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Reference
Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: new publication
Justification for type of information:
see Analogue Approach
Reason / purpose for cross-reference:
read-across source
Objective of study:
absorption
excretion
metabolism
Qualifier:
no guideline required
Specific details on test material used for the study:
Tri-(2-ethylhexyl) trimellitate (TEHTM) acquired from Sigma-Aldrich (Steinheim, Germany). TEHTM was purified in advance using silica gel chromatography. TEHTM of high purity (> 99.9%) was obtained.
The diesters 1,4-di-(2-ethylhexyl) trimellitate (1,4-DEHTM) and 2,4-di-(2-ethylhexyl) trimellitate (2,4-DEHTM) as well as the monoesters 1-mono-(2-ethylhexyl)trimellitate (1-MEHTM) and 4-mono-(2-ethylhexyl)trimellitate (4-MEHTM) were purchased from Toronto Research Chemicals (Toronto, Canada).
The diester 1,2-di-(2-ethylhexyl) trimellitate (1,2-DEHTM) and the monoester 2-mono-(2-ethylhexyl)trimellitate (2-MEHTM) were synthesized by the Institute for Thin Film and
Microsensoric Technology e.V. (Teltow, Germany) with a stated chemical purity of at least 95% each.
The following substances were synthesized by the Max Planck Institute for Biophysical Chemistry, Facility for Synthetic Organic Chemistry (Göttingen, Germany) with a chemical purity of at least 95%, and an isotopically purity of at least 98% each: tri-(2-ethylhexyl-1,1-D2) trimellitate (D6-TEHTM), 1-mono-(2-ethyl-5-hydroxyhexyl) trimellitate (5OH1-MEHTM), 2-mono-(2-ethyl-5-hydroxyhexyl) trimellitate (5OH-2-MEHTM), 1-mono-(2-ethyl-5-oxohexyl) trimellitate (5oxo-1-MEHTM), 2-mono-(2-ethyl-5-oxohexyl (5oxo-2-MEHTM), 1-mono-(2-ethyl-5-carboxypentyl) trimellitate (5cx-1-MEPTM), 2-mono-(2-ethyl-5-carboxypentyl) trimellitate (5cx-2-MEPTM), 2-mono-(2-carboxymethylhexyl) trimellitate (2cx-2-MMHTM), 1-mono(2-carboxymethylhexyl) trimellitate (2cx-1-MMHTM), a mixture of the isomers of 1-mono-(2-ethyl-D5-hexyl) trimellitate (D5-1-MEHTM) and 2-mono-(2-ethyl-D5hexyl) trimellitate (D5-2-MEHTM) (D5-1-MEHTM/ D5-2-MEHTM, 40/60, w/w) as well as a mixture of the isomers of 1,4-di-(2-ethylhexyl-1,1-D2) trimellitate (D4-1,4-DEHTM) and 2,4-di-(2-ethylhexyl-1,1-D2) trimellitate (D4-2,4-DEHTM), 1-mono-(2-ethyl-D5-5-hydroxyhexyl) trimellitate (D5-5OH-1-MEHTM), 1-mono-(2ethyl-D5-5-oxohexyl) trimellitate (D5-5oxo-1-MEHTM), 2-mono-(2-ethyl-D5-5-oxohexyl) trimellitate (D5-5oxo-2-MEHTM), 1-mono-(2-ethyl-D5-5-carboxypentyl) trimellitate (D5-5cx-1-MEPTM) and 2-mono-(2-ethylD5-5-carboxypentyl) trimellitate (D5-5cx-2-MEPTM).
Radiolabelling:
other: non-radiolabelled deuterium isotopes
Species:
other: humnan volunteers
Sex:
male/female
Details on test animals or test system and environmental conditions:
Four healthy volunteers (two men and two women, mean age 46 ± 8 years, mean body weight 78 ± 13 kg)
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
70–105 mg (mean 87.5 ± 15.5 mg) of purified TEHTM were prepared together with a teaspoon of butter on bread and ingested by each volunteer.
Dose / conc.:
1.12 mg/kg diet
Remarks:
mean dose amounted
No. of animals per sex per dose / concentration:
2 m/2 w
Control animals:
no
Positive control reference chemical:
no
Details on study design:
For the in vivo study, four healthy volunteers (two men and two women, mean age 46 ± 8 years, mean body weight 78 ± 13 kg) were selected. Prior to oral exposure, each of the four volunteers collected one urine sample. Additionally, one pre-exposure blood sample of each volunteer was drawn using EDTA-monovettes. Subsequently, the volunteers were orally exposed to TEHTM (> 99.9 %). For oral exposure, 70–105 mg (mean 87.5 ± 15.5 mg) of purified TEHTM were prepared together with a teaspoon of butter on bread and ingested by each volunteer. The mean dose amounted to 1.12 mg/kg body weight for the four individuals. The oral exposure of each volunteer took place in the early morning.
Details on dosing and sampling:
The mean dose amounted to 1.12 mg/kg body weight for the four individuals.
The doses were thus selected that they did not exceed the DNEL of TEHTM, which was set within the ECHA registration of TEHTM to 1.13 mg/kg body weight per day for oral exposure of the general population.

