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Genetic toxicity: in vitro

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Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported, published 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
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:
not reported, published 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not specified
Principles of method if other than guideline:
The description of the test method is insufficient to compare in details with the OECD guideline (because it is a publication).
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Gens of Histidine Operons (hisG46, hisC3076, hisD3052, hisG428 and hisD6610)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
DNA polymerase A deficient
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 liver
Test concentrations with justification for top dose:
0.01, 0.03, 0.1, 0.3, 1.0, 3.0 and 10.0 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [water]
- Justification for choice of solvent/vehicle: the test substance is soluble in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
not specified
Positive controls:
yes
Remarks:
The activation-independent positive controls
Positive control substance:
sodium azide
Remarks:
further substances: 4-nitrophenylenediamine and 9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
not specified
Positive controls:
yes
Remarks:
The activation-dependent positive control
Positive control substance:
other: 2-aminoanthracen
Details on test system and experimental conditions:
METHOD OF APPLICATION (briefly described in the present paper): in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration: for 48-72 h at 37°C in the dark
- Expression time (cells in growth medium): no
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): direct after exposure


Evaluation criteria:
The numbers of colonies per plate were counted and the background lawn was examined. If toxicity was observed as an inhibition of growth of the background lawn, the plate was not counted, and was recorded as toxic. A test chemical was considered a bacterial mutagen if the number of revertant colonies was at least twice the solvent control for at least one concentration and there was evidence of a concentration-related increase in the number of revertant colonies.
Statistics:
not reported
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS not reported
RANGE-FINDING/SCREENING STUDIES: The highest concentration was selected from a cytotoxicity test in strain TA100 which produced either a significant (> 50%) reduction in the number of revertant colonies or a significant inhibition of background lawn growth. COMPARISON WITH HISTORICAL CONTROL DATA: not reported
ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Table 1

Results of the Salmonella mutagenicity test

Chemical

mg/plate

TA98

TA100

TA1535

TA1537

TA1538

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Water

100

21 ± 8

31± 5

98 ± 20

90 ± 15

13 ± 4

8 ± 3

8 ± 3

10 ± 4

9 ± 4

19 ± 3

4-NPD

0.01

960 ± 26

 

 

 

 

 

 

 

1228 ± 14

 

NaN3

0.01

 

 

1525 ± 114

 

1269 ± 101

 

 

 

 

 

9-AA

0.06

 

 

 

 

 

 

121 ± 53

 

 

 

2-AA

0.01

 

669 ± 106

 

388 ± 67

 

64 ± 50

 

60 ± 30

 

103 ± 59

DMEA

0.01

17± 1

 

88 ± 5

 

17 ± 1

 

5 ± 4

 

7 ± 2

 

DMEA

0.03

24 ± 6

 

90 ± 9

 

14 ± 2

 

7 ± 3

 

9 ± 4

 

DMEA

0.1

22 + 6

 

102 ± 6

 

20 ± 6

 

7 ± 2

 

7 ± 1

 

DMEA

0.3

24 ± 6

32 ± 9

97 ± 8

91 ± 6

11 ± 3

11± 2

8 ± 3

11 ± 4

6 ± 2

19 ± 4

DMEA

1

16 ± 4

27 ± 4

84 ± 13

99 ± 9

14 ± 3

10 ± 3

7 ± 2

8 ± 3

6 (S,T)

22 ± 3

DMEA

3

 

16 ± 4

 

82 ± 6

 

8 ± 2

 

8 (S)

 

12 ± 6

DMEA

10

 

12 ± 4

 

S, T

 

16 (T)

 

4 (T)

 

16 (T)

4-NPD, 4-nitrophenylenediamine; NaN3, sodium azide; 9-AA, 9-aminoacridine; 2-AA, 2-aminoanthracene.

T - Toxic: clearing of background lawn or average number of colonies less than half of the solvent control value.

S - sparse growth of background lawn in one or two of the triplicates tested. Counts not included in the calculation of mean and standard deviations not calculated.

T, toxic to one or two of the triplicates tested.

