Trinonyl benzene-1,2,4-tricarboxylate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Type:

absorption

Results:

Half-life - 0.7 hours

Type:

excretion

Results:

Half-life - 42 hours

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

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

Recovery of radioactivity was 94.1% of the administered dose.

Conclusions:

In this in vivo study low bioaccumulation potential for [Hexyl-2-14C] tri-(2-ethylhexyl)trimellitate (TOTM) was identified.

Executive summary:

The study used as source investigated absorption, metabolism and excretion of [Hexyl-2 -14C] tri-(2 -ethylhexyl)trimellitate (TOTM, 97.1% purity). 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. Justification and applicability of this data is outlined in the read-across report in section 13 or find a link in cross-reference “assessment report”.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Sex:

male

Type:

other: hydrolysis

Results:

no hydroylsis (metabolism) of TOTM in rat homogenate within 30 minutes

Retention time of TOTM was excessively long (>30 minutes), analysis was consequently only performed for metabolites. No evidence for the release of 2 -ethylhexanol within the 30 minutes incubation with rat gut homogenate was found.

The authors suggested that this might be either a result of the very limited water solubility of TOTM or another hydrolysis limiting factor.

Conclusions:

In this in vitro study, potential hydrolysis of TOTM was investigated using freshly prepared rat gut (small intestines) homogenate. Under the condistions of the study, no hydrolysis of TOTM was observed, while other phthalates were hydrolysed significantly (e.g. di(2-ethylhexyl)phthalate).

Executive summary:

The study used as source investigated the potential of hydrolysis in an in vitro study using rat gut homogenate. In this assay four plasticisers were compared, including TOTM. Within the 30 minutes incubation period no evidence of any hydolysis of TOTM was observed. In contrast significant hydrolysis was observed with the other test substances (e.g. di(2 -ethylhexyl)phthalate). Justification and applicability of this data is outlined in the read-across report in section 13 or find a link in cross-reference “assessment report”.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Type:

distribution

Results:

see table below

Type:

distribution

Results:

peak levels were found in liver, spleen and lungs (further details see table below)

Type:

excretion

Results:

After 14 days 16.9% and 3.3% were found in faeces and urine, respectively (further details see table below)

Details on distribution in tissues:

The distribution half life, disposition half life, apparent distribution volume & plasma clearance were 46.2 mins, 5.34d , 7.49 l/kg and 40.5 ml/kg respectively. These data reflect a fairly rapid initial distribution and slow clearance of TOTM from the body. The large apparent distribution volume indicates extensive uptake of the compound by the tissues.

48h after administration: Recovery from the liver, lungs & spleen was 66.8, 13.1 & 3.8%, respectively.
The liver was the major organ in terms of TOTM uptake peak radio activity 71.6% after 24h, declining to 44.8% after 14d.
The lungs accounted for 18.6% ( a significant portion) of the administered dose after 1h which declined from 12.6% after 72h to 0.6% after 7d.
The levels in the spleen remained constant throughout the 14d period.
Small amounts of radioactivity were found in the heart, kidney, pancreas & adrenals & all levels had declined to less than 0.1% of the radioactivity after 7d.
The concentration of the parent compound was low due to the rapid initial distribution into the extravascular compartment that was not kinetically analysed.

Details on excretion:

After 14 days 16.9% and 3.3% of the administered dose was found in the faeces and urine, respectively. The renal clearance of 14C TOTM was 13.ml/kg.h while plasma clearance was 40.5 ml/kg.h indicating that the rapid disappearance from plasma was due to uptake by tissues rather than excretion.
Elimination of radioactivity via faeces indicates biliary excretion of TOTM and/or its metabolites.