The volunteers were instructed to collect urine samples in hourly intervals for the first 8 h at least. Throughout 72 h, all urine samples were collected from each volunteer and the sampling times and the excretion volumes were recorded. All in all, each volunteer delivered between 25 and 44 urine samples. The total 72 h urine volume ranged from 5.0 to 9.7 L (mean 7.8 L). The samples were stored frozen at − 20 °C until analysis. Additionally, blood samples were drawn from each volunteer after 1, 3, 5, 7, 24, and 48 h post-exposure. Thus, altogether seven blood samples were collected from each of the volunteers. Oral exposure and sample collection were approved by the local ethics committee of the University of Erlangen/Nuernberg. All subjects were informed about the aims of the study and gave their written informed consent to their participation. All subjects stated that they did not have any known occupational exposure to TEHTM or to any other plasticizers. Volunteers that received infusions or transfusions during the past 6 weeks prior to this study, were excluded.
Preliminary studies:
Comparison of human metabolism of TEHTM and DEHP
The present metabolism study is based on the hypothesis that TEHTM and DEHP are metabolized similarly. Indeed, that theory was confirmed, as all the here postulated TEHTM metabolites were identified in the blood and urine samples of the four volunteers, respectively. Nevertheless, the additional ester side chain of TEHTM at position 4 of the aromatic ring leads to a distinctly extended and more complex metabolism of TEHTM compared to DEHP. Moreover, TEHTM metabolism is characterized by a pronounced regioselective formation of metabolites. Additionally, in direct comparison, rather different amounts of the respective urinary metabolites of DEHP and TEHTM in relation to the respective oral dose were found in humans. In a study of Koch et al. (2005), three different doses of D4-DEHP were administered independently, and orally to a volunteer, and it was observed that about 74% of the orally administered dose was recovered in urine as metabolites, 48 h after dosage. For TEHTM, however, only 5.8% of the orally administered dose was recovered in urine, even 72 h after dosage, indicating a distinctly lower and slower urinary excretion rate of TEHTM compared to DEHP. Presumably, the additional ester side chain of TEHTM provides a stereochemical barrier, causing lower resorption and lower enzyme affinity. This is in line with a previous study (Höllerer et al. 2018a), where TEHTM itself was observed to be relatively stable towards enzymatic hydrolysis. DEHP and the regioisomers of DEHTM, however, showed a pronounced and fast metabolism. Furthermore, the elimination kinetics of TEHTM and DEHP appear to be rather different: in the case of D4-DEHP 90% of the total recovered urinary dose was already excreted 24 h after dosage (Koch et al. 2005). For TEHTM, only 66% were recovered at that time, indicating again a significantly slower excretion process of TEHTM. For both plasticizers, biphasic elimination kinetics was observed. Interestingly, the metabolite spectrum of 1-MEHTM and its oxidized metabolites is rather similar to that of MEHP and the secondary DEHP metabolites, while the metabolite spectrum of 2-MEHTM and its oxidized metabolites proved to be quite different. Presumably, this is due to the catalytic effectivity of enzymes that is dependent on the molecular structure of the substrates. In fact, the molecular structure of 1-MEHTM is rather congruent to MEHP with the exception of an additional acid moiety. For 2-MEHTM, however, the ester-side chain is located at a different position, which might lead to a poorer oxidation efficiency by CYP 450.
TEHTM is hydrolyzed with a lower effectivity than DEHTM that is evidently more easily accessible for enzymatic cleavage than the parent compound TEHTM. This is in line with a previous in vitro study (Höllerer et al. 2018a), demonstrating that TEHTM is rather stable towards enzymatic hydrolysis, whereas the hydrolysis of DEHTM leads efficiently to the formation of the monoesters 2-MEHTM and 1-MEHTM. In that study, the isomer 4-MEHTM was also generated at small amounts only (< 1%). Another explanation concerns the tissue distribution of TEHTM in a multi-compartment model (Martis et al. 1987; Derendorf et al. 2011). Since TEHTM holds a rather high molecular weight associated with a distinctive lipophilicity (log P o w = 8.88 at 55 °C) (Sigma-Aldrich 2018), it might be accumulated in lipid depots. Hence, it may be initially withdrawn from metabolism and is subjected to the enterohepatic cycle leading to a repeated TEHTM release and resorption. This consideration is in line with an animal study, where biliary excretion was found to be the major route of elimination for TEHTM (Martis et al. 1987). As biliary excreted substances underlie the enterohepatic cycle, TEHTM might be returned to the intestine with bile after passing the liver and be resorbed again, resulting in a delayed tmax of TEHTM in blood and an extended elimination half-life. In fact, TEHTM showed the highest mean elimination half-life (t1/2 = 27 h) of all the here investigated analytes (Table 1). By contrast, the elimination half-lives of 1,2-DEHTM (t1/2 = 3.8 h) and 2,4-DEHTM (t1/2 = 4.1 h) indicate a rapid metabolization and elimination, presumably to the isomers of MEHTM. For the hydrolysis product 1-MEHTM, an elimination half-life of 6 h was calculated, also indicating a relatively fast elimination from blood. The blood concentration of 2-MEHTM, however, decreased distinctly slower with an elimination half-life of about 14 h.
Derendorf H, Gramatté T, Schäfer HG, Staab A (2011) Pharmakokinetik kompakt, 3 edn. Wissenschaftliche Verlagssgesellschaft mbH, Stuttgart

