Tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate

Endpoint summary

Description of key information

Read-across, OECD Guideline 401, Acute Oral Toxicity to rat: LD50 > 2000 mg/kg bw

Read-across, Federal Insecticide, Fungicide, and Rodecticide Act, Part 163, Title 40; Code of the Federal Regulations 40 CRF 163.81., Acute Dermal Toxicity to rabbit: LD50 > 2000 ml/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

INTRODUCTION
In order to fulfil the information requirements according to Annex VIII to Regulation (EC) No1907/2006 which applies for tonnages in the range between 10 and 100 tonnes/year, data on physico-chemical, toxicity, ecotoxicity and environmental fate properties of a chemical must be submitted. For the registration of tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate (EC No 221-508-0, CAS No 3126-80-5), available experimental data are confined to some physical-chemical and environmental fate information. Further information, notably for toxicicological and ecotoxicological endpoints, can be obtained from studies using the source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate, with the abbreviation TOTM (EC No 222-020-0, CAS No 3319-31-1). Data on structurally related ester compounds with other aliphatic alcohols can strengthen the read-across approach. In this regard, trioctyl benzene-1,2,4-tricarboxylate is considered as supporting compound.

The read-across justification relies on the principles detailed in the Guidance on information requirements and chemical safety assessment, Chapter R.6: QSARs and grouping of chemicals (ECHA, 2008), and the Read-Across Assessment Framework document (ECHA, 2017). The read-across hypothesis implies that different, but structurally similar compounds produce the same type of effects, or both are characterized by the absence of effects (analogue approach - Scenario 2). The properties of the target substance are predicted to be quantitatively equal or lower when compared to those of the source substance (worst-case prediction). Similar but not identical (bio-)transformation products or metabolites with the same type of functional groups may occur but are not exclusively the basis for the read-across hypothesis.

The source and the supporting compounds are characterized by a low-toxicity profile, with minor concerns arising from repeated dose and reproduction toxicity testing of the source substance. Due to the low systemic toxicity, information on the mode of action is limited which otherwise could be used to improve the read-across hypotheses. On the other hand, regarding the environmental fate, the source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate is known to be not readily biodegradable.

It will have to be shown that tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate (TOTM) and tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate do not share the properties, especially the mammalian toxicity, of phthalates such as bis(2-ethylhexyl) phthalate, with the abbreviation DEHP (EC No 204-211-0, CAS No 117-81-7), which proved to be an endocrine disruptor. Toxicokinetic studies in mammals indicate significant differences in gastrointestinal hydrolysis, metabolism and absorption between TOTM and DEHP which can explain the dissimilarity in the toxicity profile.


1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rationale for the hypothesis is described in the Guidance on information requirements and chemical safety assessment, Chapter R.6: QSARs and grouping of chemicals (ECHA, 2008), and the Read-Across Assessment Framework document (ECHA, 2017). The read-across hypothesis implies that different, but structurally closely related compounds produce the same type of effects, or are characterized by the absence of effects, due to similar, biological active or inactive structural characteristics (analogue approach - Scenario 2).

Here, the properties of the target substance are predicted to be quantitatively equal or lower when compared to those of the source substance which represent the worst-case. This assumption is based on the observation that the toxicity decreases with an increasing number of formic acid residues attached to the phenyl ring (2 in phthalates, 3 in the source, 4 in the target compound). An explanation for this observation would be that the hydrolysis rate decreases with an increasing number of formic acid residues which are in an ester bond with 2-ethylhexan-1-ol. At the same time, resorbable mono-esters are formed to a lower extend. The sub-structure feature, which is shared by phthalates as well as the source and target compound, is phthalic acid, synonym 1,2-benzenedicarboxylic acid. Except for the hydrolysis product 2-ethylhexan-1-ol, which seems to be of low toxicological relevance, the biotransformation products or metabolites are similar as they show the same type but not the same number of functional groups. Details are reported in sections 4.1-3.

Moreover, lower toxicity of the target is also expected since the phthalate DEHP is specified as an impurity of the source but not of the target compound. Actually, the low concentrations of DEHP present as an impurity showed no significant influence on the outcome of toxicity studies. Thus, the hazard values established for the source substance constitute a worst-case because the target substance is less potent in terms of biological effects (“inert”).