Plasma concentration of radioactivity following i.v. administration (10.5 mg/kg) of [C^14-carbonyl] – tri-(2 ethylhexyl) trimellitate to rats

Time (h)	Plasma concentration (dpm/ml) X 10^3for animal numbers
	2	4	6	11	22
0.5	473.8	526.95	469.03	773.6	491.15
1.0	409.81	365.37	297.83	291.45	465.31
3.0	231.48	199.16	70.54	281.10	351.48
6.0	16.9	14.31	11.06	36.55	376.60
12.0	3.95	4.02	4.52	5.51	15.91
24.0	1.85	2.79	1.83	2.11	3.04
48.0	1.96	2.44	2.40	1.65	2.63
72.0	2.40	-	2.74	1.83	2.30
168.0	2.34	1.54	1.22	2.11	1.91
336.0	1.56	1.17	0.97	2.16	1.79

Distribution of radioactivity following i.v. administration (15.6 mg/kg) of [C^14-carbonyl] –tri-(2 ethylhexyl) trimellitate to rats

Tissue	Mean^a(± SD) % of dose recovered
	1 h	6 h	24 h	48 h	72 h	168 h	336 h^b
Liver	51.3 ± 15.9	63.9 ± 7.5	71.6 ± 9.6	66.8 ± 10.8	62.8 ± 5.8	55.7 ± 9.0	44.8 ± 1.6
Spleen	3.8 ± 1.0	4.3 ± 0.6	5.3 ± 1.9	3.8 ± 0.7	4.1 ± 1.0	4.9 ± 0.3	4.7 ± 0.6
Lungs	18.6 ± 14.3	12.9 ± 6.7	8.5 ± 6.8	13.1 ± 8.0	12.6 ± 8.9	0.8 ± 0.1	0.6 ± 0.2
Kidneys	0.6 ± 0.1	0.2 ± 0.05	0.2 ± 0.0	0.2 ± 0.05	0.2 ± 0.05	<0.1	<0.1
Heart	0.4 ± 0.1	0.3 ± 0.06	0.2 ± 0.0	0.1 ± 0.05	0.1 ± 0.05	<0.1	<0.1
Pancreas	0.1 ± 0.0	<0.1	<0.1	<0.1	<0.1	<0.1	<0.1
Adrenal glands	<0.1	0.1 ± 0.05	<0.1	<0.1	<0.1	<0.1	<0.1
Urine	<0.1	0.4 ± 0.1	1.0 ± 0.3	1.2 ± 0.9	1.0 ± 0.3	2.4 ± 0.8	2.2 ± 1.2
Faeces	None	None	2.0 ± 0.8	5.6 ± 2.0	10.0 ± 1.3	18.2 ± 4.6	25.6 ± 7.8
Plasma(/10 ml)	35.3 ± 9.8	0.57 ± 0.14	0.2 ± 0.006	0.064 ± 0.018	0.066 ± 0.022	0.025 ± 0.006	0.022 ± 0.002
GI tract & contents	1.3 ± 0.3	2.8 ± 0.4	2.4 ± 0.5	2.3 ± 0.6	2.7 ± 0.6	0.6 ± 0.2	0.5 ± 0.0
Total recovery (without plasma)	76.1 ±	84.9 ±	91.2 ±	93.1	93.5	81.0	79.4

^a_{N = 4 except when otherwise noted;}^b_{N = 3}

Excretion of radioactivity in the urine and faeces following i.v. administration (10.5 mg/kg) of [C^14-carbonyl] – tri-(2 ethylhexyl) trimellitate to rats

Rat number	% of dose excreted at each timepoint (h)
	0-24		24-48		48-72		72-168		168-336		Total
	urine	faeces	urine	faeces	urine	faeces	urine	faeces	urine	faeces	urine	faeces
2	1.09	0.78	0.72	0.92	1.08	1.33	1.17	5.72	0.47	6.45	4.53	15.20
4	0.79	1.52	0.24	3.40	0.13	2.26	0.70	5.38	0.75	13.50	2.61	26.31
6	0.97	1.77	0.63	1.65	0.41	2.51	0.47	4.24	0.63	3.04	3.11	13.01
11	1.31	-	0.38	3.31	0.40	0.69	1.09	4.08	0.29	5.64	3.47	13.92
22	1.02	0.66	0.51	1.90	0.19	2.26	0.63	5.74	0.25	4.33	2.60	15.89
Mean ± SD	1.04 ± 0.19	1.20 ± 0.55	0.50 ± 0.19	2.24 ± 1.06	0.44 ± 0.38	1.81 ± 0.30	0.81± 0.30	5.03 ± 0.81	0.48 ± 0.21	6.63 ± 4.06	3.26 ± 0.80	16.87 ± 5.40

