Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No data is available with TMAO. Results obtained with the source substance TMA are reported.

- bacterial reverse mutation assay (Ames test, OECD TG 471): negative

- in vitro gene mutation study in mammalian cells (OECD TG 476): negative

- in vitro chromosome aberration study in mammalian cells (OECD TG 473): negative

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
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
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 / 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.
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.
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

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

Treatment

(µg/mL)

-S-9

% RS

+S-9

% RS

0

100

100

18.47

98

83

36.94

128

117

73.88

124

89

147.8

93

71

295.5

67

54

591

0

0

% RS               Percentage Relative Survival

Table 3: Experiment 1 (3-hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

2.93

 

0

 

100

4.91

 

100

 

90

5.47

NS

100

 

89

3.27

NS

200

 

79

3.91

NS

200

 

78

4.34

NS

250

 

55

4.08

NS

250

 

83

3.11

NS

350

 

50

2.69

NS

300

 

85

5.75

NS

400

 

25

5.03

NS

350

 

51

3.87

NS

 

 

 

 

 

400

 

38

5.30

NS

 

 

 

 

 

450

 

5

4.91

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

66

36.99

 

2

 

59

63.49

 

0.15

 

41

55.41

 

3

 

22

79.34

 

 

Table 4: Experiment 2 (3-hour treatment in the absence and presence of S-9)

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

 

% RS

MF§

 

% RS

MF§

0

 

100

4.31

 

0

 

100

6.78

 

100

 

97

4.02

NS

200

 

90

4.26

NS

200

 

64

3.78

NS

300

 

75

5.01

NS

250

 

72

4.61

NS

350

 

71

3.65

NS

300

 

66

4.25

NS

400

 

59

4.95

NS

350

 

50

4.43

NS

425

 

54

6.38

NS

375

 

53

3.79

NS

450

 

30

5.62

NS

400

 

47

4.51

NS

475

 

23

3.43

NS

425

 

30

4.63

NS

500

 

33

6.92

NS

450

 

23

5.43

NS

 

 

 

 

 

500

 

20

4.72

NS

 

 

 

 

 

Linear trend

NS

Linear trend

NS

NQO

 

 

 

 

B[a]P

 

 

 

 

0.1

 

75

21.42

 

2

 

37

25.78

 

0.15

 

69

24.06

 

3

 

19

82.32

 

§                     6TG resistant mutants/106 viable cells 7 days after treatment

% RS               Percent relative survival adjusted by post treatment cell counts

NS                   Not significant

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).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiated on 15 December 2009 and completed (final report issued) on 11 May 2010. Experimental work started on 17 December 2009 and was completed on 16 February 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted in accordance with current testing guidelines and GLP-compliant.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells. The objective of this study was to evaluate the clastogenic potential of Trimethylamine (TMA-opl in water) by examining its effects on the chromosomes of cultured human peripheral blood lymphocytes treated in the absence and presence of S9.
Trimethylamine (TMA-opl in water) was tested in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals. The test article was formulated in purified water and the highest concentration used in the Main Experiments, 591.1 µg/mL, (equivalent to 10 mM) was determined following a preliminary cytotoxicity Range-Finder Experiment.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: lymphocytes: duplicate human lymphocyte cultures prepared from the pooled blood of three male donors
Details on mammalian cell type (if applicable):
Blood from three healthy, non-smoking male volunteers.
Range-finder: Donor identity= 5553, 9597, 9683
Experiment 1: Donor identity= 5553, 9597, 9683
Experiment 2: Donor identity= 6747, 7100, 9023
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 591.1 µg/mL was selected for the cytotoxicity Range-Finder Experiment.