For the source chemical, a comprehensive database is available which shows that the substance has a low toxicity profile. Irritating/corrosion effects on skin and eye, and skin sensitization were not observed. An OECD 422 screening test did not indicate developmental toxicity effects. Only marginal effects on reproductive organs were considered as not adverse. Observed changes in clinical chemistry parameters and liver weights of lower toxicological relevance could have been caused by an adaptive response. The target chemical is expected to share the low toxicity profile due to similar structural features.

Although the target and source chemicals are proposed by OECD Toolbox (v. 4.1) profiling to be attributed to the OECD HPV Chemical Category of High molecular weight phthalate esters this not constructive for the prediction of (eco-) toxicological effects. Well established differences in toxicokinetics (absorption, metabolism) of the source chemical and supporting compounds in comparison to phthalate esters such as bis(2-ethylhexyl) phthalate (DEHP, EC No 204-211-0, CAS No 117-81-7) can endorse this view and provide an explanation for the divergence in the biological activity found in toxicity and ecotoxicity studies. All these outlined arguments will be elucidated in more detail in the following sections.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The compound to be registered, tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate (EC No 221-508-0, CAS No 3126-80-5), with the IUPAC name 1,2,4,5-tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, is a mono constituent substance in the physical form of a liquid (also identified as tetra(2-ethylhexyl) pyromellitate, common name tetraoctyl pyromellitate, abbreviation TOPM). The purity grade is ≥ 99 % (w/w), with water and ethanol as main impurities.

The source, specified as tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate (EC No 222-020-0, CAS No 3319-31-1), is also a mono constituent chemical. The following alternative names are known:1,2,4-tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate, TOTM, tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate, and tris(2-ethylhexyl) trimellitate. The common name is trioctyl trimellitate. According to information on the appearance, physical state, and colour of the registered source substance, the substance is liquid at standard temperature and pressure with pale yellow colour and faint odour. The purity grade is in the range between 98.29% and 100% (w/w). Chemical analysis revealed bis(2-ethylhexyl) phthalate (abbreviation DEHP, EC No 204-211-0, CAS No 117-81-7) as an impurity which is found in the concentration range from 0.0 to 0.07% (w/w), with a typical concentration of 0.05% (w/w). The identity of other impurities is unknown.

Identity of the source and target substance:

Chemical Target substance Source substance
EC number 221-508-0 222-020-0
EC name Tetrakis(2-ethylhexyl) Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate
benzene-1,2,4,5-tetracarboxylate
CAS number 3126-80-5 3319-31-1
IUPAC name 1,2,4,5-tetrakis(2-ethylhexyl) Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate
benzene-1,2,4,5-tetracarboxylate
Other names Tetra(2-ethylhexyl) pyromellitate, 1,2,4-tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate, TOTM, tris(2-ethylhexyl)
tetraoctyl pyromellitate, trimellitate, trioctyl trimellitate.
abbreviation TOPM
Molecular formula C33H54O6 C33H54O6
Smiles CCCCC(CC)COC(=O)c1cc(C(=O)OCC CCCCC(CC)COC(=O)c1ccc(C(=O)OCC(CC)CCCC)c(c1)C(=O)OCC(CC)CCCC
(CC)CCCC)c(cc1C(=O)OCC(CC)CCCC)C(=O)OCC(CC)CCCC
Molecular weight 702.507 g/mol 546.7783
Description Discrete chemical, mono constituent, organic Discrete chemical, mono constituent, organic
Physical form Liquid at 25°C Liquid at 25°C
Purity grade ≥ 99 % (w/w) 98.29%-100% (w/w)
Impurities Water and ethanol 0.0-0.07% (w/w) bis(2-ethylhexyl) phthalate (EC No 204-211-0),
and impurities of unknown identity

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

Functional groups and substituents
The chemical structures of the target and the source substances can be described as esters of 2-ethylhexanol with pyromellitic acid and trimellitic acid, respectively. Four or three formic acid residues which show an ester bond with branched alkane substituents are attached to an aromatic ring (benzol), respectively. The organic functional groups are specified in Table 3 (see attached 'Read-across justification' in IUCLID section 13.2) which shows that the target and the source chemical are characterized by the same type of organic functional groups. Only the number of ‘carboxylic acid esters’ with ‘alkanes, branched with tertiary carbon’ is varying (4 vs 3, respectively). The sub-structure feature, which is shared by phthalates as well as the source and target compound, is phthalic acid, synonym 1,2-benzenedicarboxylic acid. The conformation, i.e. spatial arrangement of atoms in the molecules of the target and the source substance, is flexible.

