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

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

publication

Title:

Human metabolism and kinetics of tri-(2-ethylhexyl)trimellitate (TEHTM) after oral administration

Author:

Hollerer C, Becker G, Goen T and Eckert E

Year:

2018

Bibliographic source:

Archives of Toxicology https://doi.org/10.1007/s00204-18-2264-2

Materials and methods

Objective of study:

metabolism

toxicokinetics

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Human volunteers were administered a single oral dose of the substance. The substance and its theoretical primary metabolites were investigated in blood samples (up to 48 hours after exposure) and in urine samples (collected until 72 hours after exposure).

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

Details on test material:

- Name of test material (as cited in study report): Not reported
- 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): c1(c(ccc(c1)C(OC[C@@H](CCCC)CC)=O)C(OC[C@@H](CCCC)CC)=O)C(OC [C@@H](CCCC)CC)=O
- 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
- Structural formula attached as image file (if other than submission substance): see 3319-31-1 structure.png

Radiolabelling:

no

Test animals

Species:

other: human

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Age at study initiation: 46 +/- 8 years
- Weight at study initiation: 78 +/- 13 kg

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
70 – 105 mg of purified substance (> 99.9%) were prepared together with a teaspoon of butter on bread and ingested by each volunteer.

Duration and frequency of treatment / exposure:

Single administration

Doses / concentrations

Dose / conc.:

1.12 mg/kg bw (total dose)

No. of animals per sex per dose / concentration:

2 males / 2 females

Details on study design:

- Dose selection rationale: Set not to exceed the DNEL stated in the REACH registration dossier.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, blood
- Time and frequency of sampling: Blood - pre-dose, 1, 3, 5, 7 24 and 48 hours post-dose. Urine - Pre-dose, hourly for the first 8 hours post-dose and at intervals for up to 72 hours post-dose

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, blood
- Time and frequency of sampling: Blood - pre-dose, 1, 3, 5, 7 24 and 48 hours post-dose. Urine - Pre-dose, hourly for the first 8 hours post-dose and at intervals for up to 72 hours post-dose
- Method type(s) for identification: LC-MS-MS
- Limits of detection and quantification: From 1.0 to 16.4 microgram/L, depending on medium and metabolite

Results and discussion

Metabolite characterisation studies

Metabolites identified:

yes

Any other information on results incl. tables

TEHTM and its metabolites in blood

None of the analytes was detectable in any of the volunteers prior to exposure to TEHTM. Following exposure, TEHTM was measurable in the blood samples with a mean maximum level of 158 ± 245 μg/L at 3-5 hours post-exposure. After this time the concentration of TEHTM decreased to a mean level of 44 ± 9.1 μg/L after 7 hours. TEHTM concentrations continued to slowly decrease with a finalmean concentration of 31 ± 10 μg/L measured 48 hours after exposure. The authors comment that an observed relatively high standard deviation at the 5-hour sampling time was a result of a distinctly higher TEHTM blood level of one of the four volunteers, this possibly attributed to inter-individual differences in adsorbing and distributing TEHTM.

Metabolites of TEHTM, DEHTM and MEHTM, were detectable in the blood samples of all volunteers following exposure. 1,2-DEHTM was detected with a maximum level of 83 ± 17 μg/L and 2,4-DEHTM was detected with a maximum level of 26 ± 9.7 μg/L 3-hours post-exposure. 1,4-DEHTM was not observed.

The blood concentration of the diesters rapidly decreased and could no longer be detected (< LOD) 48-hours post-exposure. Blood concentration of the monoesters, 1-MEHTM and 2-MEHTM, increased up to 5 hours after TEHTM exposure, with mean levels of 12.5 ± 0.8 μg/L 1-MEHTM and 105 ± 23.9 μg/L 2-MEHTM. 4-MEHTM was not detected. After this time MEHTM concentrations decreased with the mean blood level of 1-MEHTM being below the limit of detection after 24 hours and 2-MEHTM being quantifiable in blood samples from all volunteers after 48 hours with a mean level of 13.2 ± 7.9 μg/L.

TEHTM showed the highest mean elimination half-life (27 hours) of all the metabolites investigated. The elimination half-lives of 1,2-DEHTM (3.8 hours) and 2,4-DEHTM (4.1 hours) indicate rapid metabolism, tothe isomers of MEHTM,and/or elimination. An elimination half-life of 6 hours was calculated for the mono-ester 1-MEHTM while the blood concentration of 2-MEHTM decreased distinctly slower with an elimination half-life of approximately 14 hours.