Höllerer C, Becker G, Göen T, Eckert E (2018a) Regioselective ester cleavage of di-(2-ethylhexyl) trimellitates by porcine liver esterase. Toxicol In Vitro 47:178–185. https ://doi.org/10.1016/j. tiv.2017.11.015

Martis L, Freid E, Woods E (1987) Tissue distribution and excretion of tri-(2-ethylhexyl)trimellitate in rats. ‎J Toxicol Environ Health 20:357–366. https ://doi.org/10.1080/15287 39870 95309 89

Sigma-Aldrich (2018) GHS Material Safety Data Sheet Trioctyl trimellitate, CAS-No: 3319-31-1, Version 4.1, reviewed on 14 Jan 2015


Type:
absorption
Results:
is resorbed but comparatively low
Type:
metabolism
Results:
the substance is metabolized; slow metabolism: metabolites in blood and urine: DEHTM and MEHTM; TEHTM is hydrolyzed with a lower effectivity than DEHTM that is evidently more easily accessible for enzymatic cleavage than the parent compound TEHTM.
Type:
excretion
Results:
slow excretion rate; 5.8% of the orally administered dose was recovered in urine, even 72 h after dosage
Details on absorption:
The results of the study indicate that TEHTM is resorbed in the intestine and further metabolized to isomers of DEHTM and MEHTM, however, at low extent, because TEHTM itself was still detectable in the blood samples even 48 h after exposure.
Details on distribution in tissues:
In the blood samples of all volunteers, drawn before exposure to TEHTM, none of the analytes was detectable (< LOD). After exposure, TEHTM was found in the blood samples of each volunteer with a mean maximum level of 158 ± 245 µg/L at t = 3–5 h. Afterwards, a distinct decrease of the TEHTM blood concentration was observed to a mean level of 44 ± 9.1 µg/L after 7 h. Then, the concentration of TEHTM decreased rather slowly, with a final mean concentration of 31 ± 10 µg/L 48 h after exposure. The relatively high standard deviation at t = 5 h is due to a distinctly higher TEHTM blood level of one volunteer compared to the other three. This might be attributed to interindividual differences in adsorbing and distributing TEHTM, e.g., caused by food effects, or differences in the volume of distribution in the human body.

