Registration Dossier
Registration Dossier
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EC number: 221-508-0 | CAS number: 3126-80-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Read-across - in vitro bacterial reverse mutation assay (Ames test), OECD Guideline 471: The substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli.
Read-across - in vitro Chromosome aberration test, OECD Guideline 473: The substance does not induce chromosomal aberrations in human lymphocytes after in vitro treatment
Read-across - in vitro mammalian cell gene mutation assay (MOLY), OECD Guideline 476: It is concluded that tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- 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.
Genetic toxicity
In accordance with the information requirements for registration of the read-across chemical, 3 standard in vitro tests were performed to evaluate the genotoxic potential: A bacterial reverse mutation assay (Ames test) has shown the substance not to induce reverse mutation in Salmonella typhimurium or Escherichia coli strains. The ability to cause chromosomal damage was investigated in cultured human lymphocytes, and the substance was found not to induce chromosomal aberrations in human lymphocytes in vitro. The mutagenic activity in mammalian cells was examined by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment. The substance did not induce mutation in this cell type. Moreover, no mutagenic effects were observed in a dominant lethal assay conducted in the mouse in vivo.
Regulation (EC) No 1907/2006 (Annex VIII, 8.4 Column 2) states that appropriate in-vivo mutagenicity studies should be considered in case of a positive result in any of the in vitro genotoxicity studies. In vitro investigations were negative and in vivo studies are therefore regarded as inappropriate and not in line with current concerns regarding animal welfare and the use of animals in scientific experiments.
It is noted that profiling by OECD Toolbox v.4.1 showed a frequently reported structural alert for in vivo mutagenicity (Micronucleus) by ISS (H-acceptor-path3-H-acceptor), both for the source and target chemical. Based on experimental data, which are available for the source compound, and structural similarity, it is expected that the target compound does not induce chromosomal aberrations or gene mutations.
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:
- Toxicokinetic
- Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not determined
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In the initial toxicity test no toxicity was observed at any dose-level with any tester strain in the absence or presence of S9 metabolism at concentrations up to a maximum of 5000 ug/plate.
No precipitation was observed. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative
It is concluded that the substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions. This result can be considered the same for the target substance. - Executive summary:
A bacterial reverse mutation assay (Ames test) has been assessed following OECD Guideline 417 and EU test method B.13/14 in accordance with GLP criteria with the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate. A preliminary toxicity test was undertaken to select suitable concentrations for use, performed with –S9 and + S9 using the plate incorporation method. The main experiment consists of two assays that were performed including negative and positive controls both –S9 and + S9 with Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli WP2 uvr A.Three replicate plates were used at each test point. The first experiment was performed using a plate- incorporation method and the second experiment was performed using a pre- incubation method, both at amaximum dose-level of 5000 µg/plate and four lower concentrations of 1580, 500, 158 and 50.0 µg/plate of the test substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate.
The substance did not induce increases in the number of revertant colonies in Salmonella typhimurium or Escherichia coli in the plate incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism.
This can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- 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.
Genetic toxicity
In accordance with the information requirements for registration of the read-across chemical, 3 standard in vitro tests were performed to evaluate the genotoxic potential: A bacterial reverse mutation assay (Ames test) has shown the substance not to induce reverse mutation in Salmonella typhimurium or Escherichia coli strains. The ability to cause chromosomal damage was investigated in cultured human lymphocytes, and the substance was found not to induce chromosomal aberrations in human lymphocytes in vitro. The mutagenic activity in mammalian cells was examined by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment. The substance did not induce mutation in this cell type. Moreover, no mutagenic effects were observed in a dominant lethal assay conducted in the mouse in vivo.
Regulation (EC) No 1907/2006 (Annex VIII, 8.4 Column 2) states that appropriate in-vivo mutagenicity studies should be considered in case of a positive result in any of the in vitro genotoxicity studies. In vitro investigations were negative and in vivo studies are therefore regarded as inappropriate and not in line with current concerns regarding animal welfare and the use of animals in scientific experiments.
