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Description of key information

 The subchronic oral toxicity of a close analogue of the registered substance was investigated in a well-conducted study in Sprague Dawley rats after daily oral administration at dose levels of 50, 200 and 500 mg/kg/day for 13 weeks and recovery from any treatment-related effects during a recovery period of 4 weeks. Signs of treatment-related effects of the test item were observed in males and females dosed at 200 and 500 mg/kg/day. These effects included some changes in clinical pathology parameters and some post mortem findings. The liver was identified as the main target organ on the basis of the organ weight data and histopathological findings. However, these changes were mild and fully reversible. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum. As such, the hepatic effects were not considered to be adverse.
No significant changes were observed in the animals dosed at 50 mg/kg/day. It can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 500 mg/kg/day.
In a subacute 28-day oral toxicity study in rats some treatment-related effects were evident in males and to a minor extent in females at the high dose level of 1000 mg/kg/d. The adrenals were identified as the main target organs, based on the post-mortem examination. The observed effects were completely reversible over a 2-week recovery period in the high dose animals. Only mild effects were observed in the animals (essentially males) dosed at 300 mg/kg/d, therefore this dose is expected to be NOAEL. No effects were observed at the low dose level (NOEL = 100 mg/kg/d).
In the subacute 28-day study a dose of 500 mg/kg bw/d was not used but adverse effects were seen at 1000 mg/kg bw/d (LOAEL). Therefore for repeated dose toxicity it can be concluded in summary that the NOAEL is 500 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
A brief overview of the read-across study is reported below. Detailed information on the read-across justification is included in the read-across study report available in the "Attached justification" field. Please also refer to this report for the list of tools used in the assessment.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The present read-across study falls within the RAAF scenario 2, i.e. analogue approach based on the hypothesis that different compounds are supposed to cause the same type of effects as a result of structural similarity.
In the current study, TM8, TOTM and TM8-10 were selected as source chemicals for the read-across prediction of repeated dose toxicity for the UVCB target substance TM9. In particular, the sources TM8 and TOTM are mono-constituent substances, while the source TM8-10 and the target TM9 are UVCB substances. It is highlighted that the constituents of the target TM9, the sources TM8 and TOTM, and the constituents of the source TM8-10 belong to the same “pool” of structurally related constituents. Thus, according to the RAAF guidance for UVCB and MCSs5, structural similarity as a basis for the read-across may be assumed. Supporting information to further justify the analogue approach is provided by comparing the source and target substances in terms of mechanistic similarity, physico-chemical properties and ADME profile.
Additional details can be found in the attached read-across study report.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to section 1.2 (general information/composition) of both target and source's IUCLID dossiers, and to the attached read-across study report.

3. ANALOGUE APPROACH JUSTIFICATION
Structural Similarity: Overall, the target TM9 exhibits high structural similarity toward the source substances TM8, TOTM and TM8-10. Minimal differences were identified among the analysed compounds due to the branching and lengths of the alkyl chains, however it is acknowledged that all of the functional groups and structural fragments of the target TM9 are represented by the source substances, which possess both linear and branched alkyl chains (as the target TM9) and cover almost the entire range of chain lengths from C8 to C10 (except for TM9 constituent 3, which possesses one C11 alkyl chain). It is then concluded that the target TM9 is included in the chemical space defined by the source compounds, thus reducing the uncertainty associated with structural similarity.

Concluding, the target and source substances exhibit high structural similarity. Uncertainty associated with structural similarity was assessed to be low.

Mechanistic similarity: The target and source compounds exhibited high mechanistic similarity. No structural alerts for protein binding were identified in all target and source constituents by the OECD QSAR Toolbox OASIS and OECD profilers. All target constituents and source compounds were classified into Cramer’s Class I (Low concern). The Cramer Class I category refers to substances with simple chemical structures and for which efficient modes of metabolism exist, suggesting a low order of oral toxicity.
Finally, the OECD QSAR Toolbox HESS profiler for Repeated dose toxicity identified the phthalate ester alert in the target and the source compounds. It is further described in the profiler that it was found that toxicity induced by phthalate esters depends on their alkyl side-chain length. It was reported that phthalates with alkyl side-chain lengths from C4 to C6 produce similarly sever reproductive effects in experimental animals It is highlighted, that the profiler is characterised by a low rank of reliability (“Rank C” – i.e., toxicity mechanism not well known, category boundary experientially defined by using the repeated-dose toxicity (RDT) database test data).