Since TEHTM holds a rather high molecular weight associated with a distinctive lipophilicity (log P o w = 8.88 at 55 °C) (Sigma-Aldrich 2018), it might be accumulated in lipid depots. Hence, it may be initially withdrawn from metabolism and is subjected to the enterohepatic cycle leading to a repeated TEHTM release and resorption.
Details on excretion:
Equally to the blood analysis, also in the urine samples of all volunteers drawn prior to TEHTM exposure, none of the analytes were detectable (< LOD). After exposure, however, the monoester isomers 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 between 6 and 11 h postexposure, respectively. Then, a distinct decrease of the mean concentration levels of these metabolites was observed. Finally, 72 h post 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/h, respectively. Interestingly, none of the diester metabolites 1,2-DEHTM, 1,4DEHTM, and 2,4-DEHTM were found in any of the urine samples (< LOD). This may be explained by their rapid degradation to MEHTM and further metabolites. Furthermore, it has to be considered that a renal excretion of DEHTM is indeed rather unlikely due the high molecular weight of the DEHTM isomers and their elevated lipophilicity.

Renal excretion kinetics
The monoesters 1-MEHTM, 2-MEHTM, and 4-MEHTM showed maximum urinary levels at 7 h after TEHTM exposure, which is 2 h later than the tmax of 1-MEHTM and 2-MEHTM in blood. The mean urinary concentration levels at that time were 15 ± 3.7, 115 ± 56, and 1.6 ± 0.7 µg/h, respectively. Thus, by analogy to the blood samples, 2-MEHTM was found in the urine samples in distinctly higher levels than 1-MEHTM. These findings demonstrate again the distinct regioselective formation of the TEHTM monoesters, particularly with regard to the isomer 4-MEHTM, that was found in the urine samples of all volunteers at a very low excretion rate and at a total amount of < 0.1%, related to the oral dose of TEHTM. As a consequence, no oxidative metabolites of 4-MEHTM were investigated in this study. Nevertheless, all chromatograms were analyzed for unidentified peaks showing the respective mass fragments of potential secondary metabolites of 4-MEHTM. However, no significant unidentified peaks were observed, so that the formation and urinary excretion of oxidative metabolites of 4-MEHTM after oral TEHTM exposure may be regarded as rather unlikely. The urinary concentration levels of the secondary metabolites showed a similar course of excretion in time, compared to the monoesters 1-MEHTM and 2-MEHTM, with maximum levels between 6 and 7 h, with the exception of 5cx-2-MEPTM, 2cx-1-MMPTM and 2cx-2-MMPTM that showed delayed urinary maximum levels at 11 h after exposure. In fact, biphasic elimination kinetics was observed for all urinary metabolites. In general, for the first and the second elimination phases, elimination half-lives between 4 and 6 h, and between 10 and 33 h, were calculated for almost all analytes, respectively. The metabolite 2cx-2-MMPTM was found in urine in very low levels, so that no mean urinary excretion curve could be reliably assessed, and hence, no elimination half-life was calculated. For 5cx-2-MEPTM and 2cx1-MMPTM, the urinary excretion rate was also rather low, so that only one elimination half-life (t1/2 = 17 and 15 h, respectively) could be reliably assessed for these metabolites, probably reflecting combined elimination halflives of phase 1 and phase 2. The urinary level of 4-MEHTM was already found to be below the limit of detection 24 h after TEHTM ingestion, so that only the elimination half-life of phase 1 could be assessed (t1/2 = 5 h). With regard to the secondary metabolites of TEHTM, differences with respect to the extent of oxidation were observed: 2-MEHTM was mainly excreted unchanged in urine, while the secondary oxidative metabolites of 1-MEHTM (namely 5cx-1-MEPTM and 5OH-1-MEHTM) showed distinctly elevated excretion rates. All in all, the main urinary metabolite was 2-MEHTM followed by two secondary metabolites of 1-MEHTM (5cx-1-MEPTM and 5OH-1-MEHTM). Only then a secondary metabolite of 2-MEHTM (5OH-2-MEHTM) and 1-MEHTM itself follows. The slower elimination kinetics of the secondary metabolites of 2-MEHTM compared to the oxidation products of 1-MEHTM are also reflected by the respective elimination half-lives. The monoester 1-MEHTM is evidently rapidly metabolized to its oxidative metabolites, causing relatively short elimination half-lives of 1-MEHTM, as well as of its consecutive hydroxy- and oxo-metabolites. 2-MEHTM, by contrast, is apparently slower processed to oxidative metabolites, illustrated by elevated elimination half-lives of 2-MEHTM and its metabolites. The slower excretion rate of the oxidized 2-MEHTM metabolites might be caused by the slower oxidation of 2-MEHTM itself, and is probably due to sterical reasons. This also explains the fact that 2-MEHTM was still detectable in blood and urine even 48 h and 72 h after exposure, respectively. Moreover, this is in accordance with the observed different elimination halflives of 1-MEHTM (t1/2 = 5.8 h) and 2-MEHTM (t1/2 = 14 h) in blood (Table 1). Furthermore, the total excretion rate of the metabolites in urine was analyzed. For that purpose, the total cumulative amount of excreted metabolites (expressed in µmol) as a function of time after TEHTM exposure. The urinary excretion of several TEHTM metabolites, namely 2-MEHTM, 5cx-1-MEPTM, and 5OH-2-MEHTM is apparently not yet complete, even 72 h after exposure, since their cumulative courses show a steady increase over time. By comparison, the cumulative excretion rate of 1-MEHTM already reaches a plateau, approximately 50 h after TEHTM exposure, indicating a rather complete elimination process.