It is noted that profiling by OECD Toolbox v.4.1 showed a frequently reported structural alert for in vivo mutagenicity (Micronucleus) by ISS (H-acceptor-path3-H-acceptor), both for the source and target chemical. Based on experimental data, which are available for the source compound, and structural similarity, it is expected that the target compound does not induce chromosomal aberrations or gene mutations.
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:
- Toxicokinetic
- Key result
- Species / strain:
- lymphocytes: Human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Vehicle controls validity:
- not applicable
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- One hundred metaphase spreads were scored for chromosomal aberrations from each culture.
In the first experiment, following treatment in the absence of S9 metabolism, no remarkable toxicity was observed at any dose-level selected for treatment. In the presence of S9 metabolism, mild or slight toxicity was observed over the whole dose-range (mitotic indexes between 73-92% of the control) with the exception of the two lower dose levels of 78.1 and 39.1 micrograms/ml where no remarkable toxicity was observed.
In the second experiment, following the continuous treatment in the absence of S9 metabolism, moderate toxicity was observed at the three higher dose levels (5000, 2500 and 1250 microg/ml) where the mitotic indexes were reduced in the range between 64-68% of the control. Mild toxicity was observed at the dose levels of 625, 313, 156 and 78.1 micrograms/ml where the mitotic indexes were reduced in the range between 74-78% of the control. Slight toxicity was observed at the lowest dose of 39.1 micrograms/ml where the mitotic index was 86% of the control.
The highest dose level selected for the scoring of aberrations should be a concentration causing moderate toxicity (ideally the reduction of mitotic index should be approximately 50%) and treatments reducing the mitotic index to below 20% should not be scored. In the absence of toxicity the highest treatment level will be selected as the highest dose for scoring. On the basis of the observed toxicity the treatment-levels selected for the scoring of aberrations were 5000, 2500 and 1250 micrograms/ml
Following treatment with the substance, no relevant increase in the incidence of cells bearing aberrations including or excluding gaps over the control values, was observed at any dose level in the absence or presence of S9 metabolism.
Marked increases in the frequency of cells bearing aberrations (including and excluding gaps) were seen in the cultures treated with the positive control substance, Mitomycin-C and Cyclophosphamide indicating the correct functioning of the assay system. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative
It is concluded that the source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate does not induce chromosomal aberrations in human lymphocytes after in vitro treatment, under the reported experimental conditions. This result can be considered the same for the target substance. - Executive summary:
The read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate was assayed for the ability to cause chromosomal damage in cultured human lymphocytes according to OECD Guideline 473 and in compliance with GLP criteria. In-vitro treatment in two independent assays for chromosomal damage were performedin the absence and presence of S9 metabolic activation at amaximum dose-level of 5000 micrograms/plate and four lower concentrations of 2500, 1250, 625 and 313 micrograms/plate.
The source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate did not induce chromosomal aberrations in human lymphocytes after in-vitro treatment.
This can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- 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.
Genetic toxicity
In accordance with the information requirements for registration of the read-across chemical, 3 standard in vitro tests were performed to evaluate the genotoxic potential: A bacterial reverse mutation assay (Ames test) has shown the substance not to induce reverse mutation in Salmonella typhimurium or Escherichia coli strains. The ability to cause chromosomal damage was investigated in cultured human lymphocytes, and the substance was found not to induce chromosomal aberrations in human lymphocytes in vitro. The mutagenic activity in mammalian cells was examined by assaying for the induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment. The substance did not induce mutation in this cell type. Moreover, no mutagenic effects were observed in a dominant lethal assay conducted in the mouse in vivo.
Regulation (EC) No 1907/2006 (Annex VIII, 8.4 Column 2) states that appropriate in-vivo mutagenicity studies should be considered in case of a positive result in any of the in vitro genotoxicity studies. In vitro investigations were negative and in vivo studies are therefore regarded as inappropriate and not in line with current concerns regarding animal welfare and the use of animals in scientific experiments.
