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EC number: 214-675-6 | CAS number: 1184-78-7
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Study initiated on 15 December 2009 and completed (final report issued) on 17 May 2010. Experimental work started on 18 December 2009 and was completed on 8 March 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Conducted in accordance with current testing guidelines and GLP compliant
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 010
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Principles of method if other than guideline:
- The objective of this study was to evaluate the potential of Trimethylamine (TMA-opl in water) to induce forward mutation at the hprt locus (the hypoxanthine guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance)) in mouse lymphoma L5178Y cells using a fluctuation protocol in the absence and presence of a rat liver metabolising system (S9). The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post mitochondrial fraction (S9).
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- Trimethylamine
- EC Number:
- 200-875-0
- EC Name:
- Trimethylamine
- Cas Number:
- 75-50-3
- Molecular formula:
- C3H9N
- IUPAC Name:
- N,N-dimethylmethanamine
- Details on test material:
- Trimethylamine (TMA-opl in water), alternative names: Methylamine, N,N dimethyl; Methanamine, N,N-dimethyl; N,N-Dimethyl methanamine), batch number G7280 (see Appendix 8), was a clear colourless liquid with a molecular weight of 59.11 (CAS number for Trimethylamine: 75-50-3). It was received on 1 December 2009 and stored at 15 25C in the dark. The test article was provided as a 1.17% v/v solution of Trimethylamine (TMA-opl in water) in purified water and the expiry date was given as 29 November 2011.
Determinations of the stability and characteristics of the test article were the responsibility of the Sponsor.
The test article was supplied by the Sponsor as a 1.17% v/v (i.e. 11.7 mg/mL) solution of Trimethylamine (TMA-opl in water) in purified water.
Test article stock solutions were prepared under subdued lighting by formulating Trimethylamine (TMA-opl in water) in purified water (with the aid of vortex mixing as required) immediately prior to assay to give the maximum required treatment solution concentration. The stock solutions were membrane filter-sterilised (Pall Acrodisc 32, 0.2 m pore size) and subsequent dilutions made using purified water. The test article solutions were protected from light and used within 1.5 hours of initial formulation of the test article.
- Name of test material (as cited in study report): Trimethylamine (TMA-opl in water)
- Molecular formula: Not applicable
- Molecular weight: 59.11
- Physical state: Clear colourless liquid
- Analytical purity: 1.17%v/v solution in purified water
- Purity test date: Not specified
- Lot/batch No.: G7280
- Expiration date of the lot/batch: 29 November 2011
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature 20 ± 5ºC in the dark
- Other: Solubility limit in culture media was 1170 µg/mL
Constituent 1
Method
- Target gene:
- The mutation systems work by placing cells under selective pressure so that only mutant cells are able to survive. Resistance to the toxic analogue 6-thioguanine (6TG) results from lack of hypoxanthine-guanine phosphoribosyl transferase (HPRT) activity. Thus, the hprt- mutants are unable to use 6TG and survive in its presence. The hprt gene is X-linked and mainly point mutations are detected at this locus. However, hprt is a large gene and changes of 30 to 40 kilobases have also been reported.
Species / strain
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- L57178 tk +/- (3.7.2C) mouse lymphoma cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells were purged of tk- mutants, checked for spontaneous mutant frequency and that it was mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Arocolor 1254 induced rat liver post-mitochondrial fraction (S-9)
- Test concentrations with justification for top dose:
- A maximum concentration of 591 µg/mL was selected for the cytotoxicity Range-Finder Experiment
Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range Finder Experiment.
Range-finder (with and without S-9) - 18.47, 36.94, 73.88, 147.8, 295.5 and 591.0 µg/mL Trimethylamine (TMA-opl in water).
Experiment 1 (with and without S-9) - 100, 200, 250, 300, 350, 400, 450, 500, 550 and 591.1 µg/mL Trimethylamine (TMA-opl in water).
Experiment 2 (without S-9) - 100, 200, 250, 300, 350, 375, 400, 425, 450 and 500 µg/mL Trimethylamine (TMA-opl in water).