Concentrations for cytogenic analysis:
Range-finder: 0, 2.144, 3.574, 5.957, 9.928, 16.55, 27.58, 45.96, 76.61, 127.7, 212.8, 354.7, 591.1 µg/mL
Experiment 1: 0, 37.50, 75.00, 150.0, 250.0, 350.0, 450.0, 591.1 µg/mL
Experiment 2: 0, 50.00, 100.0, 150.0, 200.0, 230.0, 260.0, 290.0, 300.0, 320.0, 350.0, 400.0, 500.0, 591.1 µg/mL
Vehicle / solvent:
Vehicle used: purified water
Justification for choice of vehicle: The test article was supplied as a 1.17% v/v solution of trimethylamine in purified water. Test article solution was prepared by dilution with further purified water.
Untreated negative controls:
yes
Remarks:
Sterile purified water was added to cultures designated as negative controls as described in the methods section of this report
Negative solvent / vehicle controls:
yes
Remarks:
sterile purified water
Positive controls:
yes
Positive control substance:
other: 4-Nitroquinoline 1 oxide (NQO) or Cyclophosphamide (CPA)
Details on test system and experimental conditions:
Metabolic activation system
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it is prepared from male Sprague Dawley rats induced with Aroclor 1254. The batches of MolToxTMS-9 were stored frozen in aliquots at -80°C nominal 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-catalyzed enzyme activities (alkoxyresorufin-O-dealkylase activities). The quality control statements, relating to the batches of S-9 preparation used, are included in Appendix 6 of this report.
The S-9 mix was prepared in the following way:
Glucose-6-phosphate (G6P: 180 mg/mL),b-Nicotinamide adenine dinucleotide phosphate (NADP: 25 mg/mL), Potassium chloride (KCl: 150 mM) and rat liver S-9 were mixed in the ratio 1:1:1:2. For all cultures treated in the presence of S-9, an aliquot of the mix was added to each cell culture to achieve the required final concentration of the test article in a total of 10 mL. The final concentration of liver homogenate in the test system was 2%.
Cultures treated in the absence of S-9 received an equivalent volume of 150 mM KCl.

Blood cultures
Blood from three healthy, non-smoking male volunteers was used for each experiment of this study. No volunteer was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. The measured cell cycle time of the donors used at Covance falls within the range 13 +/- 1.5 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within one day of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.
Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 9.0 mL (Range-Finder and Experiment 1) or 8.1 mL (Experiment 2) HEPES-buffered RPMI medium containing 20% (v/v) heat inactivated foetal calf serum and 50mg/mL gentamycin, so that the final volume following addition of S-9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37°C±1°C for approximately 48 hours and rocked continuously.
Where appropriate, one hundred metaphases from each code were analysed for chromosome aberrations. Where 10 cells with structural aberrations (excluding gaps) were noted on a slide, analysis may have been terminated. Only cells with 44 to 46 chromosomes were considered acceptable for analysis. Any cell with more than 46 chromosomes (that is, polyploid, hyperdiploid or endoreduplicated cells) observed during this evaluation was noted and recorded separately.
Evaluation criteria:
Categorisation:
1.cells with structural aberrations including gaps
2.cells with structural aberrations excluding gaps
3.polyploid, endoreduplicated or hyperdiploid cells.
Acceptance criteria
The assay was considered valid if all the following criteria were met:
1. binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures
2. proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the normal range
3.At least 160 cells out of an intended 200 were suitable for analysis at each concentration, unless 10 or more cells showing structural aberrations (per slide) other than gaps only were observed during analysis
4.The positive control chemicals induced statistically significant increases in the proportion of cells with structural aberrations.
Evaluation criteria
Test article was considered to induce clastogenic events if:
1.proportion of cells with structural aberrations at one or more concentrations that exceeded the normal range was observed in both replicate cultures
2.statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) was observed (p ≤ 0.05)
3.There was a concentration-related trend in the proportion of cells with structural aberrations (excluding gaps).
The test article was considered as positive if all of the above criteria were met.
The test article was considered as negative if none of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case by case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result.
Statistics:
mitotic index (MI) determined i.e. the percentage of cells in mitosis. This is defined as a clear decrease in mitotic index compared with negative controls, (based on at least 1000 cells counted, where possible), and is preferably concentration-related.
The statistical method used was Fisher's exact test. Probability values of p ≤ 0.05 were accepted as significant.
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
lymphocytes: human lymphocyte cultures prepared from the pooled blood of three male volunteers.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
not applicable
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

1.     The binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures.

2.     The proportion of cells with structural aberrations (excluding gaps) in negative control cultures fell within the normal range.

3.     At least 160 cells out of an intended 200 were suitable for analysis at each test article concentration and vehicle controls. In Experiment 2, <10 cells showing structural aberrations other than gaps were observed on one slide from the ‘B’ replicate of the CPA (12.5 µg/mL) positive control (from a total of 26 metaphases analysed). However, a clear positive response was observed, therefore this did not affect the interpretation of the data.