It is noted that profiling by OECD Toolbox v.4.1 showed a frequently reported structural alert for in vivo mutagenicity (Micronucleus) by ISS (H-acceptor-path3-H-acceptor), both for the source and target chemical. Based on experimental data, which are available for the source compound, and structural similarity, it is expected that the target compound does not induce chromosomal aberrations or gene mutations.
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:
- Toxicokinetic
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Cytotoxicity test
Both in the absence and presence of S9 metabolic activation, the test substance was assayed at a maximum dose level of 5000 µg/ml and at lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 µg/ml.
Slight decreases in relative survival were observed at intermediate dose levels, both in the absence and presence of S9 metabolic activation, using a 3 hour treatment time. No relevant toxicity was observed using a long treatment time in the absence of S9 metabolic activation.
Mutation assays
Two independent assays for mutation to trifluorothymidine resistance were performed.
The mutant frequencies in the solvent control cultures fell within the normal range (5E7 2E8 viable cells). The positive control chemicals induced clear increases in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).
The cloning efficiencies at Day 2 in the negative control cultures fell within the range of 65-120% and the control growth factor over 2 days fell within the range of 8 – 32 in both experiments.
In the absence of S9 metabolic activation, using the 3-hour treatment time, slight toxicity was observed at the highest dose level (2500 µg/ml) reducing survival to 72% of the concurrent negative control value. The relative total growth was reduced to 78% at the same concentration. Using a long treatment time, no relevant toxicity was observed at any dose level.
In the presence of S9 metabolic activation, no relevant toxicity was observed in the first experiment, while in the second experiment, a slight decrease in relative total growth (60%) was observed only at the intermediate dose level of 313mg/ml.
In Experiment 1, in the presence of S9 metabolic activation, the heterogeneity between replicate cultures for survival at the end of treatment, at the concentration of 625 µg/ml, was higher than usual and thus excluded from the determination of overall heterogeneity. In addition, in the second experiment, in the absence of S9, a slight but statistically significant difference between replicate plates was observed for culture A treated at 625 µg/ml.These results have not affected the validity of the study.
No statistically significant increases in mutant frequency were observed in the absence or presence of S9 metabolic activation, following treatment with the test substance at any concentration level.
Osmolality and pH
The test substance did not have any obvious effect on the osmolality or pH of the treatment medium. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative
It is concluded that teh source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions. This result can be considered the same for the target substance. - Executive summary:
The read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate substance has been examined for mutagenic activity by assaying for the induction of 5‑trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.The study has been conducted in accordance with OECD Guideline 476 and EU Method B.17 and in compliance with GLP criteria.
The source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate does not induce mutation in mouse lymphoma L5178Y cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
This can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies
Referenceopen allclose all
In Assay 1, using the plate incorporation method, the substance was tested at the maximum concentration of 5000 μg/plate and at four lower concentrations. No relevant toxicity was observed with any tester strain at any dose-level. No relevant increase in revertant numbers was observed at any concentration tested. A pre-incubation step was included for all treatments of Assay 2 in which the same concentrations were employed. No toxicity was observed at any dose-level with any tester strain.
No precipitation of the test item was observed at the end of the incubation period at any concentration in the plate incorporation experiment or in the pre-incubation experiment.
The substance did not induce increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism.
