then further extracted with methylene chloride (three times) and the extracts counted in scintillation fluid I.

Analysis of TOTM and its metabolites in excreta - Radioactive components in faecal extracts were separated using HPLC (from Waters Assoc., Milford, AM, USA with Dupont C18 analytical reverse phase columns), and utilising both spectrophotometric (uv/vis; Varian Instruments, Waltham, MA, USA) and radiochemical detection (by counting eluent fractions with Multirac fraction collector from LKB, Bromma, Sweden). Standards used for metabolite characterisation were prepared from [Hexyl 2-14C] tri(2-ethylhexyl)trimellitate and from synthetic mixtures prepared by the incomplete esterification of trimellitic acid with 2-ethylhexanol. These mixtures were formed from 1:1 and 2:1 molar ratios of 2-ethyhexanol and trimellitic acid and were regarded as being mono- and di-esters, mono(2-ethylhexyl)trimellitate and di(2-ethylhexyl)trimellitate. Faecal extracts were analysed directly using HPLC (25 x 0.45 cm C18 column, 10 minute linear gradient from 50% water/methanol to 100% methanol, solvent flow 1.5 mL/min). Urinary metabolites were analysed by GC/MS following solvent extraction in diethyl ether (acidification and re-extration , derivatised with diazomethane; GC separation on Hewlett-Packard model HP5987A gc/ms witht 10 m 0.3 mm DRS column and temperature gradient 130 to 300 °C (10 °C/min)).

Analysis of Kinetic Data - Excretion half-lives were estimated from the rates of excretion of radioactivity in urine and in expired air.

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Body fluids sampled: urine, faeces, cage washes, expired air
- Time and frequency of sampling: 4, 8, 12, 24, 36, 48, 72, 96, 120 and 144 hours post dose

- Tissues sampled: brain, heart, lungs, liver, spleen, kidneys, small and large intestines, testes, abdominal fat (remaining carcasses were stored too)
- Time and frequency of sampling: at the end of the study only (144 hours post dose)

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: 4, 8, 12, 24, 36, 48, 72, 96, 120 and 144 hours post dose
- From how many animals: 4 individual animals (no details on pooling of samples reported)
- Method type(s) for identification: GC-MS, HPLC-UV-vis, Liquid scintillation counting
- Limits of detection and quantification: no data

Statistics:

Students' t-test. Values of p < 0.05 considered as significant

Results and discussion

Main ADME results

Main ADME results 1

Type:

absorption

Results:

Half-life - 0.7 hours

Main ADME results 2

Type:

excretion

Results:

Half-life - 42 hours

Toxicokinetic / pharmacokinetic studies

Details on absorption:

The excretion of radioactivity by male Sprage-Dawley rats following gavage administration of a single dose (100 mg TOTM/kg bw) occurred mainly in the faeces, accounting for approximately 75% of the administered dose. In urine and expired air 16.3% and 1.9% were found, respectively.
For expired air, two peaks in the plot of the rate of excretion of 14CO2 were observed in the treated rats. The first occuring 2-3 hours post dose and the second 8-12 hours post dose, the authors suggesting this to be a result of metabolism of 2-ethylhexanol occurring from its release by hydrolysis from the tri-ester followed by a further hydrolysis step releasing 2-ethyl hexanol from the di-ester. The half-life for initial absorption was estimated to be approximately 0.7 hours.

Details on distribution in tissues:

At the end of the study (144 hours post dose) radioactivity remaining in tissues and carcasses was low (0.6% f the administered dose). Tissues analysed for radioactivity revealed only liver (5x) and adipose tissue (3x) showing concnetrations of radioactivity greater than the carcass average.