These findings suggest that TEHTM is absorbed via the intestine and further metabolised to isomers of DEHTM and MEHTM. It is presumed that TEHTM is hydrolysed at a lower rate than DEHTM, the latter being more easily accessible for enzymatic cleavage than the parent compound.

The data show that the ester side chains located on the aromatic ring of TEHTM are regioselectively hydrolysed, preferably at position 1, and with lower efficiency at position 4. Thus 2,4-DEHTM is presumably exclusively cleaved at position 4 to the metabolite 2-MEHTM while the diester 1,2-DEHTM is apparently additionally cleaved at position 2, resulting in the monoester 1-MEHTM. The mechanism of ester hydrolysis is thus connected to, and influenced by, the ester side chain at position 4 of the aromatic ring of TEHTM. The authors comment that this is probably due to a stereochemical barrier as the enzyme appears to be able to access and catalyze hydrolysis of the ester moiety at position 2, only if the side chain at position 4 is hydrolysed.

 

Characteristics of the kinetics of TEHTM and its metabolites in blood following oral exposure to 1.12 mg/kg body weight TEHTM (mean value;n= 4)

 

	Cmax(μg/L)	tmax(h)	t1/2(h)
TEHTM	159 ± 250	3 - 5	27
1,2-DEHTM	26 ± 9.7	3	3.8
2,4-DEHTM	83 ± 17	3	4.1
1,4-DEHTM	<LOD	-	-
1-MEHTM	13 ± 0.8	5	5.8
2-MEHTM	105 ± 23	5	14
4-MEHTM	<LOD	-	-

 

 

TEHTM metabolites in urine

Noneof the analytes were detectable in pre-exposure urine samples from the volunteers. After exposure the monoesters 1-MEHTM, 2-MEHTM, and 4-MEHTM, as well as several secondary oxidation products were identified in the urine samples of all volunteers with peak concentrations noted between 6 and 11 hours post-exposure. A distinct decrease of the mean concentration levels of these metabolites was then observed. 72 Hours following exposure, the levels of all analytes were below the limit of detection, with the exception of 2-MEHTM, 5cx-1-MEPTM, and 5OH-2-MEHTM

that were still quantifiable in urine, with mean levels of 5.1 ± 1.3, 1.7 ± 0.6, and 1.6 ± 1.3 μg/hour, respectively. The diesters, 1,2-DEHTM, 1,4-DEHTM and 2,4-DEHTM were not found in any of the urine samples, this possibly explained by their rapid degradation to MEHTM. It should also be considered that renal excretion of DEHTM may be unlikely due the high molecular weight and their elevated lipophilicity.

The monoesters 1-MEHTM, 2-MEHTM, and 4-MEHTM showed maximum urinary levels 7 hours after TEHTM exposure, this being 2 hours later than thetmax of 1-MEHTM and 2-MEHTM in blood. The mean urinary concentration levels at that time were 15 ± 3.7 μg/hour for 1-MEHTM, 115 ± 56 μg/hour for 2-MEHTM and 1.6 ± 0.7 μg/hour for 4-MEHTM. 2-MEHTM was found in the urine in distinctly higher levels than 1-MEHTM, demonstrating the distinct regioselective formation of the TEHTM monoesters. This is particularly notable with regard to 4-MEHTM which was found in the urine at a very low excretion rate and at a total amount of < 0.1% relative to the administered oral dose of TEHTM. While the study did not investigate oxidative metabolites of 4-MEHTM, chromatograms were examined for unidentified peaks showing the respective mass fragments of potential secondary metabolites of 4-MEHTM. No significant unidentified peaks were observed and it was concluded that the formation and urinary excretion of oxidative metabolites of 4-MEHTM after oral TEHTM exposure may be regarded as unlikely.

The urinary concentration levels of the secondary metabolites of 1-MEHTM and 2-MEHTM generally showed a similar time course of excretion as the monoesters themselves, with maximum levels occurring between 6 and 7 hours post-exposure. The exceptions were 5cx-2-MEPTM, 2cx-1-MMPTM and 2cx-2-MMPTM that showed urinary maximum levels 11 hours after exposure.

