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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

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

In vitro hydrolysis
The rate of hydrolysis of radiolabeled plasticisers such as [hexyl 2-14C] tris(2-ethylhexyl) trimellitate and [hexyl 2-14C] di (2 -ethylhexyl) phthalate has been examined using gut homogenates from male Sprague-Dawley rats (Fox et al., 1984). The plasticisers were tested at concentrations to yield an equimolar concentration of 2-ethylhexanol (66 µM tris(2-ethylhexyl) trimellitate, and 100 µM di (2 -ethylhexyl) phthalate, each in methanol). Homogenates were incubated with the substance for up to 30 minutes and samples taken at intervals during this time. Following inactivation of enzymes, solvent extraction was undertaken to provide samples for analysis. The kinetics of 2-ethylhexanol formation was examined. There was no evidence of hydrolysis occurring with tris(2-ethylhexyl) trimellitate while the corresponding phthalate, di (2 -ethylhexyl) phthalate, was significantly hydrolysed. The authors comment that probably either due to a hydrolysis limiting process, or the low water solubility of tris(2-ethylhexyl) trimellitate, no hydrolysis was observed in this test.

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.

Target substance: Tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, EC No 221-508-0, CAS No 3126-80-5
Hydrolysis simulator (acidic/basic) Compounds predicted by OECD Toolbox (v. 4.1)
7 metabolites: 2-ethylhexan-1-ol, tris(2-ethylhexyl) pyromellitate, di-(2-ethylhexyl) pyromellitate (3 isomers), mono-(2-ethylhexyl) pyromellitate (1 isomer), pyromellitic acid
Microbial metabolism simulator 94 metabolites
Source substance: Tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate, EC No 222-020-0, CAS No 3319-31-1
Hydrolysis simulator (acidic/basic) 8 metabolites: 2-ethylhexan-1-ol, di-(2-ethylhexyl) trimellitate (3 isomers), mono-(2-ethylhexyl) trimellitate
(3 isomers), trimellitic acid
Microbial metabolism simulator 166 metabolites

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.

Algal growth inhibition has been investigated in a 72-hour test according to OECD test methods. Dissolution/dispersion agents were used to increase concentration of the substance in the test medium. The EC50 (72 h) was determined to be >100 mg/L and the NOEC was 100 mg/L, the highest concentration examined.
In short-term toxicity tests on fish and aquatic invertebrates (Daphnia sp.), the alga growth inhibition and activated sludge respiration inhibition assays, no effects of the source chemical up to the highest concentration, which has been examined, were detected. An activated sludge respiration inhibition test using the target compound has already been performed. Due to structural similarity, it is expected that the target compound will also not show significant influence on the function and morphology of environmental organisms. On this basis, the assessment option ‘acceptable with medium confidence’ is chosen for the Assessment Elements 2.3 and 2.4 according to the RAAF-Document (ECHA, 2017) with regard to similarities in underlying mechanism, both from a qualitative and quantitative point of view.

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

Reason / purpose for cross-reference:

read-across source

Analytical monitoring:

yes

Remarks:

by HPLC analysis

Vehicle:

yes

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

not applicable, OECD medium used

Test temperature:

23±2 °C

pH:

pH=7.3-7.4 at start and 8.3-8.8 at end of the test (72h) in at least one replicate of each concentration

Dissolved oxygen:

Not reported

Salinity:

not applicable

Nominal and measured concentrations:

Nominal concentrations : 0, 100 (mg/L) and dispersant control

Measured concentrations : At start of the test (0 hours), <1.0, 80.6, <1.0 (mg/L); at end of the test (72 hours), <1.0, 68.7, <1.0 (mg/L) respectively

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: comparison of area under growth curve

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: comparison of area under growth curve

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes, For control mean cell density increased to 2.70E6 cells/mL (270-fold increase) after 72 hr; for dispersant control mean cell density increased to 2.75E6 cells/mL (275-fold increase)
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: No, 100mg/L group showed normal and similar growth to that of control (283 fold increase after 72 h)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

EC50 of potassium dichromate was 0.41 mg/L.

Reported statistics and error estimates:

Bartlett test for homogeneity in variances and One-way Anova (EcoTox-Statistics Ver.1.0 beta-edition R1.4) were used for EC50, LC50 and NOEC determination (p=0.05).
Method of calculating mean measured concentrations: Geometric mean.

No significant difference in the growth curve was observed between values in the 100 mg/L tris(ethylhexyl)benzene-1,2,4-tricarboxylate group and in each of the control groups.

Cell density at each flask at each measuring point:             

Nominal Concentration (mg/L)	Cell Density         (x10^4cells/mL)
	0 hr	24 hr	48 hr	72 hr
Control	1.0 ± 0.00	6.5 ± 0.50	50.5 ± 3.48	270.5 ± 23.50
Dispersant Control	1.0 ± 0.00	9.3 ± 1.66	57.5 ± 9.39	275.2 ± 17.22
100	1.0 ± 0.00	16.1 ± 7.82	65.1 ± 12.82	283.3 ± 7.98

 (Each value represents the mean of three sample counts.)

Growth curves: Logarithmic growth until end of the test (72 h).

Percent biomass/growth rate inhibition per concentration: Not described.

Observations: Test group(100mg/L) showed normal and similar growth to that of control (283 fold increase after 72 hr).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the algal growth inhibition is by comparison of area under growth curve EC50 (0-72 h) > 100 mg/L and NOEC (0-72 h) 100 mg/L. The growth inhibition by comparison of growth rates is EC50 (24-48h) > 100 mg/L; EC50 (24-72h) > 100 mg/L; NOEC (24-72h) 100 mg/L and these results can be considered the same for the target substance.

Executive summary:

Algal growth inhibition has been investigated in a 72 hour test according to OECD Guideline 201 and in compliance with GLP criteria with the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate.

The concentration of the test substance in the test medium was increased by generating a supersaturated solution, which was considered to be substantially homogeneous, with the aid of a solubilizer (HCO-40). The nominal test concentrations of the source test item were 0 and 100 mg/L in OECD medium in triplicate, incubated for 72 hours under static conditions. The EC50 (72h) of Pseudokirchneriella subcapitata of the source test item tris(2 -ethylhexyl)benzene-1,2,4 -tricarboxylate was determined to be >100 mg/L (nominal) and the NOEC was determined to be 100 mg/L, the highest concentration examined.

These results can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies.

Description of key information

Read-across, OECD Guideline 201, Pseudokirchneriella subcapitata, static: EC50 (72h) > 100 mg/L (nominal), NOEC (72h) = 100 mg/L (nominal)

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

Algal growth inhibition has been investigated in a 72 hour key study (METI Japan, 1998c) according to OECD Guideline 201 and in compliance with GLP criteria with the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate.

The concentration of the test substance in the test medium was increased by generating a supersaturated solution, which was considered to be substantially homogeneous, with the aid of a solubilizer (HCO-40). The nominal test concentrations of the source test item were 0 and 100 mg/L in OECD medium in triplicate, incubated for 72 hours under static conditions. The EC50 (72h) of Pseudokirchneriella subcapitata of the source test item tris(2 -ethylhexyl)benzene-1,2,4 -tricarboxylate was determined to be >100 mg/L (nominal) and the NOEC (72h) was determined to be 100 mg/L, the highest concentration examined.

These results can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies.

Supporting information has been derived using QSAR model. The predicted short-term toxicity effect to aquatic invertebrates using ECOSAR model is (96hr) EC50 = 9.45e-8 mg/L. The substance is not soluble enough to measure this predicted effect. No effects at saturation (NES) are reported.