Concluding, the target and source substances exhibited high mechanistic similarity. However, the uncertainty associated with mechanistic similarity was assessed as medium, due to limitations associated with the endpoint type.

Physico-chemical similarity:From the analysis and comparison of experimental and in silico predicted physico-chemical data, no major differences are noted between target and source substances. As expected from their chemical structure, which is characterised by rather long chains of carbon atoms, all of the analysed structures were both experimentally and predicted to be highly hydrophobic and insoluble, with LogKow values greater than 8 and water solubility (experimental and predicted) below 1 mg/L. Since no dissociating groups are found in both structures, pH has no influence on LogKow, water solubility, and ADME properties. Lastly, volatilisation is not expected to occur for all substances (boiling point greater than 150 °C, vapour pressure lower than < 500 Pa).
Concluding, the target and the source compounds exhibited high similarity in terms of their physico-chemical profile. Specifically, very high hydrophobicity, low water solubility, and no volatilisation potential were observed in target and source compounds. The uncertainty associated with PC similarity was assessed to be low.



ADME similarity: From the main results of the in-silico ADME analysis, a rather similar ADME profile was noted between the target TM9 and the sources TM8, TOTM, and TM10 (as for TM8-10).
Based on the predicted ADME data, target and source compounds exhibited a similar profile of highly lipophilic and poorly soluble substances, with a good passive absorption, a negligible oral bioavailability, a moderate first-pass metabolism, and high affinity to extravascular tissues. The experimental data available for the source TOTM showed that little of this substance was absorbed in a toxicokinetic study, since 75% of the initial dose was excreted in faeces, and 86% of which was TOTM unchanged. TOTM was found to be partially hydrolyzed in the gastro-intestinal tract to 2-ethylhexanol (2-EH) and the corresponding di-ester and, following further hydrolysis, the mono-ester. A low accumulation potential was also concluded for TOTM in the same study.
Based on the predicted physico-chemical and ADME properties, no major variations in the ADME profile of the target TM9 are expected due to interactions among target’s constituents.
Concluding, target and source compounds exhibited high similarity in terms of their predicted ADME profile. Overall, the uncertainty associated with ADME similarity was assessed to be low.



Potential metabolic products:Following the analysis of the simulated first level metabolites it is anticipated that all UVCB TM9 target’s constituents and source TM8, TOTM, TM10 (as for TM8-10) compounds have common biotransformation products resulting from Phase I metabolic reactions, including aromatic carboxylation and hydrolysis of the carboxylic acid esters. Predicted metabolites mainly include di-esters (benzene-1,2,4-tricarboxylic acid esterified by two alkyl chains (C8-C11)) and linear or branched alcohols (C8-C11) derived from the hydrolysis of one of the alkyl rich linear or branched substituents. In the 2nd level metabolism further hydrolysis and Phase II metabolism was predicted. With a very low odds ration primary alcohol oxidation was predicted resulting in e.g. a nonanol converted into nonanoic acid. Finally, predicted metabolites of the target and source compounds were profiled within the OECD QSAR toolbox (structural- and mechanistic-based profilers). The assessment of their structural and mechanistic similarity was performed in order to possibly understand whether some differing metabolites could give rise to new properties. By considering the mechanistic profiling, no differences were identified among the targets’ metabolites and the metabolites of the source as no alerts for protein binding were found by both the OASIS and OECD profilers. According to the Toxic hazard classification by Cramer, all metabolites of target and sources were classified as Cramer class I (Low toxicity). Based on the Repeated Dose (HESS) profiler, all hydroxylated and diester metabolites from the target constituents and the source compounds were identified as phthalate esters relating to testicular toxicity (Rank C). The major difference between the target constituents’ metabolites and the sources’ metabolites, identified by the Repeated Dose (HESS) profiler, were two hepatotoxicity alerts: Valproic acid and Perhexiline. Further analysis of te Perhexiline alert led to the conclusion that the alert is not relevant. As for Valproic acid alert it was considered a relevant alert as the simulation of metabolism presented above demonstrated that alcohols could undergo oxidation to acids, the alert appears to be relevant for 4 target metabolites.
Concluding, the target and source substances exhibited high similarity in terms of their potential metabolites. In particular, the fact that all substances are likely to undergo ester hydrolysis which finds confirmation in experimental toxicokinetic studies. The predicted metabolites showed high structural similarity but moderate mechanistic similarity. The main uncertainty relates to the valproic acid alert identified in a common metabolite of 4 target constituents. Overall, uncertainty associated with the similarity in potential metabolic products was assessed to be medium.