Biphasic elimination kinetics was observed for all the urinary metabolites. Elimination half-lives between 4 and 6 hours and between 10 and 33 hours were calculated for the first and the second elimination phases for almost all analytes. The metabolite 2cx-2-MMPTM was found in urine at very low levels, preventing the calculation of an elimination half-life. The urinary excretion rate of 5cx-2-MEPTM and 2cx-1-MMPTM was also low, allowing the calculation of only a single elimination half-life (17 and 15 hours, respectively), this probably reflecting combined elimination half-lives of the two phases. Levels of 4-MEHTM in the urine were already below the limit of detection 24 hours post-exposure so that only the elimination half-life of phase 1 could be calculated (5 hours).

Differences with respect to the extent of oxidation were observed with regard to the monoester metabolites. The main urinary metabolite was 2-MEHTM followed by two secondary metabolites of 1 -MEHTM (5cx-1 -MEPTM and 5OH-1-MEHTM). It appears that 1-MEHTM may be rapidly metabolised to its oxidative metabolites, resulting in relatively short elimination half-lives of 1-MEHTM,

as well as of its consecutive hydroxy- and oxo-metabolites. 2-MEHTM is apparently processed more slowly to oxidative metabolites, this illustrated by elevated elimination half-lives of 2-MEHTM and its metabolites. The authors comment that the slower excretion rate of the oxidized 2-MEHTM metabolites might be caused by the slower oxidation of 2-MEHTM itself, which may also explain the fact that 2-MEHTM was still detectable in blood even 48 hours and in urine 72 hours after exposure.

 

The total excretion rate of the metabolites in urine was assessed by examining the total cumulative amount of excreted metabolites as a function of time after exposure to TEHTM. The urinary excretion of several metabolites: 2-MEHTM, 5cx-1-MEPTM and 5OH-2-MEHTM appears to be incomplete even 72 hours after exposure, since their cumulative courses show a steady increase over time. In contrast, the cumulative excretion rate of 1-MEHTM reaches a plateau approximately 50 hours after TEHTM exposure, indicating a more complete elimination process.

  

Characteristics of renal excretion kinetics of TEHTM metabolites after oral exposure to 1.12 mg/kg body weight TEHTM (mean value;n= 4)

 

	REmax(μg/h)	tmax(h)	t1/2(h)^a	t1/2(h)^b	Fraction of administered dose (%)
1-MEHTM	15 ± 3.7	7 ± 0.2	4	10	0.3 ± 0.005
2-MEHTM	115 ± 56	7 ± 0.2	5	20	3.3 ± 0.9
4-MEHTM	1.6 ± 0.7	7 ± 0.2	5	-	0.04 ± 0.01
5cx-1-MEPTM	29 ± 18	7 ± 0.3	4	33	0.7 ± 0.09
5cx-2-MEPTM	3.7 ± 0.2	11 ± 0.2	17		0.2 ± 0.05
2cx-1-MMPTM	1.0 ± 0.5	11 ± 0.3	15		0.04 ± 0.01
2cx-2-MMPTM	0.2 ± 0.2	11 ± 0.2	-	-	0.02 ± 0.01
5OH-1-MEHTM	27 ± 9.5	6 ± 0.3	4	12	0.5 ± 0.03
5OH-2-MEHTM	18 ± 11	6 ± 0.3	6	27	0.6 ± 0.3
5oxo-1-MEHTM	5.2 ± 1.6	6 ± 0.3	4	10	0.1 ± 0.01
5oxo-2-MEHTM	2.9 ± 2.1	6 ± 0.3	4	16	0.09 ± 0.05

 

RE_max= maximum renal excretion rate (mean ± standard deviation)

t_max= time at which RE_maxwas found (mean ± standard deviation)

t_1/2is the elimination half-life of the respective metabolite

a Elimination phase 1

b Elimination phase 2

c Cumulative calculation over 72 hours after oral dosage of TEHTM (mean ± standard deviation)

 

 

A total of 5.8% of the administered TEHTM dose was recovered in urine 72 hours after exposure. The monoester 2-MEHTM was the main metabolite over this time with 3.3% of the applied dose recovered as this metabolite. This was followed by the

oxidative metabolites 5cx-1-MEPTM (0.66% of the applied dose), 5-OH-2-MEHTM (0.59% of the applied dose) and 5-OH-1-MEHTM (0.52% of the applied dose) as well as 1-MEHTM (0.30% of the applied dose) The monoester 4-MEHTM, together with the other investigated oxidative metabolites 5cx-2-MEPTM, 5oxo-1-MEHTM, 5oxo-2-MEHTM, 2cx-1-MMPTM, 2cx-2-MMPTM were found to be minor metabolites (total of 0.45% of the applied dose). Within the first 24 hours post-exposure, the TEHTM metabolites accounted for approximately 3.54% of the recovered urinary metabolites related to the oral dose of TEHTM and between 24 and 72 hours post-exposure a further 1.84% were excreted. A total of 5.38% of the administered

dose was excreted in urine in form of the main metabolites after 72 hours.