Experiment 2 (with S-9) - 100, 200, 250, 300, 350, 400, 425, 450, 475 and 500 µg/mL Trimethylamine (TMA-opl in water). - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: The solubility limit in culture medium was in excess of 1170 µg/mL. A maximum concentration of 591 µg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a concentration equivalent to approximately 10 mM (an acceptable maximum concentration as stated in current regulatory guidelines). Concentrations selected for the Mutation Experiments were based on the results of this cytotoxicity Range Finder Experiment.
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Purified water diluted 10-fold in the treatment medium
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- 4-nitroquinoline-N-oxide (NQO), Benzo[a]pyrene (B[a]P, +S-9) (Both prepared in DMSO)
- Remarks:
- NQO at 0.10 and 0.15 µg/mL final concentration, B[a]P at 2.00 and 3.00 µg/mL final concentration.
- Details on test system and experimental conditions:
- Mouse lymphoma L5178Y (master stock of L5178Y tk +/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. ).
The mammalian liver post-mitochondrial fraction (S9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA, where it was prepared from male Sprague Dawley rats, induced with Aroclor 1254. The batches of MolToxTM S9 were stored frozen at –80ºC prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
Metabolic activation system
Rat liver S-9 fraction from male Sprague Dawley rats induced with Aroclor 1254. Batches were stored frozen at -80°C prior to use. Each batch was checked by the manufacturere for sterility, protein content, ability to convert kown promutagens to bacterial mutagens and cytochrome P-450-catalysed enzyme activities.
Treatment was carried out both in the presence and absence of S-9, and was prepared in the following way:
Glucose-6-phosphate (G6P: 180 mg/mL), β-Nicotinamide adenine dinucleotide phosphate (NADP: 25 mg/mL), Potassium chloride (KCl: 150 mM) and rat liver S-9 were mixed in the ration 1:1:1:2.. For all cultures treated in the presence of S-9, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. Cultures treated in the absence of S-9 received 1 mL KCl (150 mM). The final concentration of the liver homogenate in the test system was 2%. - Evaluation criteria:
- Acceptance criteria
The assay was considered valid if the following criteria were met:
1.the mutant frequencies in the negative (vehicle) control cultures fell within the normal range (not more than three times the historical mean value)
2.at least one concentration of each of the positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).
Evaluation criteria
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1.the mutant frequency at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2.there was a significant concentration relationship as indicated by the linear trend analysis (p≤0.05)
3.the effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. - Statistics:
- Statistical significance of mutant frequencies was carried out according to the UKEMS guidlines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Results and discussion
Test results
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- The highest concentration to provide >10% RS was 295.5 µg/mL, which gave 67% and 54% RS in the absence and presence of S-9, respectively.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
Any other information on results incl. tables
Table1: Trimethylamine (TMA-opl in water) Concentrations Tested
Experiment |
S‑9 |
Concentration of treatment solution (mg/mL) |
Final concentration (µg/mL) |
|
|
|
|
Range-finder |
-and + |
0.1847 0.3694 0.7388 1.478 2.955 5.910 |
18.47 36.94 73.88 147.8 295.5 591.0 |
|
|
|
|
1 |
-and + |
1.00 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 5.91 |
100 200 250 300 350 400 450 500 550 591.1 |
|
|
|
|
2 |
- |
1.00 2.00 2.50 3.00 3.50 3.75 4.00 4.25 4.50 5.00 |
100 200 250 300 350 375 400 425 450 500 |
|
|
|
|
|
+ |
1.00 2.00 2.50 3.00 3.50 4.00 4.25 4.50 4.75 5.00 |
100 200 250 300 350 400 425 450 475 500 |
|
|
|
|
Experiment 1: ten concentrations, ranging from 100 to 591.1 µg/mL, were tested in the absence and presence of S‑9. Following the treatment incubation period, the highest three concentrations tested in the absence of S-9 (500 to 591.1 µg/mL) and the highest two concentrations tested in the presence of S-9 (550 and 591.1 µg/mL) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest remaining concentrations in the absence and presence of S‑9 (450 and 500 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, an intermediate concentration (300 µg/mL) in the absence of S-9 was not selected as there were sufficient concentrations to define an appropriate toxicity profile. All other concentrations in the absence and presence of S-9 were selected. The highest concentrations selected were 400 µg/mL in the absence of S‑9 and 450 µg/mL in the presence of S‑9, which gave 25% and 5% RS, respectively (seeTable8). In the absence and presence of S-9, no concentration gave 10-20% RS. In the absence of S‑9, cultures treated at 350 and 400 µg/mL, gave 50% and 25% RS, respectively and in the presence of S-9, cultures treated at 400 and 450 µg/mL, gave 38% and 5% RS, respectively. Both concentrations were therefore analysed under each treatment condition.
