2,4,6-trimethylbenzene-1,3-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test according to OECD 471 is already available for the test substance (LPT 2016). Under the test conditions, the test item caused a pronounced concentration-related mutagenic effect in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation in test strain TA 100. No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. The results point to a base-pair substitution. The mutagenic effect was only seen in one strain.

To gather more data on the test substance, the QSAR tools Sarah (statistical based) and Derek (expert knowledge based) to predict mutagenicity seem appropriate.
The in silico assessment by using Sarah Nexus v. 3.1 (provided by Lhasa limited) seems to be appropriate as the (quantitative) structure-activity relationship ((Q)SAR) methodology gives an overall transparent prediction about Ames Mutagenicity of a structure, and a confidence rating in the prediction. The confidence score is based on each fragment’s contribution to the overall prediction and the weight placed upon each fragment. Furthermore, Sarah Nexus is able to predict the mutagenicity individually for the different bacterial stains (QSAR_Sarah_2021). The compound is predicted to be positive with 100% confidence for the 'Mutagenicity in vitro' endpoint in the model: 'Sarah Model - 2020.1'. This is based on an exact match with a compound found in the training dataset. 3 supporting hypotheses were also found and are displayed for information in the attached report.

Derek Nexus is a proprietary, rule-based expert system for the prediction of toxicity (QSAR_Derek_2021). Its knowledge base is composed of alerts, examples and reasoning rules which may each contribute to the predictions made by the system. Each alert in Derek describes a chemical substructure believed to be responsible for inducing a specific toxicological outcome (often referred to as a toxicophore). Alerts are derived by experts, using toxicological data and information regarding the biological mechanism of action. 

Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain> probable>plausible>equivocal. In Summary the test compound is likely to be mutagen based on its structural alert "aromatic amine".

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Remarks:

Sarah Nexus v. 3.1 (provided by Lhasa limited), predicted on 04 February 2021

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

An Ames test according to OECD 471 is already available for the test substance (LPT 2016). Under the test conditions, the test item caused a pronounced concentration-related mutagenic effect in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation in test strain TA 100. No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. The results point to a base-pair substitution. The mutagenic effect was only seen in one strain. To gather more data on the test substance, a QSAR tool to predict mutagenicity seems appropriate.
Therefore , the in silico assessment by using Sarah Nexus v. 3.1 (provided by Lhasa limited) seems to be appropriate as the (quantitative) structure-activity relationship ((Q)SAR) methodology gives an overall transparent prediction about Ames Mutagenicity of a structure, and a confidence rating in the prediction. The confidence score is based on each fragment’s contribution to the overall prediction and the weight placed upon each fragment. Furthermore, Sarah Nexus is able to predict the mutagenicity individually for the different bacterial stains.

Principles of method if other than guideline:

Sarah Nexus (provided by Lhasa limited) is an in silico tool. Sarah Nexus is a proprietary, statistical system for the prediction of
mutagenicity. It employs a self-organising hypothesis network (SOHN) of structural fragments to make predictions for mutagenicity [Hanser et al, 2014]. The fragments in the SOHN (referred to as hypotheses) are associated with activity or inactivity depending on the distribution of compounds containing this fragment in the training set of compounds with associated bacterial reverse mutation data.
The SOHN is derived automatically from the training data using a set of rules relying on the statistical distribution of positive and negative results for each structural fragment in the training set. An overall prediction for a query compound is derived based on resolving the results from the different hypotheses it activates. A quantitative confidence value is also associated with each hypothesis based on the activity of the nearest neighbours in the training set to the query compound. An overall confidence in the prediction is then derived by combining these confidences for individual hypotheses. Predictions are supported by displaying the relevant hypotheses associated with the query compound as well as compounds in the training set used to derive these hypotheses in order of similarity to the query compound. Detailed strain information on each training set compound along with CAS identification numbers and references to the primary literature are also provided where available. A strain profile for each hypothesis is generated based on the strain
information from the individual compounds belonging to it. Sarah Nexus has a domain of applicability. A compound is deemed to be within the
applicability domain of the model if all of the fragments present in the query structure have been adequately represented in the training set of
the model. If they have not then the relevant fragment will be highlighted in the query structure and it will be assigned as out of
domain. As well as positive and negative Sarah Nexus can also give equivocal results. These are produced when a prediction of only a low
confidence can be made by the model. By reporting this information to the user, Sarah provides highly transparent predictions.

Key result

Species / strain:

not specified

Remarks:

no detailed information on strains

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

The compound is predicted to be positive with 100% confidence for "Mutagenicity in vitro" endpoint in the model.