PubChem substructure similarity features
The structural similarity of the target and source read-across substances has in addition been verified by application of PubChem substructure similarity features which are implemented in OECD Toolbox (v. 4.1).

Method: The PubChem generates a substructure fingerprint for each chemical structure. These fingerprints are used for similarity neighboring. In this context, a substructure is a fragment of chemical structure. A fingerprint is an ordered list of binary (1/0) bits. Each bit represents a Boolean determination of specific atom or test features. 7 groups of PubChem features have been defined:
• Hierarchical element counts;
• Rings;
• Simple atom pairs;
• Simple atom nearest neighbors;
• Detailed atom neighbors;
• Simple SMARTS patterns (SMART is a language that allows specifying substructures by using rules that are straightforward extensions of SMILES);
• Complex SMARTS patterns.

Results: The target compound shares 111 out of 112 substructure features with the source whereas the source compound shares 111 out of 113 substructure

The assessment of similarities in compounds the organism is exposed to relies on experimental data regarding the toxicokinetics of tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate as well as the prediction of hydrolysis products and metabolites by OECD Toolbox (v. 4.1) both for the source and target read-across substance. Hydrolysis of ester bonds by intestinal and liver esterases is also well established (Younggil, 2001). Moreover, substantial information on in vivo metabolism of phthalates such as mono(2-ethylhexyl) phthalate (MEHP, CAS No 4376-20-9, EC No 224-477-1) is considered since differences in metabolism can endorse the view that tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate and tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate are less toxic than low molecular weight phthalates.

Please refer to IUCLID section 7.1.1 'Basic toxicokinetis' and IUCLID section 13.2 'read-across justification' for detiailed information about absorption, distribution, metabolism and excretion of the source and target substance.

Prediction of metabolites by the hydrolysis simulator (acidic) and hydrolysis simulator (basic) resulted in an identical set of 7 metabolites in each case for tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, and a set of 8 metabolites for tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate. Obviously, the prediction is based on the assumption that the ester bonds of trimellitic acid and pyromellitic acid, respectively, are successively hydrolysed, with the result that all of the possible isomers are released. An overview of predicted hydrolysis products and compounds yielded by the metabolism simulators is presented below, along with experimental data.

Summary of toxicokinetics with conclusions on similarities:

Hydrolysis by esterases is considered to be an important first step in the oral absorption of ortho-phthalates. The potential for such hydrolysis to occur with the source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate has been examined in an in-vitro study using rat gut homogenate. There was no evidence of hydrolysis occurring wheras the corresponding phthalate, bis(2-ethylhexyl) phthalate (abbreviation DEHP, EC No 204-211-0, CAS No 117-81-7), was significantly hydrolysed.
The absorption, distribution, metabolism and elimination of the radiolabeled source substance have been investigated in the rat following oral administration of a single dose. Recovery of the administered dose was 94%, with approximately 75% eliminated unchanged in the faeces, 16.3% found in the urine and 1.9% in expired air. Residual radioactivity in the carcass after 6 days was <0.6% of the administered dose. Findings indicate that the compound may be partially hydrolysed in the gastro-intestinal tract to 2-ethylhexan-1-ol and the corresponding di-ester and, following further hydrolysis, the mono-ester. Only 2-ethylhexanol and a single isomer of the monoester (i.e. mono-(2-ethylhexyl) trimellitate) appear to be absorbed. Following absorption, 2-ethylhexanol was extensively metabolised with metabolites eliminated in the urine and as expired 14CO2. There was no evident metabolism of mono-(2-ethylhexyl) trimellitate, this being eliminated unchanged. Urinary excretion of radioactivity was bi-phasic with half-lives of 3.1 and 42 hours.
When barriers to absorption are by-passed by intravenous administration, the source chemical has been found to distribute mainly in the liver, lungs and spleen. Excretion of the substance or its metabolites over 14 days was slow with 21.3% and 2.8% of the administered dose found in the faeces and urine, respectively, suggesting a half-life of approximately 40 days. While data from the intravenous route may suggest a possible concern for potential bioaccumulation, the substance is poorly absorbed by the oral route, and the kinetics of urinary elimination suggest a far shorter half-life, indicating a lower potential for bioaccumulation.
An in vitro dermal absorption study using full-thickness skin samples in a Franz diffusion cell system showed that the compound is not systemically bioavailable after dermal exposure. This finding is supported by the results of the IH SkinPerm model. Due to the low vapor pressure, a significant respiratory uptake from airborne vapors can be excluded.