Source experimental data: Repeated dose toxicity experimental studies (oral) were available for the source compounds TM8, TOTM and TM8-10. Study reports were reported to be reliable without or with restrictions (Klimisch score 1 or 2), most of them being GLP-compliant, and having followed the main guideline, i.e. Repeated Dose 90-Day Oral Toxicity in Rodents, Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test and some supporting studies were also provided by the registrant i.e. short-term repeated dose toxicity studies.

4. DATA MATRIX
In the read-across analysis, TM8, TOTM and TM8-10 was selected as source chemical for the target TM9. The similarity assessment, which consisted of a comparison of structural, mechanistic, physicochemical, ADME and metabolism profiles, showed that the sources TM8, TOTM and TM8-10 and the target TM9 are sufficiently similar to justify the read-across approach.
The data matrix is included in the attached report.
Reason / purpose:
read-across source
Remarks:
TM8
Reason / purpose:
read-across source
Remarks:
TM8
Reason / purpose:
read-across source
Remarks:
TOTM
Reason / purpose:
read-across source
Remarks:
TOTM
Reason / purpose:
read-across source
Remarks:
TOTM
Reason / purpose:
read-across source
Remarks:
TM8-10
Reason / purpose:
read-across source
Remarks:
TM8-10
Qualifier:
according to
Guideline:
other: ECHA, 2015: Read-Across Assessment Framework (RAAF).
Qualifier:
according to
Guideline:
other: ECHA, 2017: Read-Across Assessment Framework (RAAF). Considerations on multi-constituent substances and UVCBs.
Specific details on test material used for the study:
EC: 941-303-6
Type: UVCB
Key result
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
other: Source TM8
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Read-across
Key result
Dose descriptor:
NOAEL
Effect level:
225 mg/kg bw/day (nominal)
Based on:
other: Source TOTM
Sex:
male/female
Basis for effect level:
other: Lack of significant treatment related effects
Remarks on result:
other:
Remarks:
Read-across
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
other: Source TM8-10
Sex:
male/female
Basis for effect level:
other: Lack of significant treatment related effects
Remarks on result:
other:
Remarks:
Read-across
Critical effects observed:
not specified
Conclusions:
Repeated dose experimental studies in rodents were available for the source TM8, TOTM and TM8-10. The studies were assessed as adequate for the read-across prediction for the target TM9. Based on the read-across study presented, it is concluded that the repeated dose toxicity data available for the source TM8, TOTM and TM8-10 could be used to support the assessment and classification of TM9.
Executive summary:

This study was designed to generate read-across predictions ofrepeated dose toxicityfor the UVCB TM9. The target TM9 is an esterification products of 1,3-dioxo-2-benzofuran-5-carboxylic acid and nonan-1-ol, C9 linear alkyl rich and is to be used for its safety assessment in the regulatory framework of REACH.

The target is composed by nine main constituents (one mono-constituent and eight UVCB substances for about 50 structures), which share a common core structure, i.e. benzene-1,2,4-tricarboxylic acid, esterified by linear and/or branched C8-C11 alkyl chains (C9 rich). In the present assessment, 15 of these substances were selected as representative compounds of the target TM9.