Conclusions:
Interpretation of results: negative in all strains tested
N,N-dimethylethanolamine (DMEA) was considered to be not mutagenic under the conditions of this in vitro mutagenicity test.
Executive summary:

N,N-dimethylethanolarnine was evaluated for potential genotoxic activity using the Salmonella/microsome reverse gene mutation test, the CHO/HGPRT forward gene mutation test, a sister chromatid exchange test in cultured CHO cells, and an in vivo peripheral blood micronucleus test in Swiss-Webster mice (see also Endpoints 7.6.1.003, 7.6.1.004 and 7.6.2.001).

No mutagenic activity was observed in any of the 5 strains tested (in the Salmonella/microsome reverse gene mutation test). In the absence or the presence of S9 activation, either by evidence of a dose-response relationship or a doubling of the mean number of colonies over the vehicle control value (Table 1 in "Remarks on results").

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
not reported, published 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
not specified
Principles of method if other than guideline:
The description of the test method is insufficient to compare in details with the OECD guideline (because it is a publication).
GLP compliance:
no
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hypoxanthine-guanine phosphoribosyl transferase gene
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Having a very low chromosome number (2n = 22) for a mammal, the Chinese hamster is an ideal model for radiation cytogenetics and tissue culture.
The cells grow as a cultured monolayer and require the amino acid proline in their culture medium. CHO cells do not express the Epidermal growth factor receptor (EGFR) (from Wikipedia).
Metabolic activation:
with and without
Metabolic activation system:
S9 liver
Test concentrations with justification for top dose:
1.Without metabolic activation: 0.1, 0.2, 0.4, 0.6, 0.8 and 1.5 mg/mL
2. With metabolic activation: 0.3, 0.6, 1.0, 1.3 and 1.5 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [water]
- Justification for choice of solvent/vehicle: the test substance is soluble in water
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
culture medium
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium. Modified F12 cell-culture medium (Gibco, Grand Island, NY) supplemented to 5% (v/v) with heat-inactivated fetal bovine serum was used as the growth medium for test chemicals without metabolic activation. Identical medium without serum was used for treatments incorporating an S9 metabolic activation system.

DURATION
- Preincubation period: 20-24 h
- Exposure duration: 5 h
- Expression time (cells in growth medium): 18-24 h before chemical-induced cytotoxicity was determined, 7 days for expression of the mutant phenotype
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): exposure duration plus expression time of mutant phenotypes

SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not reported

NUMBER OF REPLICATIONS: not reported
NUMBER OF CELLS EVALUATED: 100-200 for determination of cytotoxicity

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
The colony-forming ability determined by the viable fraction of the plated cells was used to correct the mutant frequency for the individual treated cultures and to detect variations in the growth ability of the cells.

OTHER EXAMINATIONS: No
Evaluation criteria:
not reported
Statistics:
Statistical analysis of the mutation data for this test has been described by Slesinski et al (cited in the present paper).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
up to 1 mg/mL without S9 and up to 3 mg/mL with metabolic activation (about 90% inhibition of cell culture growth)
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS (not reported)
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was conducted to select the appropriate dose ranges (Table 1 in "Remarks on results").

COMPARISON WITH HISTORICAL CONTROL DATA: All the mutant indices were within the typical range of variation for this test based on historical negative control variability.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Table 1

Cytotoxicity determination for the Chinese hamster ovary gene mutation and sister chromatid exchange tests

Chemical (mg/ml)

% Survival relative to control (final cell density X 105)

DMEA

- S9

+ S9

Culture medium

100 (45.8)

100 (44.0)

0.0003

 

 

0.001

 

 

0.003

 

 

0.01

96.2

93.7

0.03

89.1

77.1

0.1

72.4

89.9

0.3

84.2

87.7

0.6

 

 

1

cytotoxic

67.5

3

cytotoxic

cytotoxic

5

 

 

10

cytotoxic

cytotoxic

Cell density was determined approximately 18-24 h after treatment. The initial cell density was 1 x105cells/flask. Cytotoxic: all cells lysed or detached from monolayer.