Regarding similarities in chemical structures, physico-chemical properties (see below), and in addition results of OECD Toolbox profiling, similar toxicokinetic characteristics are expected for the target substance. This means that the ester bonds of tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate will probably be successively hydrolysed at a low rate in the gastrointestinal tract. The hydrolysis products 2-ethylhexan-1-ol and mono-(2-ethylhexyl) pyromellitate will likely be absorbed to a low extend. 2-Ethylhexan-1-ol but not the mono-ester will undergo further phase-I metabolism. There is no evidence that carboxyl groups of the source and target compounds are modified during phase I-metabolism, or metabolites are produced which play a role for the toxicological behavior of phthalates.
In conclusion, except for the hydrolysis product 2-ethylhexan-1-ol, the biotransformation products or metabolites of the source and target compound are similar as they show the same type but not the same number of functional groups. Available toxicokinetic data may imply that the extend of monoester formation in the gastrointestinal tract, as well as the absorption and systemic bioavailability decreases with an increasing number of ester bonds (2 ester bonds occur in phthalates, 3 in the source substance, 4 in the target substance). Therefore, it seems to be plausible that the gastrointestinal absorption of hydrolysis products of the target substance, i.e. the potential metabolites 2-ethylhexan-1-ol and mono-(2-ethylhexyl) pyromellitate, is very low. The latter metabolite will possibly not undergo a phase-I metabolism but rather be excreted unchanged in the urine, similarly to mono-(2-ethylhexyl) trimellitate. Especially due to low water solubility < 1 mg/L, the target substance is predicted by the IH SkinPerm model to be not absorbed through the skin. The low volatility of the target substance as well as the octanol-water partition coefficient (log Po/w of 6.01) will obviate a significant respiratory uptake


Synopsis of physico-chemical properties
Pyromelliate (Target) TOTM (Source)
Physical state Liquid at 20 °C and 101.3 kPa Liquid at 20 °C and 101.3 kPa
Melting / freezing point -34°C at 101,3kPa (exp.) -43 °C at 101.3 kPa (exp.)
Boiling point 573.5 °C (QSAR, SPARC by ARChem) 355 °C (exp.)
Relative density 0.9908 g/cm3 at 25 °C 0.9885 g/cm3
Granulometry Not applicable (liquid state) Not applicable (liquid state)
Vapour pressure 2.09E-9 hPa (QSAR, MPBPWIN™ 6.8E-10 hPa at 25 °C (exp.)
by EPI Suite v4.1)
Partition coefficient
n-octanol/water (log value) 6.01 (exp.) 8.00 at 25 °C and pH 4.8 (exp.)
Water solubility < 1 mg/L at 30 °C (exp.) 3.06 µg/L at 25 °C (exp.)
(identical to the cut-off value for insolubility
in water according to ECHA guidance)
Surface tension Study scientifically not necessary in accordance with ECHA guidance
Flash point 262 °C (exp.) 224 °C (exp.)
Autoflammability / self-ignition temperature Study scientifically not necessary, flash point >200 °C at 101.3 kPa
Flammability Study scientifically not necessary, flash point >200 °C at 101.3 kPa
Explosive properties Study scientifically not necessary - the substance contains no chemical groups associated with explosive properties.
Oxidising properties Based on a consideration of the chemical structure of the substance, oxidising properties do not
need to be assessed.
Stability in organic solvents and
identity of relevant degradation products The stability of the substance in organic solvents is not considered to be critical.
Dissociation constant Study scientifically not necessary - the substance does not contain any functional groups that dissociate.
Viscosity 1.7 cm2/s at 40°C 0.87 cm2/s at 40 °C (kinematic viscosity, exp.)
(kinematic viscosity, exp.)
*Published data, cf. UNEP (2002) and ECHA (2013-2017)