Urinary excretion factors (FUE)
Additionally, urinary excretion factors (FUE) of the investigated urinary TEHTM metabolites, as percentages of the applied dose, were calculated. All in all, 5.8% of the administered TEHTM dose was recovered in urine 72 h after exposure. The monoester 2-MEHTM was observed to be the main metabolite over the whole time with 3.3% of the applied dose recovered in urine after 72 h, followed by the specific oxidative metabolites 5cx-1-MEPTM with 0.66%, 5-OH-2-MEHTM with 0.59%, and 5-OH-1-MEHTM with 0.52%, as well as 1-MEHTM with 0.30%. The monoester 4-MEHTM as well as the other investigated oxidative metabolites 5cx-2-MEPTM, 5oxo-1-MEHTM, 5oxo-2-MEHTM, 2cx-1-MMPTM, 2cx2-MMPTM were found to be minor metabolites, making up in total for 0.45% of the applied dose within 72 h. Within 24 h, the aforementioned main TEHTM metabolites made up for about 3.54% of the recovered urinary metabolites related to the oral dose of TEHTM. Then, between 24 and 72 h further 1.84% were excreted. In total, 5.38% of the administered TEHTM dose was excreted in urine in form of the main metabolites after 72 h. In general, the total share of metabolites recovered in urine up to 72 h after exposure related to the oral dose is rather low (5.8%), and the results might pose the question if there are further unidentified metabolites of TEHTM that still have to be explored. However, this can be considered as rather unlikely as the observed low urinary excretion rates are presumably caused by an equally low resorption rate of TEHTM due to its high molecular weight and its lipophilicity, leading to low solubility, and thus low permeability, probably causing a poor resorption of TEHTM in the intestine.


Metabolites identified:
yes
Remarks:
1,2- and 2,4-DEHTM (di-2-(ethylhexyl) trimellitates) and 1-and 2-MEHTM (mono-2-(ethylhexyl) trimellitates) in blood and urine
Details on metabolites:
The TEHTM hydrolysis products DEHTM and MEHTM were also detectable in the blood samples of all volunteers post exposure. Here, 1,2-DEHTM and 2,4-DEHTM were detected with a maximum level of 83 ± 17 and 26 ± 9.7 µg/L, respectively, 3-h post-exposure, while the isomer 1,4-DEHTM was not observed in any of the samples. Then, in contrast to TEHTM, their blood concentration rapidly decreased until they could no longer be detected (< LOD), 48-h post-dosage. In comparison, the blood concentration of the monoesters, 1-MEHTM and 2-MEHTM, showed a steep increase up to 5 h after TEHTM exposure, with mean levels of 12.5 ± 0.8 and 105 ± 23.9 µg/L, respectively. Here again, one possible regioisomer, namely 4-MEHTM, was not detectable at all. Then, the MEHTM concentration decreased again with the mean blood level of 1-MEHTM being below the limit of detection after 24 h, whereas 2-MEHTM was still quantifiable in blood samples of all volunteers, even 48 h after TEHTM exposure, with a mean level of 13.2 ± 7.9 µg/L.