Table 2

Mutant induction in Chinese hamster ovary gene mutation test

Chemical

DMEA

- S9

+ S9

C.E.

M.F.

C.E.

M.F.

Culture medium

 

15.8

 

1.0

 

12.3

 

7.1

EMS

85

189.8

 

 

DMN

 

 

5

228.4

0.1 mg/ml

95

6.5

 

 

 

23.5

 

 

0.2

97

26.3

 

 

 

16

 

 

0.3

 

 

85

8.8

 

 

 

16.2

0.4

71

0

 

 

 

37.8

 

 

0.6

85

2.6

88

0

 

6.4

 

6.0

0.8

78

0

 

 

 

8.1

 

 

1.0

 

 

84

12.3

 

 

 

14.9

1.3

 

 

71

12.4

 

 

 

15.1

1.5

 

 

T

 

14.4

 

 

 

2.0

 

 

 

 

 

 

 

 

33.0

 

 

 

 

 

 

 

 

EMS, ethylmethane sulfonate (0.2 mg/ml); DMN, dimethylnitrosamine (0.2 mg/ml for DMEA). C.E., cloning efficiency (% of combined negative controls). About 100 cells inoculated into each plate. M.F., mutation frequency = total number of mutants per million clonable cells corrected for the viable fraction. 2 x105cells inoculated in each of 5 plates (1 x106total cells). T, cytotoxic (all cells lysed or detached from monolayer).
Statistical difference above control: no superscript indicatesp>0.05; data for positive and negative controls were compared to historical ranges, but were not analyzed statistically. Historical negative control is 3-4 mutants/106viable cells, but a 95 percentile range of 0-25 mutants/106viables cells is typical.

Conclusions:
Interpretation of results: negative both with and without metabolic activation
DMEA is not mutagenic in this in vitro gene mutation assay.
Executive summary:

N,N-dimethylethanolarnine was evaluated for potential genotoxic activity using the Salmonella/microsome reverse gene mutation test, the CHO/HGPRT forward gene mutation test, a sister chromatid exchange test in cultured CHO cells, and an in vivo peripheral blood micronucleus test in Swiss-Webster mice (see also Endpoints 7.6.1.002, 7.6.1.004 and 7.6.2.001 )

DMEA did not produce any statistically significant increases in the incidence of mutations of CHO cells with or without an S9 metabolic activation system, although there were some increases which were 2-3 times greater than the concurrent controls. These increases, however, were not dose-related and not repeatable in a duplicate culture at the same corresponding doses. All the mutant indices were within the typical range of variation for this test based on historical negative control variability. Given the random nature of these increases, DMEA was not judged to be positive in this in vitro gene mutation test (Table 2 in "Remarks on results").

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not reported, published 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication which meets basic scientific principles.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Principles of method if other than guideline:
The description of the test method is insufficient to compare in details with the OECD guideline (because it is a publication).
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
- Age at study initiation: 6-8 weeks
- Weight at study initiation: not reported
- Assigned to test groups randomly: [yes ]
- Fasting period before study: not reported
- Housing:not reported
- Diet (e.g. ad libitum): not reported
- Water (e.g. ad libitum): not reported
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONSnot reported
- Temperature (°C):
- Humidity (%):
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: [water]
- Justification for choice of solvent/vehicle: the test substance is well soluble in water
- Amount of vehicle (if gavage or dermal): no (intraperitoneal)
- Type and concentration of dispersant aid (if powder): no (intraperitoneal)
Details on exposure:
intraperitoneal. No further details reported
Duration of treatment / exposure:
Mice were dosed by a single intraperitoneal injection and observed for mortality for 72 h.
Frequency of treatment:
single exposure
Post exposure period:
72 h.
Dose / conc.:
270 mg/kg bw/day (nominal)
Dose / conc.:
540 mg/kg bw/day (nominal)
Dose / conc.:
860 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine
- Justification for choice of positive control(s): not reported
- Route of administration: intraperitoneal
- Doses / concentrations: 0.3 mg/kg bw
Tissues and cell types examined:
polychromatic/normochromatic erythrocyte
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Three dose levels of about 80, 50 and 25% of the LD50 were selected for the micronucleus test.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Blood samples were collected from the tail vein at 24 or 30, 48 and 72 h after dosing.