Based on experimental data and QSARs, the relevant physico-chemical properties of the source and target substance are similar. This supports the view that their pattern of biological effects and the underlying mechanisms may also show analogies.

The assessment of similarities in compounds the organism is exposed to (section 4.2), with reference to the corresponding Assessment Element 2.1 of the RAAF document (ECHA, 2017), relies on experimental data for the source compound and predictions (OECD Toolbox, IH SkinPerm model) combined with theoretical considerations for the target compound. Due to a very low oral and dermal absorption rate with very low systemic bioavailability, non-common compounds (with the same type but not the same number of functional groups) are considered as not relevant in vivo. On this basis, the assessment option “acceptable with medium confidence” is chosen.

Both the source and supporting compound have shown a low toxicity profile, without local and systemic toxicity effects except for minor findings, which were regarded as non-adverse, after repeated exposure to the supporting chemical. More in detail, in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 using 1,2,4-benzenetricarboxylic acid, trioctyl ester some irregularities in clinical-chemistry parameters were noted in male and female rats at the mid and/or high dose level (125 and 500 mg/kg bw/day orally, respectively). At 500 mg/kg bw/day significant changes in liver weights of females were recorded. These findings may be attributed to treatment but could represent an adaptive response.

The information presented in paragraph 4.3 suggests that the hypothesis of common underlying biological mechanisms, both in qualitative and quantitative aspects (Assessment Elements 2.2 and 2.3 according to the RAAF-document, see ECHA, 2017) is acceptable. With respect to the fact that no toxicity studies for the target compound are available, the assessment option “acceptable with just sufficient confidence” may be appropriate. For the specific case, the Assessment Elements 2.4 (Exposure to other compounds than to those linked to the prediction) and 2.5 (Occurrence of other effects than covered by the hypothesis and justification) are considered as not relevant.

Acute oral toxicity
In a study conducted according to OECD TG 401, 5 male and 5 female Crj:CD(SD) rats were administered 2000 mg/kg bw 1,2,4-tris benzenetricarboxylic acid (2-ethylhexyl) ester (common name used in study report) by oral gavage (Fujishima, 1996). During the 14-day observation period, no death occurred, and no significant clinical signs were noted except for loose stool. A NOAEL value > 2000 mg/kg bw for acute oral toxicity was derived from this study.

In another study, a method similar to that described in Section 1500.3 of the U.S. Federal Hazardous Substances Act Regulations - 16 CFR, p. 114, was employed (Gilman and Costello, 1981). One group of ten (5 male and 5 female) albino rats of the outbred Sprague-Dawley Strain weighing between 200 and 300 g was dosed 5 g/kg of the test material into the stomach by means of a syringe and dosing needle. No deaths were noted, and a NOAEL value > 5000 mg/kg bw was established.
In conclusion, classification of the source substance for acute oral, dermal or inhalation toxicity is not required. The results of acute toxicity studies are compatible with a low toxicity profile. Both the source and target substance can be attributed to class I (low hazard) of the toxic hazard classification by Cramer (extended) which is implemented in OECD Toolbox v.4.1. The target substance will likely not exert significant acute toxicity effects, because similarly to the source substance its absorption potential is expected to be very low by all routes of exposure.

4. DATA MATRIX
see Section 13.2 'Read across justification' and 'Data Matrix'

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Remarks:

Read-across justification

Reason / purpose for cross-reference:

read-across: supporting information

Remarks:

Toxicokinetik

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

corn oil

Control animals:

yes

Preliminary study:

In preliminary study 200 and 2000 mg/kg did not cause any mortality.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No deaths occured

Mortality:

No deaths occurred of either male or female animals.