 

The total share of metabolites recovered in urine over a 72 hour period following exposure is low (5.8%), raising the question if there are further unidentified metabolites of TEHTM yet to be explored. The authors consider thisunlikely as the observed low urinary excretion rates are presumably caused by an equally low absorption rate of TEHTM via the intestine due to its high molecular weight and its

lipophilicity, leading to low solubility, and thus low permeability. The Biopharmaceutics Classification System (BPS) considers that substances of low solubility and low permeability are predominantly excreted unchanged by renal or biliary route.

  

Mean urinary excretion factors (FUE) as TEHTM dose equivalents in % ± standard deviation after 24 and 72 hours (n=4)

 

	Urinary excretion factor (FUE) as % TEHTM dose equivalents
	After 24 hours	After 72 hours
1-MEHTM	0.27 ± 0.03	0.30 ± 0.05
2-MEHTM	2.12 ± 0.57	3.31 ± 0.89
4-MEHTM	0.03 ± 0.01	0.04 ± 0.01
MEHTM (total)	2.43 ± 0.60	3.65 ± 0.94
5OH-1-MEHTM	0.45 ± 0.05	0.52 ± 0.03
5OH-2-MEHTM	0.29 ± 0.18	0.59 ± 0.33
5OH-MEHTM (total)	0.74 ± 0.23	1.11 ± 0.36
5oxo-1-MEHTM	0.09 ± 0.03	0.10 ± 0.01
5oxo-2-MEHTM	0.05 ± 0.02	0.09 ± 0.05
5oxo-MEHTM (total)	0.14 ± 0.05	0.19 ± 0.05
5cx-1-MEPTM	0.41 ± 0.09	0.66 ± 0.09
5cx-2-MEPTM	0.09 ± 0.02	0.16 ± 0.05
5cx-MEPTM (total)	0.50 ± 0.11	0.82 ± 0.14
2cx-1-MMPTM	0.02 ± 0.01	0.04 ± 0.01
2cx-2-MMPTM	0.01 ± 0.01	0.02 ± 0.01
2cx-MMPTM (total)	0.03 ± 0.01	0.06 ± 0.01
Sum of all metabolites	3.85 ± 1.01	5.83 ± 1.51
Sum of main metabolites (1-MEHTM, 2 -MEHTM, 5cx-1-MEPTM, 5OH-2-MEHTM, 5OH-1-MEHTM)	3.54 ± 0.92	5.38 ± 1.39

 

Bold = sum of metabolites

 

Applicant's summary and conclusion

Conclusions:

The metabolism and excretion kinetics of Tri-(2-ethylhexyl) trimellitate (TEHTM) following administration of a single oral dose has been studied in humans. TEHTM was shown to be absorbed and regioselectively hydrolysed to its diesters di-2-(ethylhexyl) trimellitates (1,2-DEHTM and 2,4-DEHTM) with maximum blood concentrations occurring 3-hours post-exposure, and further hydrolysis to the monoester isomers mono-2-(ethylhexyl) trimellitates (1-MEHTM, 2-MEHTM) with peak blood concentrations 5-hours post-exposure. Biphasic elimination kinetics of urinary metabolites was observed. The most dominant urinary metabolite was 2-mono-(2-ethylhexyl) trimellitate (2-MEHTM), followed by a number of specific secondary metabolites.
Approximately 5.8% of the orally administered dose was recovered in urine over a 72 hour period.

Executive summary:

The metabolism and excretion kinetics of Tri-(2-ethylhexyl) trimellitate (TEHTM) following administration of a single oral dose has been studied in humans. TEHTM was shown to be absorbed and regioselectively hydrolysed to its diesters di-2-(ethylhexyl) trimellitates (1,2-DEHTM and 2,4-DEHTM) with maximum blood concentrations occurring 3-hours post-exposure, and further hydrolysis to the monoester isomers mono-2-(ethylhexyl) trimellitates (1-MEHTM, 2-MEHTM) with peak blood concentrations 5-hours post-exposure. Biphasic elimination kinetics of urinary metabolites was observed. The most dominant urinary metabolite was 2-mono-(2-ethylhexyl) trimellitate (2-MEHTM), followed by a number of specific secondary metabolites.

Approximately 5.8% of the orally administered dose was recovered in urine over a 72 hour period.