DETAILS OF SLIDE PREPARATION: Slides of blood smears were stained with Gurr's R-66 Giemsa diluted in phosphate buffer, coded and read without knowledge of treatment group to prevent bias.


Evaluation criteria:
The polychromatic/normochromatic erythrocyte ratio for approximately 1000 total cells for each animal was calculated to provide an estimate of cytotoxicity. Excessive cytotoxicity was defined by a polychromatic/normochromatic erythrocytes ratio of 0.01 or lower. A minimum of 1000 polychromatic erythrocytes for each animal was scored for the presence of micronuclei unless the cytotoxicity of the test substance prevented this.
Statistics:
Data were compared for significant differences using the Fisher's Exact Test. A positive result was concluded if at least one statistically significant increase above vehicle control was observed with an indication of a dose-related effect.
Sex:
male/female
Genotoxicity:
negative
Remarks:
no significant increases in the incidence of micronucleated polychromatic erythrocyte were observed at any sampling time
Remarks on result:
other: there were no major gender differences in mortality responses

Table 1

Induction of micronucleus in peripheral erythrocytes of Swiss-Webster mice injected intraperitoneally with DMEA

 

Mean PCE/1000 NCE (± SD)

Mean MN-PCE per 1000 PCE (± SD)

 

Water

TEM

DMEA (mg/kg)

Water

TEM

DMEA (mg/kg)

 

 

 

270

540

860

 

 

270

540

860

30 h postdosing

 

 

 

 

 

 

 

 

 

M

20 ± 7

17 ± 1

22 ± 3

22 ± 5

22 ± 8

3.2 ± 2.3

20.8 ± 3.6b

3.8 ± 1.5

3.8 ± 2.4

1.6 ± 1.1

F

21± 8

20 ± 9

32 ± 8

27 ± 7

28 ± 9

1.4 ± 1.5

24.8 ± 8.7b

2.4 ± 2.1

1.2 ± 0.8

2.0 ± 1.4

48 h postdosing

 

 

 

 

 

 

 

 

 

M

26 ± 7

 

19 ± 5

20 ± 6

18 ± 4

3.6 ± 1.5

 

3.2 ± 1.9

4.0 ± 2.6

3.4 ± 2.3

F

36 ± 8

 

42 ± 14

30 ± 8

32 ± 9

1.4 ± 1.1

 

0.8 ± 0.8

1.4 ± 1.1

1.0 ± 1.4

72 h postdosing

 

 

 

 

 

 

 

 

 

M

32 ± 6

 

30 ± 3

33 ± 3

36 ± 15

2.8 ± 1.5

 

2.8 ± 1.3

3.2 ± 1.1

3.2 ± 1.9

F

35 ± 5

 

32 ± 8

33 ± 8

33 ± 10

1.6 ± 1.1

 

1.8 ± 1.5

1.8 ± 2.1

0.8 ± 1.1

Water (10 mL/kg); TEM, triethylenemelamine (0.3 mg/kg). PCE, polychromatic erythrocytes; NCE, normochromatic erythrocytes; MN-PCE, micronucleated polychromatic erythrocytes.
Statistical analysis employed the Fisher's Exact Test (1-tailed):ap< 0.01;bp<0.00

Conclusions:
Interpretation of results: negative
DMEA did not induce micronuclei in this assay.
Executive summary:

N,N-dimethylethanolarnine was evaluated for potential genotoxic activity using the Salmonella/microsome reverse gene mutation test, the CHO/HGPRT forward gene mutation test, a sister chromatid exchange test in cultured CHO cells, and an in vivo peripheral blood micronucleus test in Swiss-Webster mice (see also Endpoints 7.6.1.002, 7.6.1.003 and 7.6.1.004)

