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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
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.

Acute toxicity to fish has been investigated in a 96 hour test according to OECD test methods. Dissolution/dispersion agents were used to increase concentration of the substance in the test medium. The LC50 (96h) was determined to be >100 mg/L.
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
Reason / purpose for cross-reference:
read-across: supporting information
Remarks:
Read-across justification
Analytical monitoring:
yes
Vehicle:
yes
Remarks:
hydrogenated casteroil = HCO-40, 100 mg/L
Test organisms (species):
Oryzias latipes
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
25 mg/ml, as CaCO3
Test temperature:
23.5-24.1 °C
pH:
6.7-6.8
Dissolved oxygen:
5.0-9.2 mg/L
Salinity:
not applicable
Nominal and measured concentrations:
nominal concentrations: 0, 100 mg/l (measured concentrations 101 - 103% of nominal concentration)
measured concentrations at 0h: <1, 103 mg/l respectively
measured concentrations at 24h: <1, 102 mg/l respectively

Reference substance (positive control):
yes
Remarks:
Copper (II) sulphate pentahydrate
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: At 24 hours, one fish exposed at 100 mg/L showed abnormal breathing behaviour.
- Mortality of control: one fish was found dead at 96 hours.
- Other adverse effects control: No
- Abnormal responses: None other than above
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Test solution became cloudy at 100mg/L concentration, but no precipitation. occurred
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Mortality: Yes
- LC50: 0.43 mg/L (96h).
- Other: reference substance used: Copper(II)sulphate pentahydrate.
Reported statistics and error estimates:
Not applicable as a limit test.
Sublethal observations / clinical signs:

Mortality (%) of Oryzias latipes exposed to

Nominal Concentration (mg/L)      Cumulative number of dead fish (% mortality)         
 24 hour  48 hour  72 hour  96 hour
 Control  0 (0)  0 (0)  0 (0)  1 (10)
 Dispersant Control  0 (0)  0 (0)  0 (0)  0 (0)
 100  0 (0)  1 (10)  1 (10)  1 (10)

Reference substance used was copper (II) sulphate pentahydrate with LC50at 96h of 0.43 mg/L.

Symptoms of Toxicity observed in Orange killfish (Oryzias Latipes) Exposed to the sample under Semi-static test condititons

Nominal Concentration (mg/L)     Symptoms         
 24 hour  48 hour  72 hour  96 hour
 Control  0  0  0  0
 Dispersant Control  0  0  0  0
 100  B (1)  0  0  0

0 = normal

B = abnormal respiration

C = abnormal swimming behaviour

D = loss of equilibrium or swimming ability

E = other symptoms

(n) = number of fish

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the test the LC50 (96h) for fish is greater than 100mg/L for the source substance, and this result can be considered the same for the target substance.
Executive summary:

Acute toxicity to fish has been investigated in a 96 hour test according to OECD Guideline 203 and in compliance to GLP standards 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 plus dispersant control in test water for 10 fishes per group, incubated for 96 hours under semi-static conditions.

The LC50 (96h) for Oryzias latipes of the source test item tris(2 -ethylhexyl)benzene-1,2,4 -tricarboxylate was determined to be >100 mg/L (nominal) and 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 203, semi-static, Oryzias latipes: LC50 (96h) >100 mg/L (nominal)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

Acute toxicity to fish has been investigated in a 96 hour key study (METI Japan, 1998a) according to OECD Guideline 203 and in compliance to GLP standards with the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate. The concentration of the source 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 plus dispersant control in test water for 10 fishes per group, incubated for 96 hours under semi-static conditions.

The LC50 (96h) for Oryzias latipes of the source test item tris(2 -ethylhexyl)benzene-1,2,4 -tricarboxylate was determined to be >100 mg/L (nominal) 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 QSAR approach for tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate the predicted short-term toxicity effect to fish using ECOSAR model is (96hr) LC50 = 3.19e-6 mg/L. No effects at saturation (NES) are reported.