4.     The positive control chemicals induced statistically significant increases in the proportion of cells with structural aberrations.

 

Table 2 : Mitotic Index Determinations - Experiment 1

Treatment

(µg/mL)

Mitotic index (%)

3+17 hours, -S-9

3+17 hours, +S-9

 

A/C

B/D

MIH*

A/C

B/D

MIH*

 

 

 

 

 

 

 

Vehicle

8.0/8.5

7.4/9.4

-

7.3/6.9

9.9/8.3

-

37.50

NS

NS

-

NS

NS

-

75.00

NS

NS

-

NS

NS

-

150.0

NS

NS

-

8.5

9.7

0

250.0

NS

NS

-

8.5

11.1

0

350.0

10.8

10.0

0#

9.5

10.2

0#

450.0

8.5

7.8

2#

8.0

7.1

7#

591.1

9.3

12.7

0#

6.7

9.0

3#

 

NS = Not scored

NT = Not tested

*Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

# Highlighted concentrations selected for analysis

 

Table 3: Mitotic Index Determinations – Experiment 2

Treatment

(µg/mL)

Mitotic index (%)

20+0 hours, -S-9

3+17 hours, +S-9

 

A/C

B/D

MIH*

A/C

B/D

MIH*

 

 

 

 

 

 

 

Vehicle

6.4/6.6

6.7/6.0

-

9.2/10.8

10.2/8.4

-

50.00

7.6

4.9

3

NS

NS

-

100.0

4.9

5.4

20

11.3

8.5

0

150.0

5.5

4.8

20

NT

NT

-

200.0

5.6

6.0

10#

10.4

8.0

5

230.0

3.4

6.1

26

NT

NT

-

260.0

4.3

5.2

26#

NT

NT

-

290.0

5.2

5.3

18

NT

NT

-

300.0

NT

NT

-

8.7

6.9

19

320.0

3.3

3.7

46#

NT

NT

-

350.0

2.4

4.5

46

NT

NT

-

400.0

3.3

1.9

60#

7.9

5.8

29#

500.0

1.0

1.3

82

10.0

8.9

2#

591.1

1.5

0.8

82

7.6

9.3

12#

 

NS = Not scored

NT = Not tested

*Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

 # Highlighted concentrations selected for analysis

Conclusions:
Treatment of cultures with Trimethylamine (TMA-opl in water) in the absence and presence of S-9 in Experiments 1 and 2 resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within normal ranges.

No increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were generally observed in cultures treated with Trimethylamine (TMA-opl in water) in the absence and presence of S-9 in Experiments 1 and 2. The only exception to this was observed in Experiment 2 in the absence of S-9 at one intermediate concentration (320.0 µg/mL), where the frequency of cells with numerical aberrations marginally exceeded the 95th percentile of the normal range (but fell within the observed normal range in both cultures. The increases were almost entirely attributable to hyperdiploid cells but they were small and there was no evidence of a concentration-related response, therefore they were not considered biologically relevant.
It is concluded that Trimethylamine (TMA-opl in water) did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to the limit of cytotoxicity for 20+0 hours in the absence of S-9 and up to a maximum of 10 mM for 3+17 hours in the absence and presence of S-9.
Executive summary:

Trimethylamine (TMA-opl in water) was tested by Covance in 2010 in an in vitro cytogenetics assay using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals. The test article was formulated in purified water and the highest concentration used in the Main Experiments, 591.1 mg/mL (equivalent to 10 mM) was determined following a preliminary cytotoxicity Range-Finder Experiment. The test article concentrations for chromosome analysis were selected by evaluating the effect of Trimethylamine on mitotic index. In each experiment, chromosome aberration were analysed at three or four concentrations. Appropriate negative (vehicle) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within current historical vehicle control (normal) ranges. 4-Nitroquinoline 1-oxide (NQO) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

Treatment of cultures with Trimethylamine in the absence and presence of S-9 resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within normal ranges. No increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were generally observed in cultures treated with Trimethylamine in the absence and presence of S-9. The only exception to this was observed in Experiment 2 in the absence of S-9 at one intermediate concentration (320.0 µg/mL), where the frequency of cells with numerical aberrations marginally exceeded the 95th percentile of the normal range (but fell within the observed normal range) in both cultures. The increases were almost entirely attributable to hyperdiploid cells but they were small and there was no evidence of a concentration-related response, therefore they were not considered biologically relevant.