After resorption in the intestine TEHTM is further metabolized to isomers of DEHTM and MEHTM, however, at low extent, because TEHTM itself was still detectable in the blood samples even 48 h after exposure. Presumably, TEHTM is hydrolyzed with a lower effectivity than DEHTM that is evidently more easily accessible for enzymatic cleavage than the parent compound TEHTM. TEHTM seems to be rather stable towards enzymatic hydrolysis, whereas the hydrolysis of DEHTM leads efficiently to the formation of the monoesters 2-MEHTM and 1-MEHTM.

TEHTM showed the highest mean elimination half-life (t1/2 = 27 h) of all investigated analytes. By contrast, the elimination half-lives of 1,2-DEHTM (t1/2 = 3.8 h) and 2,4-DEHTM (t1/2 = 4.1 h) indicate a rapid metabolization and elimination, presumably to the isomers of MEHTM. For the hydrolysis product 1-MEHTM, an elimination half-life of 6 h was calculated, also indicating a relatively fast elimination from blood. The blood concentration of 2-MEHTM, however, decreased distinctly slower with an elimination half-life of about 14 h.

The results of the in vivo study illustrate that the ester side chains located at the aromatic ring of TEHTM are regioselectively hydrolyzed, preferably at position 1, and with lower effectivity, at position 4. Consequently, 2,4-DEHTM is presumably exclusively cleaved at position 4 to the metabolite 2-MEHTM. The diester 1,2-DEHTM, however, is apparently additionally cleaved at position 2, as the monoester 1-MEHTM was also found in the blood samples. Thus, the mechanism of ester hydrolysis is obviously connected to, and influenced by the ester side chain at position 4 of the aromatic ring of TEHTM. 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 hydrolyzed.


Table 1  Characteristics of the kinetics of TEHTM and its metabolites in blood after oral exposure to 1.12 mg/kg body weight TEHTM (mean value; n = 4 volunteers)

 metabolites/parent compound  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  -  -
Conclusions:
The results show that TEHTM is resorbed and metabolized in the human body, after oral exposure. The resorption rate of TEHTM in humans is comparatively low, combined with an apparently rather slow metabolism and excretion rate. Metabolites found in the blood are the primary diester metabolites (DEHTM) and the the primary monoester metabolites (MEHTM). In urine was found the most prominent urinary biomarker was found to be 2-MEHTM, followed by several specific secondary metabolites.
Executive summary:

The metabolism and excretion kinetics of TEHTM, a substitute for high molecular weight plasticizers such as DEHP, was investigated in four human volunteers. The results show that TEHTM is resorbed and metabolized in the human body, after oral exposure. Here, a pronounced regioselective ester hydrolysis of TEHTM in blood was observed, as well for the primary diester metabolites (DEHTM), as for the primary monoester metabolites (MEHTM). The same applies for the investigated urinary metabolites of TEHTM, where the most prominent urinary biomarker was found to be 2-MEHTM, followed by several specific secondary metabolites. All in all, approximately 5.8% of the orally administered dose was recovered in urine over a period of 72 h. The results of this study indicate that the resorption rate of TEHTM in humans is comparatively low, combined with an apparently rather slow metabolism and excretion rate as TEHTM and selected metabolites were still detectable in blood and urine after 48 h and 72 h postexposure, respectively.

Description of key information

No studies on the target substance are available.

Based on information of similar Trimellitates and phthalates the following is expected:

Accumulation in man of Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) is unlikely.