Details on excretion:

Radioactivity in the faecal extracts investigated via HPLC was identified as 86% TOTM, 7% di-(2-ethylhexyl)trimellitate and 1% mono-(2-ethylhexyl)trimellitate. Only one of the three possible mono-ester isomers was found.
Urine analysis by GC/MS revealed the presence of 2-ethylhexanol, 2-ethylhexanoic acid, 2-heptanone and mono-(2-ethylhexyl)trimellitate. Comparision to HPLC retention times indicated that the mono-ester was the identical isomer as found in faecal extracts. No isomers of di-(2-ethylhexyl)trimellitate were found. Urinary elimination of radioactivity was bi-phasic with half-lives of 3.1 and 42 hours

Metabolite characterisation studies

Metabolites identified:

yes

Details on metabolites:

Faeces (% from radioactivity): 86% TOTM, 7% di-(2-ethylhexyl)trimellitate and 1% mono-(2-ethylhexyl)trimellitate (only one isomer of three possible isomers)
Urine: 2-ethylhexanol, 2-ethylhexanoic acid, 2-heptanone and mono-(2-ethylhexyl)trimellitate (same isomere as in faeces), no isomers of di-(2-ethylhexyl)trimellitate were found

Any other information on results incl. tables

Recovery of radioactivity was 94.1% of the administered dose.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): low bioaccumulation potential based on study results
In this study four male Sprague-Dawley rats were treated with 100 mg [Hexyl-2-14C] tri-(2-ethylhexyl)trimellitate (TOTM)/ kg bodyweight via gavage. Faeces, urine and expired air were collected at different times up to 144 hours post dosing. Analysis of radioactivity revealed that 75% of the administered dose were excreted via the faeces (mostly unchanged). In urine and expired air 16.3% and 1.9% of radioactivity were found, respectively. Furthermore results indiacte that TOTM is only partially hydrolised in the gastrointestinal tract to 2-ethylhexanol, and respective di - and mono-esters. Evidence suggests that only 2-ethylhexanol and one mono-ester were absorbed. No significant accumulation in any of the analysed tissues was identified (liver and adipose tissue showed 5 and 3fold higher radioactivity than average carcass levels). At the end of the study only < 0.6% of the administered dose remained in the carcass.

Executive summary:

In this study absorption, metabolism and excretion of [Hexyl-2 -14C] tri-(2 -ethylhexyl)trimellitate (TOTM, 97.1% purity) were investigated. To this end, 100 mg TOTM/ kg bodyweight were administered via gavage to each of four fasted male Sprague-Dawley rats. Animals were placed in separate metabolism cages and faeces, urine and expired air were collected at 4, 8, 12, 24, 36, 48, 72, 96, 120 and 144 hours post dosing. At the end of the study animals were killed, various tissues were removed and radioactivity was analysed. The overall recovery of radioactivity was 94.1%.

75% of the administered dose were excreted via the faeces. In urine and expired air 16.3% and 1.9% of radioactivity were found, respectively.

In faeces, 86% of the radioactivity accounted for TOTM, and only 7% di-(2-ethylhexyl)trimellitate and 1% mono-(2 -ethylhexyl)trimellitate were indetified (remark: only one of the three possible mono-esters was identified). In urine, metabolites identified were 2-ethylhexanol, 2-ethylhexanoic acid, 2-heptanone and mono-(2-ethylhexyl)trimellitate. Comparison to HPLC retention times indicated that the mono-ester was the identical isomer as found in faecal extracts. No isomers of di-(2-ethylhexyl)trimellitate were found. Urinary elimination of radioactivity was bi-phasic with half-lives of 3.1 and 42 hours. For expired air, two peaks in the plot of the rate of excretion of 14CO2 were observed in the treated rats. The first occuring 2-3 hours post dose and the second 8-12 hours post dose. At the end of the study only <0.6% of the administered dose was remainig in the carcass. Tissues analysed for radioactivity revealed only liver (5x) and adipose tissue (3x) showing concnetrations of radioactivity greater than the carcass average.

In conclusion these results indicate that TOTM is only partially hydrolised in the gastrointestinal tract to 2-ethylhexanol, and respective di - and mono-esters. Only 2 -ethylhexanol and mono-ester were absorbed. Only 2 -ethylhexanol seems to be further metabolised and exrected in urine and to 14CO2 in expiration air. As mono-(2ethylhexyl)trimellitate was excreted unchanged no metabolism was evident.