The sterility of the S9 mix and the test solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
TABLE 1 - Mitotic index results - Without metabolic activation
MAIN ASSAY: 1
SOLVENT: ETHANOL
TREATMENT TIME: 3 hours
SAMPLING TIME: 24 hours
_____________________________________________________________________
Treatment Dose level Culture Cells Metaphases Mean Relative
(µg/ml) No. Scored MI(%) MI(%)
_____________________________________________________________________
Untreated - 1 1000 40 4.1 95
2 1000 42
_____________________________________________________________________
Solvent 1% 3 1000 44 4.3 100
4 1000 42
_____________________________________________________________________
Test Item 39.1 19 1000 45 4.3 100
20 1000 41
_____________________________________________________________________
Test Item 78.1 17 1000 40 4.2 97
18 1000 43
_____________________________________________________________________
Test Item 156 15 1000 38 4.2 97
16 1000 45
_____________________________________________________________________
Test Item 313 13 1000 37 3.9 91
14 1000 41
_____________________________________________________________________
Test Item 625 11 1000 43 4.2 97
12 1000 40
_____________________________________________________________________
Test Item 1250 9 1000 41 4.0 92
10 1000 38
_____________________________________________________________________
Test Item 2500 7 1000 40 4.3 99
8 1000 45
_____________________________________________________________________
Test Item 5000 5 1000 43 4.1 95
6 1000 39
_____________________________________________________________________
Mitomycin-C 0.50 21 1000 20 1.9 46
22 1000 18
_____________________________________________________________________
Mitomycin-C 0.75 23 1000 18 1.8 43
24 1000 17
_____________________________________________________________________
TABLE 2 - Mitotic index results - With metabolic activation
MAIN ASSAY: 1
SOLVENT: DMSO
TREATMENT TIME: 3 hours
SAMPLING TIME: 24 hours
_____________________________________________________________________
Treatment Dose level Culture Cells Metaphases Mean Relative
(µg/ml) No. Scored MI(%) MI(%)
_____________________________________________________________________
Untreated - 73 1000 56 5.6 114
74 1000 55
_____________________________________________________________________
Solvent 1% 75 1000 49 4.9 100
76 1000 48
_____________________________________________________________________
Test Item 39.1 91 1000 49 5.1 104
92 1000 52
_____________________________________________________________________
Test Item 78.1 89 1000 46 4.7 97
90 1000 48
_____________________________________________________________________
Test Item 156 87 1000 35 3.6 74
88 1000 37
_____________________________________________________________________
Test Item 313 85 1000 42 4.3 88
86 1000 43
_____________________________________________________________________
Test Item 625 83 1000 44 4.4 90
84 1000 43
_____________________________________________________________________
Test Item 1250 81 1000 35 3.6 73
82 1000 36
_____________________________________________________________________
Test Item 2500 79 1000 42 4.5 92
80 1000 47
_____________________________________________________________________
Test Item 5000 77 1000 37 3.8 78
78 1000 39
_____________________________________________________________________
Cyclophosphamide 18.0 93 1000 28 2.9 52
94 1000 30
_____________________________________________________________________
Cyclophosphamide 23.0 95 1000 22 2.3 41
96 1000 24
_____________________________________________________________________
TABLE 3 - Mitotic index results - Without metabolic activation
MAIN ASSAY: 2
SOLVENT: DMSO
TREATMENT TIME: 24 hours
SAMPLING TIME: 24 hours
_____________________________________________________________________
Treatment Dose level Culture Cells Metaphases Mean Relative
(µg/ml) No. Scored MI(%) MI(%)
_____________________________________________________________________
Untreated - 49 1000 54 5.3 98
50 1000 51
_____________________________________________________________________
Solvent 1% 51 1000 50 5.4 100
52 1000 57
_____________________________________________________________________
Test Item 39.1 67 1000 45 4.6 86
68 1000 47
_____________________________________________________________________
Test Item 78.1 65 1000 42 4.2 78
66 1000 41
_____________________________________________________________________
Test Item 156 63 1000 41 4.0 75
64 1000 39
_____________________________________________________________________
Test Item 313 61 1000 39 4.0 74
62 1000 40
_____________________________________________________________________
Test Item 625 59 1000 38 4.0 75
60 1000 42
_____________________________________________________________________
Test Item 1250 57 1000 38 3.7 68
58 1000 35
_____________________________________________________________________
Test Item 2500 55 1000 36 3.5 64
56 1000 33
_____________________________________________________________________
Test Item 5000 53 1000 35 3.6 67
54 1000 37
_____________________________________________________________________
Mitomycin-C 0.30 69 1000 27 2.9 54
70 1000 30
_____________________________________________________________________
Mitomycin-C 0.45 71 1000 22 2.3 44
72 1000 24
_____________________________________________________________________
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Read-across - in vitro gene mutation study in bacteria
In the key study (Polynt, 2008a) a bacterial reverse mutation assay (Ames test) has been assessed following OECD Guideline 417 and EU test method B.13/14 in accordance with GLP criteria. The study has been conducted with the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate. A preliminary toxicity test was undertaken to select suitable concentrations for use, performed with –S9 and + S9 using the plate incorporation method. The main experiment consists of two assays that were performed including negative and positive controls both –S9 and + S9 with Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli WP2 uvr A.Three replicate plates were used at each test point. The first experiment was performed using a plate- incorporation method and the second experiment was performed using a pre- incubation method, both at a maximum dose-level of 5000 µg/plate and four lower concentrations of 1580, 500, 158 and 50.0 µg/plate of the test substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate.
