Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

DMEA has not been found to induce genotoxic effects in gene mutation assays on bacteria and mammalian cells or chromosomal aberrations in human lymphocytes.

Gene mutation on bacteria

The genotoxic activity of Dimethylethylamine (DMEA) is evaluated in the Ames test on Salmonella typhimurium according to the OECD 471 guideline (Bichet, 1990). Doses of 100, 500, 1000, 2500, 5000 µg/plate are tested on five strains (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) according to the preincubation method (20 min at 37°C). The number of revertants colonies is evaluated 48 to 72 hours later. Cytotoxicity was observed for the highest dose (5000µg/plate) with and without metabolic activation in all strain. No genotoxic activity was observed for all doses with and without metabolic activation on the 5 tested strains.

In conclusion, Dimethylethylamine did not induce genotoxicity in the Ames test on Salmonella typhimurium in the absence and presence of metabolic activation.

Gene mutation on mammalian cells

Dimethylethylamine (DMEA) was assayed for mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9) (Stone, 2010). The study was performed according to the OEGD guideline # 476 and GLP.In Experiment 1 ten concentrations, ranging from 100 to 630 µg/mL, were tested in the absence and presence of S-9. The highest concentrations analysed were 500 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9, which gave 36% and 19% RS, respectively. In the absence of S-9, no concentration gave 10 to 20% RS due to steep toxicity (630 µg/mL gave 0% RS).

In Experiment 2 eleven concentrations, ranging from 100 to 630 µg/mL, were tested in the absence and presence of S-9.The highest concentrations analysed were 550 µg/mL in the absence of S-9 and 630 µg/mL in the presence of S-9, which gave 4% and 7% RS, respectively.Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9.No statistically significant increases in mutant frequency were observed following treatment withDimethylethylamineat any concentration tested in the absence or presence of S-9 and there were no significant linear trends.It is concluded thatDimethylethylaminedid not induce mutation at thehprtlocus of L5178Y mouse lymphoma cells assay.

Chromosomal aberration

The genotoxic activity of Dimethylethylamine (DMEA) is evaluated in the in vitro cytogenetics assay at doses from 14.43 to 730.0 µg/mL on cultured human lymphocytes according to the OECD 473 guideline (McEnaney, 1991).Treatment in absence of S9mix was continuous for 20 or 44 hours. Treatment in presence of S9mix was for 3 hours only followed by a 17 or 41hours recovery period prior to harvest. Colchicine was added 1h30 prior to harvest to stop dividing cells in metaphase. Polyploidy, endoreplication and hyperdiploid were analysed in 100 metaphases but no genotoxic effect has been highlighted with or without metabolic activation. In conclusion, Dimethylethylamine did not induce genotoxicity in the in vitro cytogenetics assay in the absence and presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

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:
from 1989-10-12 to 1989-12-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD N°471 Guideline (1983 May 26th)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix from Aroclor 1254-induced rat liver (10%)
Test concentrations with justification for top dose:
100, 500, 1000, 2500, 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: sodium azide (TA100, TA1535), 2-nitrofluorene (TA98, TA1538), 9-aminoacridine (TA1537). +S9: 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20minutes at 37°C (0.1mL of the test item solution + 0.5mL of phosphat e buffer 0.2M or S09mix + 0.1mL of bacteria)
- Fixation time (start of exposure up to fixation or harvest of cells): 48hours

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth, number of revertant colonies
Evaluation criteria:
dose dependent increase in the number of revertant colonies
Statistics:
no data
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA1538, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
negative

Under these experimental conditions, DMEA is not mutagenic in S.typhimurium in Ames test.
Executive summary:

The genotoxic activity of Dimethylethylamine DMEA is evaluated in the Ames test on Salmonella typhimurium according to the OECD 471 guideline. Doses of 100, 500, 1000, 2500, 5000 ug/plate are tested on five strains (TA 98, TA 100, TA 1535, TA 1537 and TA 1538) according to the preincubation method (20 min at 37°C). The number of revertants colonies is evaluated 48 to 72 hours later.

Cytotoxicity was observed for the highest dose (5000µg/plate) with and without metabolic activation in all strain. No genotoxic activity was observed for all doses with and without metabolic activation on the 5 tested strains.