It is concluded that Trimethylamine (TMA-opl in water) did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to the limit of cytotoxicity for 20+0 hours in the absence of S-9 and up to a maximum of 10 mM for 3+17 hours in the absence and presence of S-9.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP; Only a summary was available in English
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0 - 5000 µg
Details on test system and experimental conditions:
IUCLID4 Type: Ames test
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2 uvrA; Pre-incubation Method
Conclusions:
Interpretation of results (migrated information):
negative

Trimethylamine showed no gene mutation effects to S.typhimurium strains TA98, TA100, TA1535, and TA1537 and E.coli strain WP2 uvr A.
Executive summary:

Gene mutation properties of trimethylamine was investigated in a bacterial reverse mutation assay (Ames test). This test was performed according to the OECD guideline 471 with Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 as well as with Escherichia coli strain WP2 uvr A. The concentrations of the test substance ranged from 0 to 5000 µg and the test result was negative according to all strains, with and without metablic activation.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached document in section 13 "Assessment reports" for justification and rationale for read across analogy approach.
Reason / purpose:
read-across source
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Based on the read across analogy on the trimethylamine for assessed the trimethylamine N-oxide, the toxicity of both of the two substances is expected to be the same. Hence, according to the CLP criteria, the trimethylamine N-oxide was considered as not mutagenic for bacteria strains.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached document in section 13 "Assessment reports" for justification and rationale for read across analogy approach.
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Based on the read across analogy on the trimethylamine for assessed the trimethylamine N-oxide, the toxicity of both of the two substances is expected to be the same. Hence, according to the CLP criteria, the trimethylamine N-oxide was considered as not mutagenic for mammalian cells.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached document in section 13 "Assessment reports" for justification and rationale for read across analogy approach.
Reason / purpose:
read-across source
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

No data is available with TMAO. Results obtained with the source substance TMA are reported.

Gene mutation properties of trimethylamine was investigated in a bacterial reverse mutation assay (Ames test). This test was performed according to the OECD guideline 471 with Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 as well as with Escherichia coli strain WP2 uvr A. The concentrations of the test substance ranged from 0 to 5000 µg and the test result was negative in all strains, with and without metablic activation.

Trimethylamine (TMA-opl in water) was assayed 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 (OECD TG 476). 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 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 andthere 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 thehprtlocus 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).

Trimethylamine (TMA-opl in water) was tested in an in vitro cytogenetics assay (OECD TG 473) using duplicate human lymphocyte cultures prepared from the pooled blood of three male donors in two independent experiments. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254 induced animals.

Treatment of cultures with Trimethylamine in the absence and presence of S-9 resulted in frequencies of cells with structural aberrations that were similar to those observed in concurrent negative controls. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within normal ranges. No increases in the frequency of cells with numerical aberrations, which exceeded the concurrent controls and the normal ranges, were generally observed in cultures treated with Trimethylamine in the absence and presence of S-9. The only exception to this was observed in Experiment 2 in the absence of S-9 at one intermediate concentration (320.0 µg/mL), where the frequency of cells with numerical aberrations marginally exceeded the 95thpercentile of the normal range (but fell within the observed normal range) in both cultures. The increases were almost entirely attributable to hyperdiploid cells but they were small and there was no evidence of a concentration-related response, therefore they were not considered biologically relevant.

It is concluded thatTrimethylamine (TMA-opl in water) did not induce chromosome aberrations in cultured human peripheral blood lymphocytes when tested to the limit of cytotoxicity for 20+0 hours in the absence of S-9 and up to a maximum of 10 mM for 3+17 hours in the absence and presence of S-9.

Justification for classification or non-classification

No data is available with TMAO.

The conclusion on the classification is based on the three Klimisch-1 or 2-rated in-vitro studies on genetic toxicity available with the source substance TMA:

- bacterial reverse mutation assay (Ames test, OECD TG 471): negative

- in vitro gene mutation study in mammalian cells (OECD TG 476): negative

- in vitro chromosome aberration study in mammalian cells (OECD TG 473): negative

Based on a weight of evidence it is concluded that TMAO is not classified for genetic toxicity according to CLP criteria.