Cytotoxicity / choice of top concentrations:

not specified

Additional information on results:

The compound is predicted to be positive with 100% confidence for the 'Mutagenicity in vitro' endpoint in the model: 'Sarah Model - 2020.1'. This is based on an exact match with a compound found in the training dataset. 3 supporting hypotheses were also found and are displayed for information

Results	Number of Hypothesis
Positive	2
Negative	0
Positive (overruled by training set example)	1
Negative (overruled by training set example)	0
Total count	3

Conclusions:

As the in vitro results from LPT (2016) already indicated, the test substance, which is an aromatic amine, was also predicted to be positive with 100% confidence in the statistical based QSAR model Sarah Nexus.

Executive summary:

The mutagenicity of the test compound was predicted with the statistical based QSAR model Sarah Nexus. The compound is predicted to be positive with 100% confidence for the 'Mutagenicity in vitro' endpoint in the model: 'Sarah Model - 2020.1'. This is based on an exact match with a compound found in the training dataset. 3 supporting hypotheses were also found and are displayed for information in the attached report. In addition the strain specific analysis reveals that altogether 4 strains (TA98, TA100, TA1535 and TA97) are predicted to be positive for mutagenicity with S9 mix (see attached strain picture of the Sarah model).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

(Q)SAR

Remarks:

Derek Nexus v. 6.1.0 (provided by Lhasa limuted), predicted on 04 February 2021

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

The model is based primarily on data from the Ames test conducted
following standard test protocol (OECD TG471). If activity is observed
in a non-standard assay or protocol this will be mentioned in the
comments.

Principles of method if other than guideline:

An Ames test according to OECD 471 is already available for the test substance (LPT 2016). Under the test conditions, the test item caused a pronounced concentration-related mutagenic effect in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation in test strain TA 100. No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. The results point to a base-pair substitution. The mutagenic effect was only seen in one strain. To gather more data on the test substance, a QSAR tool to predict mutagenicity seems appropriate.


Derek Nexus is a proprietary, rule-based expert system for the prediction of toxicity. Its knowledge base is composed of alerts, examples and reasoning rules which may each contribute to the predictions made by the system. Each alert in Derek describes a chemical substructure believed to be responsible for inducing a specific toxicological outcome (often referred to as a toxicophore). Alerts are derived by experts, using toxicological data and information regarding the biological mechanism of action. Where relevant, metabolism data may be incorporated into an alert, enabling the prediction of compounds which are not directly toxicity but are metabolised to an active species. The derivation of each alert is described in the alert comments along with supporting references and example compounds where possible. By reporting this information to the user, Derek provides highly transparent predictions. The use of structural alerts for the prediction of toxicity is both widely understood and the subject of many
publications.

The Derek Nexus model for mutagenicity is developed from Ames test data in both S.typh and E.coli. Supporting data from transgenic rodent mutation assay, in vitro L5178Y TK+/- assay, in vitro HGPRT gene
mutation assay, in vitro Na+/K+ ATPase gene mutation assay has also been considered for the development of a small number of alerts. Additionally, alert writers consider both mechanistic evidence and
chemical properties (such as reactivity).

Derek Nexus v6.1 contains 148 active alerts for bacterial mutagenicity, together with reasoning rules and secondary functionality that evaluates potentially misclassified and unclassified features in compounds that do not activate bacterial mutagenicity alerts or examples.

Species / strain:

not specified

Remarks:

no detailed information on strains

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Alert matched 352 aromatic amines

Cytotoxicity / choice of top concentrations:

not specified

Conclusions:

As the in vitro results of Provivo already indicated, the test substance, which is an aromatic amine, was also predicted to be postive due to the identified structural alert of the test substance "aromatic amine".

Executive summary:

The mutagenicity of the test substance was predicted by the rule-based expert QSAR system Derek from Lhasa. The QSAR model knowledge base is composed of alerts, examples and reasoning rules which may each contribute to the predictions made by the system. Each alert in Derek describes a chemical substructure believed to be responsible for inducing a specific toxicological outcome (often referred to as a toxicophore). Alerts are
derived by experts, using toxicological data and information regarding the biological mechanism of action.

Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain> probable>plausible>equivocal. In Summary the test compound is likely to be mutagen based on its structural alert "aromatic amine".

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-03-17 to 2016-04-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The test item was not soluble in any of the solvents generally recommended: highly purified water or dimethylsulfoxide (DMSO). However, the test item was completely dissolved in ethanol, a solvent acceptable for this test system. The vehicle ethanol served as the negative control.