Conclusions:

In this in vivo study low bioaccumulation potential for [Hexyl-2-14C] tri-(2-ethylhexyl)trimellitate (TOTM) was identified.

Executive summary:

The study used as source investigated the distribution and excretion of triethylhexyl trimellitate (TOTM). Following intravenous injection into male Sprague-Dawley rats, TOTM distributes mainly in the liver, lungs and spleen. Elimination from the plasma was biphasic with half-life values of 46.2 minutes and 5.34 days. Excretion of the substance or its metabolites over 14 days was slow with 16.9 and 3.3% of the administered dose found in the faeces and urine respectively, suggesting a half-life of approximately 40 days. Justification and applicability of this data is outlined in the read-across report in section 13 or find a link in cross-reference “assessment report”.

Reference 4

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

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

Adequacy of study:

supporting study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Absorption in different matrices:

- Receptor fluid, receptor chamber, donor chamber (in vitro test system): Mouse - 0; pig - 0
- Skin preparation (in vitro test system): Mouse - 1.32 +/- 0.53 nmol/mg; pig - 0.35 +/- 0.19 nmol/mg

Dose:

5.4 mM

Parameter:

percentage

Absorption:

0 %

Remarks on result:

other: 12

Remarks:

for both mice and pigs, flux [nmol/square cm/hour] was 0

Conclusions:

In vitro skin absorption examination of triethylhexyl trimellitate (TOTM) via analytical methods in Franz cells using full-thickness skin from female nude mice and pigs revealed, that TOTM does not significantly penetrate the skin.

Executive summary:

The in vitro study used as source investigated skin absorption of triethylhexyl trimellitate using analytical methods in Franz cells with full-thickness skin samples excised from female nude mice and specific pathogen-free pigs. The receptor medium contained 40% ethanol, and the donor medium was 5.4 mM triethylhexyl trimellitate in 40% ethanol/pH 7.4 buffer. The skin samples were removed from the cells after a 12 h exposure and tape-stripped. The accumulation of triethylhexyl trimellitate was 1.32 ± 0.53 nmol/mg in nude mouse and 0.35 ± 0.19 nmol/mg in pig skin; the flux was 0 nmol/cm2/h for both mouse and pig skin. Triethylhexyl trimellitate was not found in the receptor medium after 12 h, indicating no biologically relevant availability for dermal absorption. Justification and applicability of this data is outlined in the read-across report in section 13 or find a link in cross-reference “assessment report”.

Description of key information

No information for the submission substance or the analogues substances is available. Thus only data for another structurally related substance (i.e. TOTM, a trimellitate ester containing branched alcohol of C8 chain length) are presented here as supporting evidence.
In one in vitro study, no hydrolysis of TOTM could be observed when the test material was incubated for 30 minutes with rat gut homogenate (either at 37 or 5°C). In an in vivo toxicokinetic study were 100 mg TOTM/kg bw was administered via gavage to 4 male rats, it was determined that 75% of the administered dose were excreted in the faeces (mainly unchanged). 16.3% were excreted in the urine and 1.9% of the radioactivity was exhaled as 14CO2. Metabolites identified in faeces were di-esters (7%) and on isomer of mono-ester (1%). In urine  metabolites identified were 2-ethylhexanol, 2-ethylhexanoic acid, 2-heptanone and the same mono-ester as in the faeces. Excretion in urine and expiration of 14CO2 was biphasic (urine half lifes: 3.1 and 42 hours; expiration peaks: 2-3 hours and 8-12 hours). No signifcant accumulation of the test material was noted. Overall the results indicate that there is only a low potential for bioaccumulation.
In another in vivo study investigating distribution and elimination kinetics of TOTM following intravenous administration to rats, results indicating a fairly rapid initial distribution and slow clearance of TOTM from the body over the 14-day observation period. The majority of the radioactivity was distributed in the liver (peak 71.6% after 24 h), lungs (peak 18.6% after 1 h), and spleen (5.3% after 24 h), with radioactivity declining after peaking in the liver and lungs, but in the spleen, the amount of radioactivity recovered mostly remained constant for 14 days.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Additional information