The substance did not induce increases in the number of revertant colonies in Salmonella typhimurium or Escherichia coli in the plate incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism.
This 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 (MHW Japan, 1996c) with the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate a bacterial reverse mutation assay (Ames test) has been assessed following OECD Guideline 417 and Japan Guidelines for Screening Mutagenicity Testing Of Chemicals in accordance with GLP criteria. A range finding study has been conducted with doses tested of 50-5000 µg/plate.The plate incorporation assay has been performed including negative and positive controls both –S9 and + S9 with Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100, and Escherichia coli WP2 uvr A.Three plates were used at each test point in duplicate at a maximum dose-level of 5000 µg/plate and four lower concentrations of 313, 625, 1250 and 2500 µg/plate of the test substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate.
The substance did not induce increases in the number of revertant colonies in Salmonella typhimurium or Escherichia coli in the plate incorporation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism.
This can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies.
This approach is further supported with QSAR calculations for tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate.
The prediction is the substance is NON-mutagenic using Ames test model from CAESAR.
The prediction is the substance is NON-mutagenic using ISS model from work of Benigni and Bossa. (ToxTree)
The prediction is NON-mutagenic using the model from Sarpy software from the original training set from the Mutagenicity Caesar model.
The predition is the substance is NON-mutagenic using the KNN model (VEGA).
Read-across - in vitro cytogenicity / chromosome aberration study in mammalian cells
The read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate was assayed in the key study (Polynt, 2008b) for the ability to cause chromosomal damage in cultured human lymphocytes according to OECD Guideline 473 and in compliance with GLP criteria. In-vitro treatment in two independent assays for chromosomal damage were performedin the absence and presence of S9 metabolic activation at a maximum dose-level of 5000 micrograms/plate and four lower concentrations of 2500, 1250, 625 and 313 micrograms/plate.
The source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate did not induce chromosomal aberrations in human lymphocytes after in-vitro treatment.
This 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 (MHW Japan, 2006) the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate has been assayed for its ability to cause chromosomal damage in cultured mammalian (Chinese Hamster lung (CHL/IU) cells) cells following in vitro treatment with concentrations of 0, 1.3, 2.5, 5.0 mg/ml in the absence and presence of S9 metabolic activation. The test was conducted according to Japanese Guidelines for Screening Mutagenicity Testing of Chemicals and in compliance with GLP standards.
The source chemical tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate did not induce structural chromosomal aberrations or polyploidy under the conditions of this experiment in mammalian cells.
This can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies.
Read-across - in vitro gene mutation study in mammalian cells
In the key study (Polynt, 2008c) the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate has been examined for mutagenic activity by assaying for the induction of 5‑trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method.The study has been conducted in accordance with OECD Guideline 476 and EU Method B.17 and in compliance with GLP criteria.
The source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate does not induce mutation in mouse lymphoma L5178Y cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
This 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
Reliable data is available for the read-across source substance tris(2-ethylhexyl) benzene-1,2,4-tricarboxylate on in vitro gene mutation in bacteria , in vitro cytogenicity / chromosome aberration in mammalian cells and in vitro gene mutation in mammalian cells. All results indicate that the substance is negative for gene mutation / mutagenicity. This can be similarly transferred to the target substance tetrakis(2-ethylhexyl) benzene-1,2,4,5-tetracarboxylate, due to strong structural analogies, what is also supported via QSAR predictions for the target substance.
According to Regulation (EC) No 1272/2008 classification and labelling is not indicated for genetic toxicity.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