Experiment 2: ten concentrations, ranging from 100 to 500 µg/mL, were tested in the absence and presence of S‑9. Seven days after treatment, concentrations of 100 and 250 mg/mL in the presence of S-9 were not selected as there were sufficient concentrations to define an appropriate toxicity profile. All other concentrations in the absence and presence of S-9 were selected. The highest concentration selected was 500 µg/mL, which gave 20% and 33% RS in the absence and presence of S‑9, respectively (seeTable 8). Cultures treated at 475 µg/mL in the presence of S-9 gave 23% RS, which was sufficiently close to 10‑20% RS to be considered acceptable.
Table 2: Range-Finder Experiment
Table 3: Experiment 1 (3-hour treatment in the absence and presence of S-9)
Table 4: Experiment 2 (3-hour treatment in the absence and presence of S-9)
§ 6TG resistant mutants/106 viable cells 7 days after treatment % RS Percent relative survival adjusted by post treatment cell counts NS Not significant |
Applicant's summary and conclusion
- Conclusions:
- In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Trimethylamine (TMA-opl in water) at any concentration tested in the absence and presence of S 9 and there were no significant linear trends. Under both treatment conditions, concentrations giving approximately 10 20% RS (or less) were analysed in both experiments and there was no evidence of mutagenic activity at any concentration analysed in the absence and presence of S-9 in either experiment. Furthermore, the increases in pH observed in the absence and presence of S-9 in both experiments were not associated with increases in mutant frequency, therefore they were not considered biologically relevant.
CONCLUSION
It is concluded that Trimethylamine (TMA-opl in water) did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9). - Executive summary:
Trimethylamine (TMA-opl in water) was assayed by Covance in 2010 for its potential to induce mutation at the hypoxanthine‑guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctiation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post‑mitochondrial fraction (S‑9). The test article was formulated in purified water.
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S‑9, ranging from 18.47 to 591.0 µg/mL (equivalent to approximately 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) was 295.5 µg/mL, which gave 67% and 54% RS in the absence and presence of S‑9, respectively.
Accordingly, for Experiment 1 ten concentrations, ranging from 100 to 591.1 µg/mL, were tested in the absence and presence of S‑9. Seven days after treatment, the highest concentrations selected to determine viability and 6TG resistance were 400 µg/mL in the absence of S‑9 and 450 mg/mL in the presence of S-9, which gave 25% and 5% RS, respectively. In the absence and presence of S-9, no concentration gave 10‑20% RS (in the absence of S‑9, cultures treated at 350 and 400 µg/mL, gave 50% and 25% RS, respectively and in the presence of S-9, cultures treated at 400 and 450 µg/mL, gave 38% and 5% RS, respectively). Both concentrations were therefore analysed under each treatment condition. In Experiment 2 ten concentrations, ranging from 100 to 500 µg/mL, were tested in the absence and presence of S‑9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance was 500 µg/mL, which gave 20% and 33% RS in the absence and presence of S‑9, respectively. Cultures treated at 475 µg/mL in the presence of S-9 gave 23% RS, which was sufficiently close to 10‑20% RS to be considered acceptable. In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with Trimethylamine (TMA-opl in water) at any concentration tested in the absence and presence of S‑9 and there were no significant linear trends. Under both treatment conditions, concentrations giving 10-20% RS (or less) were analysed in one experiment and there was no evidence of mutagenic activity at any concentration analysed in the absence and presence of S-9 in either experiment, therefore this did not affect data interpretation.
It is concluded that Trimethylamine (TMA-opl in water) did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to highly toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S‑9).
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