-low bioaccumulation factor of the substance
-likely that mainly the hydrolysis products will be absorbed, likely that hydrolysis is slow, therefore also oral absorption is low
-dermal and inhalative absorption are lower than oral absorption (dermal < < inhalative < oral)
- rats are far more efficient at hydrolysing the esters and, subsequently, absorbing the monoester than primates (and presumably humans)
- slowly metabolised and excreted in the urine and faeces
- accumulation is negligible (based on an oral rat study)
- minimal or no evidence of accumulation in rodent tissues.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

The metabolism and excretion kinetics of TEHTM, a substitute for high molecular weight plasticizers such as DEHP, was investigated in four human volunteers. The results show that TEHTM is resorbed and metabolized in the human body, after oral exposure. Here, a pronounced regioselective ester hydrolysis of TEHTM in blood was observed, as well for the primary diester metabolites (DEHTM), as for the primary monoester metabolites (MEHTM). The same applies for the investigated urinary metabolites of TEHTM, where the most prominent urinary biomarker was found to be 2-MEHTM, followed by several specific secondary metabolites. All in all, approximately 5.8% of the orally administered dose was recovered in urine over a period of 72 h. The results of this study indicate that the resorption rate of TEHTM in humans is comparatively low, combined with an apparently rather slow metabolism and excretion rate as TEHTM and selected metabolites were still detectable in blood and urine after 48 h and 72 h postexposure, respectively.

Accumulation in man of Esterification products of Guerbet alcohols, C24-26, branched and cyclic with benzene-1,2,4-tricarboxylic acid 1,2-anhydride (3:1) is unlikely. Reasons (including data on structurally related phthalates and the trimellitate tris(2-ethylhexyl)benzene-1,2,4-tricarboxylate):

-low bioaccumulation factor of the substance

-likely that mainly the hydrolysis products will be absorbed, likely that hydrolysis is slow, therefore also oral absorption is low

-dermal and inhalative absorption are lower than oral absorption (dermal < inhalative < oral)

- rats are far more efficient at hydrolysing the esters and, subsequently, absorbing the monoester than primates (and presumably humans)

- rapidly metabolised and excreted in the urine and faeces

- accumulation is negligible (based on an oral rat study)

- minimal or no evidence of accumulation in rodent tissues.

Experimental data are not available for the assessment of the toxicokinetic properties of the target substance and read-across for toxicokinetic property assessment is therefore a possible approach to characterise toxicokinetic endpoints for the substance. A structural related trimellitate, the Tris(2-ethylhexyl)benzene-1,2,4-tricarboxylate (TOTM) was investigated in an oral rat study. Both substances contain the same acid function, but are esterified with different alcohols. Additionally helpful information on phthalates is available. Based on structurally similarity to phthalate esters (with two instead of three carboxylic functions) a similar toxicokinetic behaviour can be expected for that substance class. Absorption and metabolism were studied for Tris(2-ethylhexyl)benzene-1,2,4-tricarboxylate (TOTM) (14C-labeled on the 2-carbon atom of 2-ethylhexyl group) mixed with corn oil and administered by gavage in a single dose of 100 mg/kg of body weight in four male Sprague Dawley rats. Rats were placed in glass metabolism cages and urine, feces and expired air were collected for 144 hrs. About 75% of the dose was excreted unchanged in the feces, 16% in the urine as metabolites and 1.9% was expired as 14CO2. Radioactivity was excreted in the feces as unchanged TOTM (85% of the fecal radioactivity), mono- and di(2-ethylhexyl) trimellitate (MOTM and DOTM, respectively,) and unidentified polar metabolites. Metabolites in the urine were identified as MOTM and metabolites of 2-ethylhexanol. Less than 0.6% of the dose remained in whole tissues. Elimination of 14CO2 was biphasic with half-lives of 4.3 and 31 hours, and excretion of radioactivity in the urine was biphasic with half-lives of 3.4 hours and 42 hours. Based on remaining labeled ratio (less than 0.6% of dose) in whole tissues at 144 hours, it is considered that the accumulation of this chemical is negligible (SIDS, Tris(2-ethylhexyl)benzene-1,2,4-tricarboxylate, 2002). The same is likely for 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters. Hydrolysis of the phthalate diester to a monoester enhances absorption. Studies have shown that for high-MW esters, breakdown (metabolism; hydrolysis) of the ester bond to liberate one alcohol and a remaining monoester greatly increases the absorption, since the monoester and alcohol are absorbed more rapidly than the diester. The more hydrolysis occurs, the more monoester is available for absorption. Once absorbed, the monoester continues to be metabolised into subsatnces that are excreted in the urine. If exposure is through skin from contact with polyvinylchloride (PVC) articles containing phthalate esters, the absorption is very slow because the ester is not hydrolysed and must be absorbed intact. Experiments with laboratory animals and human skin have demonstrated that the absorption rate of high levels of exposure of the skin to neat chemical might not result in adverse health effects because the absorption is so slow and the metabolism of the ester is minimal. For inhalation exposure, absorption is likely to be slow but faster than absorption through the skin and slower than absorption from ingestion. The route of exposure that results in the most efficient absorption of phthalate esters is ingestion. Laboratory studies have demonstrated, however, that rats are far more efficient at hydrolysing the esters and, subsequently, absorbing the monoester than primates (and presumably humans). This means that when studies of phthalate esters are conducted in laboratory animals where health effects are observed following very high doses of an ester, it is very difficult to reproduce such effects in primates (and presumably humans) because primates do not absorb phthalate esters as efficiently as other laboratory animals. Primates and humans absorb about seven times less phthalate than do rats (especially for high MW-esters). At low doses, the absorption may be more comparable (Staples, 2003). It is likely that the hydrolysis of trimellitates to the corresponding monoesters is worse simply because of the existence of three instead of two ester groups and additionally because of possible steric hinderance of hydrolysis. Hence it is likely that the absorption is also worse compared to phthalates.