Clinical signs:

other: Loosening erring of the stool attributable to the treatment with corn oil (vehicle) was observed for 3 hours from the administration for both sexes in the groups given 0 and 2,000mg/kg.

Gross pathology:

No macroscopic abnormalities that could be attributed to treatment with the test substance was seen on pathological examination.

Table 1 Mortality

Sex	Group	Dose level (mg/kg)	Number of animals	Number of deaths on the day														Mortality (%)
				1	2	3	4	5	6	7	8	9	10	11	12	13	14
Male	1	0	5	0														0
	2	2000	5	0														0
Female	3	0	5	0														0
	4	2000	5	0														0

Table 2 Clinical Signs

Sex: Female                Dose level: 0 mg/kg                              Number of animals: 5
Signs	Hours							Days
	1	2	3	4	5	6	24	2	3	4	5	6	7	8	9	10	11	12	13	14
Normal	0	0	0	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
General condition loose stool	5	5	5
Dead
Number of affected animals: 5                      Number of recovered animals: 5                  Mortality: 0/5

Sex: Female                Dose level: 2000 mg/kg                              Number of animals: 5
Signs	Hours							Days
	1	2	3	4	5	6	24	2	3	4	5	6	7	8	9	10	11	12	13	14
Normal	0	0	0	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
General condition loose stool	5	5	5
Dead
Number of affected animals: 5                      Number of recovered animals: 5                  Mortality: 0/5

Sex: Male                Dose level: 0 mg/kg                              Number of animals: 5
Signs	Hours							Days
	1	2	3	4	5	6	24	2	3	4	5	6	7	8	9	10	11	12	13	14
Normal	0	0	0	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
General condition loose stool	5	5	5
Dead
Number of affected animals: 5                      Number of recovered animals: 5                  Mortality: 0/5

Sex: Male                Dose level: 0 mg/kg                              Number of animals: 5
Signs	Hours							Days
	1	2	3	4	5	6	24	2	3	4	5	6	7	8	9	10	11	12	13	14
Normal	0	0	0	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5
General condition loose stool	5	5	5
Dead
Number of affected animals: 5                      Number of recovered animals: 5                  Mortality: 0/5

Table 3 Cross Findings

Sex: Male             Dose level: 0 mg/kg
Animal ID-No.	Classification	Days after administration	Organ	Findings and comments
1001	Sacrificed	14		Normal
1002
1003
1004
1005
Sex: Male             Dose level: 2000 mg/kg
1101	Sacrificed	14		Normal
1102
1103
1104
1105
Sex: Female             Dose level: 0 mg/kg
2001	Sacrificed	14		Normal
2002
2003
2004
2005
Sex: Female             Dose level: 2000 mg/kg
2101	Sacrificed	14		Normal
2102
2103
2104
2105

Interpretation of results:

other: EU GHS (CLP) criteria not met

Conclusions:

LD50 was established at > 2,000 mg/kg for both sexes, this result can be considered the same for the target substance.

Executive summary:

The acute oral toxicity of the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate has been determined according to OECD Guideline 401 and in compliance with GLP standards. Five rats per sex were orally exposed via gavage to a single dose of 0 and 2,000 mg/kg test item suspended in 40% w/v% corn oil.

Each rat was weighed immediately prior to treatment, 7 and 14 days after post-treatment observation period. The rats were observed each hour to 6 hours, after that, two times for one day during this time for signs of toxicity. The test substance did not cause any changes in body weight. Loosening erring of the stool attributable to the treatment with corn oil was

observed for 3 hours from the administration for both sexes in the groups given 0 and 2000 mg/kg. No deaths were recorded in treated and control group. No macroscopic abnormalities that could be attributes to treatment with the test substance were seen on pathological examination.

The LD50 for rats of the source test item tris(2 -ethylhexyl)benzene-1,2,4 -tricarboxylate was determined in this limit test to be greater than 2000 mg/kg for acute oral toxicity and can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Good quality due to OECD standard method and GLP compliance.

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1981-10-22 to 1981-11-04

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: Procedures st forth in the Federal Insecticide, Funigcide, and Rodenticide Act (FIRFA)

Version / remarks:

Federal Insecticide, Fungicide, and Rodecticide Act, Part 163, Title 40; Code of the Federal Regulations 40 CRF 163.81.