In conclusion, Dimethylethylamine did not induce genotoxicity in the Ames test on Salmonella typhimurium in the absence and presence of metabolic activation.
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD N°473 Guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 from Aroclor 1254 induced animals
Test concentrations with justification for top dose:
14.43, 20.62, 29.46, 42.08, 60.12, 85.88, 122.7, 175.3, 250.4, 357.7, 511.0 and 730.0µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide and methylmethanesulfonate
Details on test system and experimental conditions:
Treatment in absence of S9mix was continuous for 20 or 44 hours. Treatment in presence of S9 was for 3hours only followed by a 17 or41 hours recoveryperiod prior to harvest

- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): colchicine were added 1h30 prior to harvest.

SPINDLE INHIBITOR (cytogenetic assays): colchcine
STAIN (for cytogenetic assays): 5minutes in 4% filtered Giemsa in pH6.8 buffer

NUMBER OF CELLS EVALUATED: 1000 (mitotic index), 100 metaphases analyzed for each culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: hyperdiploid cells
Evaluation criteria:
the test item was considered as positive if:
a statistically significant incerase in the proportion of structurally aberrant cells occurred at one or more concentrations
the proportion of aberrant cells at such data points exceede the normal range
Species / strain:
other: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRECIPITATION CONCENTRATION: no precipitate

MITOTIC INDEX: 
- with metabolic activation:
730 µg/ml (3/20 hours): 102% of the control
730 ug/ml (3/44 hours): 112% of the control

- without metabolic activation:
511 ug/ml (20/20 hours): 44% of the control
730 ug/ml (44/44 hours): 78% of the control
Conclusions:
Interpretation of results (migrated information):
negative

Under these experimental conditions, the test item Dimethylethylamine (DMEA) was unable to induce structural chromosome aberrations in cultured human lymphocytes, when tested up to dose equivalent to 10mM, either in presence or absence of S9.
Executive summary:

The genotoxic activity of Dimethylethylamine DMEA is evaluated in the in vitro cytogenetics assay at doses from 14.43 to 730.0µg/mL on cultured human lymphocytes according to the OECD 473 guideline.

Treatment in absence of S9mix was continuous for 20 or 44hours. Treatment in presence of S9mix was for 3hours only followed by a 17 or 41hours recovery period prior to harvest. Colchicine was added 1h30 prior to harvest to stop dividing cells in metaphase.

Polyploidy, endoreplication and hyperdiploid were analysed in 100 metaphases but no genotoxic effect has been highlighted with or without metabolic activation.

In conclusion, Dimethylethylamine did not induce genotoxicity in the in vitro cytogenetics assay in the absence and presence of metabolic activation.
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 was completed (final report issued) on 11 May 2010. Experimental work started on 22 December 2009 and was completed on 23 February 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted in accordance with current testing guidelines and GLP-compliant.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxathine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance).
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y tk +/- mouse lymphoma cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of tk¿ mutants, checked for spontaneous mutant frequency and that is 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.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
Range-finder: 0, 19.69, 39.38, 78.75, 157.5, 315, and 630 µg/mL (with and without S-9);
Experiment 1: 0, 100, 150, 200, 250, 350, 400, 450, 500 and 630 µg/mL (with and without S-9);
Experiment 2: 100, 200, 300, 400, 450, 500, 525, 550, 575, 600 and 630 µg/mL (without S-9); and 100, 200, 300, 350, 400, 425, 450, 500, 550, 600 and 630 µg/mL (wiith S-9)
Vehicle / solvent:
-Vehicle(s)/solvents(s) used: purified water
Justification for choice of vehicle/solvent: Preliminary solubility data indicated that Dimethylethylamine was soluble in water for irrigation (purified water) at concentrations up to at least 2594 µg/mL as indicated by a lack of precipitation 18 hours after test article addition.Test article solutions were prepared by formulating Dimethylethylamine under subdued light conditions in purified water (with the aid of vortex mixing, as required) immediately prior to assay to give the maximum required treatment solution concentration.
Negative solvent / vehicle controls:
yes
Remarks:
Purified water diluted 10-fold in the treatment medium.
Positive controls:
yes
Remarks:
4-nitroquinoline 1-oxide (NQO, -S-9), 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:
Metabolic activation system:
Rat liver S-9 fraction from male Sprague Dawley rats induced with Aroclor 1254. Batches were stored at -80°C prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert known promutagens to bacterial mutagens and cytochrome P450-catalyzed enzyme activities.
Treatment was carried out both in the absence and presence of S-9, prepared in the following way:
Glucose-6-phosphate (180 mg/mL), ß-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, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. Cultures treated for 3 hours 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:
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 satisfy the assessment criteria above were considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. 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:
0% RS at 630µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In Experiment 1, in the absence and presence of S-9, and Experiment 2, in the absence and presence of S-9, the acceptance criteria were considered to have been met and the study was therefore accepted as valid.