Hydrolysis by esterases is regarded as an important first step in the absorption of ortho-phthalates. Thus the potential of hydrolysis has been examined in an in vitro study using rat gut homogenate. In this assay four plasticisers were compared, including TOTM. Within the 30 minutes incubation period no evidence of any hydolysis of TOTM was observed. In contrast significant hydrolysis was observed with the other test substances (e.g. di(2 -ethylhexyl)phthalate; Fox, 1984, RL2).

In one reliable and well conducted 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 a 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 (Enriquez, 1984, RL2).

In another in vivo study the distribution and excretion of triethylhexyl trimellitate (TOTM) was determined in male Sprague-Dawley rats. The study was conducted with two experimental setups each with intravenous substance administration.

Serial blood sampling was conducted with 5 rats dosed intravenously (i.v.) with 10.5 mg/kg [14C-carbonyl]triethylhexyl trimellitate (>98% radiochemically pure; 59.9 μCi/kg) in 2.5-3.5 ml of a soybean oil-water (10:90) emulsion. Blood samples were collected prior to dosing, and at 10 time points from 0.5 h to 336 h (14 days) after dosing. The animals were placed in metabolism cages, and urine and faecal samples were collected at various intervals for 14 days. The distribution half-life, disposition half-life, apparent distribution volume, and plasma clearance were 46.2 min, 5.34 days, 7.49 l/kg, and 40.5 ml/kg·h, respectively, indicating a fairly rapid initial distribution and slow clearance of TOTM from the body. Over the 14-day period, 3.3% of the radioactivity was recovered in the urine and 16.9% was recovered in the faeces; renal clearance was 13 ml/kg·h.

Twenty-eight rats were then dosed i.v. with 15.6 mg/kg [14C-carbonyl]triethylhexyl trimellitate (28.0 μCi/kg) in 2.6-3.6 ml of the vehicle; groups of 4 rats were killed at 1, 6, 24, 48, 72, 168, and 336 h after dosing. Blood samples, urine, and faeces were collected, and at necropsy, several organs were removed and analyzed for radioactivity. The majority of the radioactivity was distributed in the liver, lungs, and spleen. The peak radioactivity in the liver was 71.6% of the dose at 24 h, in the lungs 18.6% at 1 h, and in the spleen 5.3% at 24 h; the radioactivity in the liver and lungs declined after peaking, and in the spleen, the amount of radioactivity recovered mostly remained constant for 14 days (Martis, 1987, RL2).

The in vitro skin absorption of triethylhexyl trimellitate (TOTM) was determined via analytical methods in Franz cells using full-thickness skin samples excised from female nude mice and specific pathogen-free pigs. The receptor medium contained 40% ethanol, and the donor medium was 5.4 mM triethylhexyl trimellitate in 40% ethanol/pH 7.4 buffer. The skin samples were removed from the cells after a 12 h exposure and tape-stripped. The accumulation of triethylhexyl trimellitate was 1.32 ± 0.53 nmol/mg in nude mouse and 0.35 ± 0.19 nmol/mg in pig skin; the flux was 0 nmol/cm2/h for both mouse and pig skin. Triethylhexyl trimellitate was not found in the receptor medium after 12 h, indicating no biologically relevant availability for dermal absorption (Pan, 2014, RL2).