Various reviews of different phthalate esters by the Australian National Industrial Chemicals Notification and Assessment Scheme (NICNAS) are available. There are at least some toxicokinetic data available for many of the 24 phthalates, with the majority of testing conducted via the oral and dermal route. Limited data on absorption are available. Data, mostly from rats and with one human study on DEHP, suggest phthalates are readily absorbed via the oral route. A clear trend was noted in dermal absorption with data collected from five phthalates, DEP, BBP, DEHP, DINP and DIDP. Studies indicated a decrease in dermal absorption with increasing side chain length. The only information available on inhalation is on DIDP, which was readily absorbed from the lung. There is minimal or no evidence of accumulation in rodent tissues (NICNAS, Phthalates Hazard Compendium, 2008). Studies on several phthalates indicate that they are rapidly metabolised and excreted in the urine and faeces. They undergo phase I biotransformation, that is, primary metabolism into their hydrolytic monoesters by hydrolysis of one of their ester bonds. Further enzymatic oxidation of the alkyl chain occurs in some of the phthalates, resulting in more hydrophilic oxidative metabolites. Monoesters and the oxidative metabolites of phthalates may continue to undergo phase II biotransformation to produce glucuronide conjugates with increased water solubility. The data on the toxicokinetics indicate that phthalates in general are likely to be rapidly absorbed as the monoester from the gut and excreted via the urine ((NICNAS, Phthalates Hazard Compendium, 2008). For example following ingestion, di-n-octyl phthalate (DnOP) is rapidly metabolised and absorbed from the gastrointestinal tract as the monoester mono-n-octylphthalate (MnOP). Half-life of the monoester in the blood is approximately 3 hours. The liver is capable of metabolising DnOP. Elimination occurs via the urine with levels of MnOP exceeded after 24 hours by the other oxidative metabolite mono-(3-carboxypropyl) phthalate (MCPP) (NICNAS, 2008).

Literature

NICNAS, 2008; Existing Chemical Hazard Assessment Report, di-n-octyl phthalate, Australian Government of Health and Ageing NICNAS

NICNAS, 2008; Phthalate Hazard Compendium, A summary of physicochemical and human health hazard data for 24 ortho-phthalate chemicals, Australian Government of Health and Ageing NICNAS, June 2008

SIDS, 2002, SIDS Initial Assessment Report For SIAM 14, Paris, France, 26-28 March 2002, Tris(2-ethylhexyl)benzene-1,2,4-tricarboxylate Staples, C.A., 2003, Phthalate Esters, The Handbook of Environmental Chemistry, Springer Verlag Berlin, Heidelberg, New York

Staples, C.A., 2003, Phthalate Esters, The Handbook of Environmental Chemistry, Springer Verlag Berlin, Heidelberg, New York

Various reviews of different phthalate esters by the Australian National Industrial Chemicals Notification and Assessment Scheme (NICNAS) are available. The data on the toxicokinetics indicate that phthalates in general are likely to be rapidly absorbed as the monoester from the gut and excreted via the urine.

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