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EC number: 221-456-9 | CAS number: 3102-70-3
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Genetic toxicity: in vitro
Administrative data
- 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
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- 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
Test material
- Reference substance name:
- 2,4,6-trimethylbenzene-1,3-diamine
- EC Number:
- 221-456-9
- EC Name:
- 2,4,6-trimethylbenzene-1,3-diamine
- Cas Number:
- 3102-70-3
- Molecular formula:
- C9H14N2
- IUPAC Name:
- 2,4,6-trimethylbenzene-1,3-diamine
- Test material form:
- other: crystalline powder
- Details on test material:
- 2,4,6-trimethylbenzene-1,3-diamine supplied by Evonik Fibres GmbH (Lenzing, Austria), batch 20150703 (Dragon), purity 99.82 % w/w
Constituent 1
- 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.
Method
- Target gene:
- mutated gene loci resposible for histidine auxotropy
Species / strain
- 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.
Controls
- 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 = 0.05, U-test according to MANN and WHITNEY) compared to the
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
Results and discussion
Test resultsopen allclose all
- 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 = 0.05) was observed in test strain TA 100.
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.
Any other information on results incl. tables
see attchached document
Applicant's summary and conclusion
- 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.
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