In theread-across analysis,three sources of experimental data were selected, the mono-constituents TM8 and TOTM, and the UVCB TM8-10. The target and source substances belong to the same “pool” of structurally related constituents, since they share a common core structure, i.e. benzene-1,2,4-tricarboxylic acid, esterified by linear and/or branched C8-C11 alkyl chains. Thus, according to the RAAF guidance for UVCBs, structural similarity as a basis for the read-across may be assumed.The similarity assessment, which consisted of a comparison of structural, mechanistic, physicochemical, ADME and metabolism profiles, showedthat the three sourcesand the targetTM9are sufficiently similar to justify the read-across approach.

Experimental toxicity data were available for the sourcesTM8 and TOTM, and the UVCB TM8-10 i.e.Repeated Dose 90-Day Oral Toxicity Study in Rodents and as supporting studies, Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test and Repeated Dose 28-Day Oral Toxicity in Rodents. Studieson repeated dose toxicity were assessed as adequate for the read-across prediction of the repeated dose toxicity endpoint for the target TM9.

In the present read-across study, an overall medium uncertainty was associated with similarity justification. However, the following issue was highlighted: i) the endpoint to read-across does not have yet well-defined mechanistic bases (high-tier endpoint, ii) ADME assessment was mainly based on simulated data; iii) identification of potential metabolites and related structural and mechanistic profiles was mainly based onin silicopredictions iv) a metabolite related to hepatotoxicity was identified for the target but not for the source compounds.. The main uncertainties associated with the read-across argument were related to the following issues: i) endpoint-type, ii) no bridging studies among the source and target substances.

Concluding, the read-across from the repeated doseexperimental studies in rats, which areavailable forthe source substance TM8 and TOTM and TM8-10, are likely to predict the repeated dose toxicity potential of the target substance TM9 and are considered as adequate to fulfil the information requirement of Annex X 8.6 of the REACH Regulation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A number of studies are available on two structural analogues of the registered substance, two being high-quality subchronic studies.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The subchronic oral toxicity of 1,2,4 -Benzenetricarboxylic acid, decyl octyl ester, a close structural analogue of the registered substance, was investigated in a well-conducted study in Sprague Dawley rats after daily oral administration at dose levels of 50, 200 and 500 mg/kg/day for 13 weeks and recovery from any treatment-related effects during a recovery period of 4 weeks. Signs of treatment-related effects of the test item were observed in males and females dosed at 200 and 500 mg/kg/day. These effects included some changes in clinical pathology parameters and some post mortem findings. The liver was identified as the main target organ on the basis of the organ weight data and histopathological findings. However, these changes were mild and fully reversible. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum. As such, the hepatic effects were not considered to be adverse. No significant changes were observed in the animals dosed at 50 mg/kg/day. It can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 500 mg/kg/day. In the study there were not seen any indications for reproductive effects, those investigated were spermatogenic staging, oestrus cycle, weights of ovaries and testes, including microscopic and macroscopic observations.In a subacute oral toxicity study in rats some treatment-related effects were evident in males and to a minor extent in females at the high dose level of 1000 mg/kg/d. The adrenals were identified as the main target organs, based on the post-mortem examination. The observed effects were completely reversible over a 2-week recovery period in the high dose animals. Only mild effects were observed in the animals (essentially males) dosed at 300 mg/kg/d, therefore this dose is expected to be NOAEL. No effects were observed at the low dose level (NOEL = 100 mg/kg/d). In the subacute 28 -day study, a dose of 500 mg/kg bw/d was not used but adverse effects were seen at 1000 mg/kg bw/d (LOAEL). The NOAEL for repeated dose toxicity was therefore regarded as being 500 mg/kg body weight/day.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
A number of studies are available on two structural analogues of the registered substance, the selected study being on the closest structural analogue to the registered substance.

Justification for classification or non-classification

The results of the repeated dose oral toxicity studies show that classification according to the criteria of EU regulations (Directive 67/548/EEC and Regulation (EC) No 1272/2008 is not warranted