Target gene:

mutated gene loci resposible for histidine auxotropy

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

- obtained from Trinova Biochem according to Dr. Bruce N. AMES,

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver S9; male rats, obtained from Trinova Biochem

Test concentrations with justification for top dose:

Plate incorporation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate;
Preincubation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate;

Vehicle / solvent:

The test item was completely dissolved in ethanol, a solvent acceptable for this test system. The vehicle ethanol served as the negative control.

Untreated negative controls:

yes

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. No signs of cytotoxicity were observed up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction.
ADMINISTRATION
- Dosing:
* Plate incorporation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate;
* Preincubation test: 31.6, 100, 316, 1000, 3160 and 5000 µg per plate;
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
* sodium azide in highly purufied water for TA 1535 and TA 100, 10 µg/plate
* 2-nitroflurene in DMSO for TA 98, 10 µg/plate
* 9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
* Mitomycin C in highly purifies water for TA 102, 10 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100 and TA 1535, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, TA 102 and 1537, 10 µg/plate
- negative control: the vehicle DMSO was used as negative reference item (all test strains).
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

NUMBER OF REPLICATIONS: 3 per concentration and experiment

NUMBER OF CELLS EVALUATED: approximately 10E8 viable cells in the late exponential or early stationary phase

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Rationale for test conditions:

The study was performed in compliance with:
- Regulation (EC) No. 440/2008 method B.13/14: Mutagenicity: Reverse Mutation Test Using Bacteria, adopted May 30, 2008;
- OECD Guideline for Testing of Chemicals, No. 471: Bacterial Reverse Mutation Test, adopted July 21, 1997;

Evaluation criteria:

A test item is considered to show a positive response if
- the number of revertants is significantly increased (p solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation
coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Acceptance Criteria
The results of the negative and positive control cultures have to be within the range of the historical data generated by LPT.

Statistics:

According to the OECD Guideline 471, a statistical analysis of the data is not mandatory

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

The results point to a base-pair substitution.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

GENTOXIC EFFECTS:
- With and without metabolic activation: In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation a pronounced concentration-related mutagenic effect (significant at p No mutagenic effect was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test.

CYTOTOXICITY EFFECTS:
No signs of cytotoxicity were observed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in all test strains.

see attchached document

Conclusions:

In conclusion, under the present test conditions, the test item caused a pronounced concentration-related mutagenic effect in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at concentrations of 3160 and/or 5000 µg test item/plate in test strain TA 100.
No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
The results point to a base-pair substitution.

Executive summary:

The purpose of this study was to evaluate the test substance for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and (1983).

The potential of the test item to induce gene mutations was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item was not soluble in any of the solvents generally recommended: highly purified water or dimethylsulfoxide (DMSO). However, the test item was completely dissolved in ethanol, a solvent acceptable for this test system.The vehicle ethanol served as the negative control.

Preliminary test

Test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. No signs of cytotoxicity were observed up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations ranging from 31.6 to 5000 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were observed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation up to the top concentration of 5000 µg test item/plate in all test strains.

Mutagenicity

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation a pronounced concentration-related mutagenic effect (significant at p </= 0.05) was observed in test strain TA 100.

All criteria for a positive response (a concentration (log value)-related effect and a 2-fold increase in revertant colony numbers compared with control counts, significant at p </= 0.05) were met in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at a concentration of 5000 µg test item/plate and, in addition, in the experiments without S9 mix at the concentration of 3160 µg/plate in test strain TA 100.

No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test.

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and confirmed the validity of the test conditions and the sensitivity of the test system.

 

In conclusion, under the present test conditions, the test item caused a pronounced concentration-related mutagenic effect in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at concentrations of 3160 and/or 5000 µg test item/plate in test strain TA 100. No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. The results point to a base-pair substitution.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Under the present test conditions, the test item caused a pronounced concentration-related mutagenic effect in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation at concentrations of 3160 and/or 5000 µg test item/plate in test strain TA 100 (LPT, 2016). No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for strains TA98, TA102, TA1535 and TA1537 in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation. The results point to a base-pair substitution.

Due to the fact that the test item is a monomer of a polymer and no free test item is in the polymer no further genetic toxicity studies in vitro are performed.

Justification for classification or non-classification

Based on the available vitro genotoxicity data according to Ames and based on the results of the QSAR prediction tools (Derek and Sarah), the test item must be classifed as Muta. 2 (H341) according to the criteria of EC Regulation 1272/2008.