Table 2: Range-finder (3-hour treatment)

 

Treatment (µg/mL)

-S-9
(% RS)

+S-9
(% RS)

0

100

100

19.69

84

128

39.38

67

87

78.75

87

146

157.5

57

57

315

30

62

630

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

7.50

 

0

100

5.11

 

150

93

4.37

NS

100

91

5.43

NS

200

82

5.16

NS

150

84

4.64

NS

300

72

4.87

NS

250

71

4.50

NS

350

55

6.46

NS

350

69

5.50

NS

400

38

4.84

NS

400

57

4.07

NS

450

24

4.09

NS

450

19

5.47

NS

500

36

5.81

NS

 

 

 

 

Linear trend

NS

Linear trend

NS

NQO

 

 

B[a]P

 

 

0.1

65

19.16

2

18

44.25

0.15

56

18.88

3

8

73.32

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

% RS -Percent relative survival adjusted by post treatment cell counts

NS -Not significant

 

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

2.17

 

0

100

1.49

 

100

89

2.97

NS

200

81

2.20

NS

200

86

2.37

NS

350

70

2.40

NS

400

56

2.22

NS

400

67

2.42

NS

450

48

3.71

NS

450

65

1.59

NS

500

37

1.87

NS

500

49

4.42

NS

525

24

1.74

NS

550

32

2.45

NS

550

4

3.44

NS

600

25

2.57

NS

 

 

 

 

630

7

3.24

NS

Linear trend

NS

Linear trend

NS

NQO

 

 

B[a]P

 

 

0.1

74

9.95

2

56

40.63

0.15

64

19.36

3

36

66.30

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

% RS -Percent relative survival adjusted by post treatment cell counts

NS -Not significant

 

 

Conclusions:
Dimethylethylamine 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).
Executive summary:

Dimethylethylamine was assayed for mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation 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 19.69 to 630 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) was 315 µg/mL, which gave 30% and 62% RS in the absence and presence of S-9, respectively.

 

In Experiment 1 ten concentrations, ranging from 100 to 630 µg/mL, were tested in the absence and presence of S-9. The highest concentrations analysed were 500 µg/mL in the absence of S-9 and 450 µg/mL in the presence of S-9, which gave 36% and 19% RS, respectively. In the absence of S-9, no concentration gave 10 to 20% RS due to steep toxicity (630 µg/mL gave 0% RS). The data were accepted as valid and more closely spaced concentrations were employed in Experiment 2.

 

In Experiment 2 eleven concentrations, ranging from 100 to 630 µg/mL, were tested in the absence and presence of S-9.The highest concentrations analysed were 550 µg/mL in the absence of S-9 and 630 µg/mL in the presence of S-9, which gave 4% and 7% RS, respectively. In the absence and presence of S-9, no concentration gave 10 to 20% RS. Cultures treated at 525 µg/mL in the absence of S-9 and 600 µg/mL in the presence of S-9 gave 24% and 25% RS, respectively. Therefore, both concentrations in the absence and presence of S-9 were analysed.

 

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies in negative control cultures fell within normal ranges and clear increases in mutation were induced by the positive control chemicals 4 -nitroquinoline 1-oxide (without S-9) and benzo(a)pyrene (with S-9). Therefore the study was accepted as valid.

 

In Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment withDimethylethylamine at any concentration tested in the absence or presence of S-9 and there were no significant linear trends. It may be noted that in Experiment 1 in the absence of S-9 and Experiment 2 in the absence and presence of S-9 no concentration tested gave 10-20% RS. However, under both treatment conditions there was clearly no evidence of mutagenic activity at any concentration analysed and the toxicity and mutation data were well reproduced between experiments in both the absence and presence of S-9, therefore all data were considered valid.

 

It is concluded that Dimethylethylaminedid 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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to CLP and GHS criteria, no classification is warranted for germ cells mutation.