GLP compliance:

yes

Remarks:

Good Laboratory Practices Regulations, Part 58, Title 21. Code of the Federal Regulations 40 CRF 163.81*2.

Test type:

other: Limit test

Limit test:

yes

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young adult male and female New Zealand albino white rabbits were supplied by Becker, Centralia, Kansas. Upon receipt of the animals, each was identified by means of a numbered ear tag. All animals were housed in temperature and humidity controlled rooms. Prior to treatment, all animals were acclimated to laboratory conditions for a minimum of 7 days.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

Five male and five female rabbits weighing 2.3 to 3.2 kg were used to determine the acute dermal toxicity of test-compound.. Twenty-four hours prior to treatment the hair on the back of each rabbit was clipped so as to expose approximately 10% of the body surface area. Before dosing, epidermal abrasions were made longitudinally over the exposure area. The abrasions were sufficiently deep to penetrate the stratum corneum but not so deep as to cause bleeding. A dosage of 2.0 ml/kg was applied to the exposure area of three male and three female rabbits. The other two male and two female rabbits served as control animals. A 2- x 2-in. gauze pad was placed on the exposure area to prevent seepage of the compound from the area. Each rabbit was then wrapped with a rubber dam. After 24 hours of exposure, the rubber dam and gauze pad were removed, and the exposure area was wiped to remove any remaining test material. The rabbits were observed for a total of 14 days; no toxic signs were noted. A gross necropsy was performed on all animals at the end of the 14-day observation period; no gross pathology was observed. The acute dermal LD50 was> 2.0 ml/kg. Results are summarized in Table 1. Individual animal body weights are listed in Table 2.

Duration of exposure:

24 hours

Doses:

2.0 mL/kg

No. of animals per sex per dose:

3 male and 3 female rabbits with test dose
2 male and 2 female rabbits for control

Control animals:

yes, concurrent no treatment

Details on study design:

The rabbits were observed for a total of 14 days; no toxic signs were noted. A gross necropsy was performed on all animals at the end of the 14-day observation period. Body weight was measured on day 1, 7 and 14 of observation period.

Statistics:

not specified (single dosage used)

Preliminary study:

no data

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 mL/kg bw

Based on:

test mat.

Remarks on result:

other: No confidence limits calculable as no mortality

Mortality:

none

Clinical signs:

other: No toxic signs were noted. A gross necropsy was performed on all animals at the end of the 14-day observation period; no gross pathology was observed.

Gross pathology:

A gross necropsy was performed on all animals at the end of the 14-day observation period; no gross pathology was observed.

Table 1 Acute Dermal Toxicity of the sample

Dose (mL/kg)	No. of Animals (all abraded)	Mortality No. Dead / No. Dosed
0	2 M, 2 F	0 / 4
2.0	3 M, 3 F	0 / 6

Table 2 Individual Animal Body weights

Animal No.	Sex	Dose (ml/kg)	Body Weights (kg)
			Day 1	Day 7	Day 14
RB50	Male	0.0	3.2	3.4	3.6
RB35	Male	0.0	3.2	3.4	3.6
RB56	Female	0.0	2.7	3.0	3.1
RB26	Female	0.0	2.9	3.1	3.3
RB93	Male	2.0	2.3	2.3	2.5
RB77	Male	2.0	2.4	2.4	2.5
RB75	Male	2.0	2.3	2.2	2.4
RB78	Female	2.0	2.3	2.5	2.7
RB84	Female	2.0	2.4	2.6	2.7
RB80	Female	2.0	2.4	2.5	2.6

Interpretation of results:

other: EU GHS (CLP) criteria not met

Conclusions:

Under the conditions of this test the sample has an acute dermal LD50 in rabbits of >2.0 ml/kg.

Executive summary:

The acute dermal toxicity test was conducted as a limit test according to Federal Insecticide, Fungicide, and Rodecticide Act, Part 163, Title 40; Code of the Federal Regulations 40 CRF 163.81 and in compliance with GLP criteria.

A dosage of 2.0 mL/kg of the test item (unchanged) was applied to the exposure area (approximately 10% of the body surface area) of 3 male and female rabbits. The other two male and two female rabbits served as control animals. A 2x2-inch gauze pad was placed on the exposure area to prevent seepage of the compound from the area. Each rabbit was then wrapped with a rubber dam. After 24 hour of exposure, the rubber dam and gauze pad was removed and the exposure area was wiped to remove any remaining test material. The rabbits were observed for a total of 14 days; no toxic signs were noted.

The results indicate that the test item has an acute dermal LD50 in rabbits of > 2.0 ml/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Good quality due to guideline studies or test procedures in accordance with national standard methods and in compliance with GLP.

Additional information

Read-across - Acute oral toxicity

The acute oral toxicity of the test item has been determined in the key study (MHV Japan, 1996) according to OECD Guideline 401 and in compliance with GLP standards with the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate. Five rats per sex were orally exposed via gavage to a single dose of 0 and 2,000 mg/kg test item suspended in 40% w/v% corn oil.

Each rat was weighed immediately prior to treatment, 7 and 14 days after post-treatment observation period. The rats were observed each hour to 6 hours, after that, two times for one day during this time for signs of toxicity. The test substance did not cause any changes in body weight. Loosening erring of the stool attributable to the treatment with corn oil was

observed for 3 hours from the administration for both sexes in the groups given 0 and 2000 mg/kg. No deaths were recorded in treated and control group. No macroscopic abnormalities that could be attributes to treatment with the test substance were seen on pathological examination.

The LD50 for rats of the source test item tris(2 -ethylhexyl)benzene-1,2,4 -tricarboxylatewas determined in this limit test to be greater than 2000 mg/kg for acute oral toxicity and can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies.

In a supporting study (Gilman, 1981a) the acute oral toxicity in rats was assessed using test method similar to that described in section 1500.3 Federal Hazardous Substances Act Regulations, 16 CFR, p 114, and apparently similar to OECD Guideline 401 and Directive 84/449/EEC, B.1 and in compliance with GLP criteria. The study has been conducted with the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate. Five rats of each sex were observed for 14 days following gavage administration of 5000 mg/kg bw of the test item (unchanged). No deaths, or behavioural or gross pathological effects were seen.

The LD50 in rats for the acute oral toxicity of the source test item tris(2 -ethylhexyl)benzene-1,2,4 -tricarboxylatewas determined to be greater than 5000 mg/kg bodyweight and can be similarly ransferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies.

Acute inhalation toxicity - waiving

The study need not be conducted because of exposure of humans via inhalation is not likely taking into account the vapour pressure of the substance and the possibility to exposure to aerosols or droplets of an inhalable size.

Read-across - Acute dermal toxicity

The key acute dermal toxicity test (Shellenberger, 1981) was conducted as a limit test according to Federal Insecticide, Fungicide, and Rodecticide Act, Part 163, Title 40; Code of the Federal Regulations 40 CRF 163.81 and in compliance with GLP criteria with the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate.

A dosage of 2.0 mL/kg of the test item (unchanged) was applied to the exposure area (approximately 10% of the body surface area) of 3 male and female rabbits. The other two male and two female rabbits served as control animals. A 2x2-inch gauze pad was placed on the exposure area to prevent seepage of the compound from the area. Each rabbit was then wrapped with a rubber dam. After 24 hour of exposure, the rubber dam and gauze pad was removed and the exposure area was wiped to remove any remaining test material. The rabbits were observed for a total of 14 days; no toxic signs were noted.

The results indicate that the test source item tris(2 -ethylhexyl)benzene-1,2,4 -tricarboxylatehas an acute dermal LD50 in rabbits of > 2.0 ml/kg, which can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies

Justification for classification or non-classification

Read-across data of the source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate for acute oral and dermal toxicity with adequate reliability is available. The LD50oral in rats has been determined to be > 2000 mg/kg bw and the LD50dermal in rabbits was > 2.0 ml/kg bw. Taking these results into account and assuming that a read-across aproach is justified, the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate does not meet the requirements for classification as acute toxic set forth in Regulation (EC) No 1272/2008 (CLP Regulation).