In the mouse micronucleus test, there were no major gender differences in mortality responses. The LD50 (combined sexes) DMEA was about 1074 mg/kg. Selection of the top test dose was based on the lethality response rather than on bone marrow suppression. There were no significant differences in the polychromatic erythrocyte to normochromatic erythrocyte ratios at any dosages for all three alkylalkanolamines tested (Table 1 in "Remarks on results"). Furthermore, no significant increases in the incidence of micronucleated polychromatic erythrocyte were observed at any sampling time (Table 1). Therefore, DMEA was not considered to be inducer of micronuclei under the condition of this in vivo test.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Deviations:
not specified
Principles of method if other than guideline:
The description of the test method is insufficient to compare in details with the OECD guideline (because it is a publication).
GLP compliance:
no
Type of assay:
sister chromatid exchange assay in mammalian cells

Test material

Constituent 1
Chemical structure
Reference substance name:
2-dimethylaminoethanol
EC Number:
203-542-8
EC Name:
2-dimethylaminoethanol
Cas Number:
108-01-0
Molecular formula:
C4H11NO
IUPAC Name:
2-(dimethylamino)ethanol
Details on test material:
- Name of test material (as cited in study report): N,N-dimethylethanolamine obtained from the Union Carbide Corporation, South Charleston, WV
- Substance type: organic
- Physical state: liquid
- Analytical purity: 99.9%
- Impurities (identity and concentrations): no, All samples were analyzed and their chemical purity determined by capillary gas chromatography.

Method

Target gene:
Differentially labelled sister chromatids, which can be achieved e.g. by incorporation of bromodeoxyuridine (BrdU) into chromosomal DNA for two cell cycles.
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The cells grow as a cultured monolayer and require the amino acid proline in their culture medium. CHO cells do not express the Epidermal growth factor receptor (EGFR) (from Wikipedia).
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver
Test concentrations with justification for top dose:
1. Without metabolic activation: 0.1, 0.2 and 0.4 mg/mL
2. With metabolic activation: 0.6, 1.0 and 1.5 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [water]
- Justification for choice of solvent/vehicle: the test substance is soluble in water
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 40-48 h
- Exposure duration: Cells were incubated for 2 and 5 h for testing chemicals with and without a rat S9 metabolic activation system, respectively.
- Expression time (cells in growth medium): 24h
- Selection time (if incubation with a selection agent): 2h or 5h plus additional 24h
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): 3 µg/mL BrdU
SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 µg/ml)
STAIN (for cytogenetic assays): Hoechst 33258 dye. Irradiated chromosomes were stained in Gurr's giemsae
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 25 cells/dose
DETERMINATION OF CYTOTOXICITY: Method: mitotic index (in Table 2 in Endpoint 7.6.1.003)
OTHER EXAMINATIONS: no
Evaluation criteria:
The criteria for evaluation of a positive or negative response depended on the level of statistical significance and subjective analyses of concurrent and historical control data. Clearly positive responses would include any of the following: (1) doubling in the SCE frequency by any single concentration; (2) statistically significant responses of p < 0.01 with one or more consecutive concentrations; and (3) a statistically significant, concentration-related increase in the number of SCE. Equivocal results were defined as random statistical indications of positive increases, but which did not meet the criteria defined as a positive test result.
Statistics:
The data for quantitative continuous variables were intercompared for the dose and control groups by Levene's test for equality of variances, analysis of variance (ANOVA), and Mests. The Mests were used following a significant ANOVA to delineate which groups differed from the control group. If Levene's test indicated homogeneous variances, the groups were compared by an ANOVA for equal variances followed, when appropriate, by pooled variance f-tests. If Levene's test indicated heterogeneous variances, the groups were compared by an ANOVA followed by separate variance f-tests.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
up to 1 mg/mL without S9 and up to 3 mg/mL with metabolic activation (about 90% inhibition of cell culture growth)
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS not reported
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was conducted to select the appropriate dose ranges (Table 1 in "Remarks on results").

COMPARISON WITH HISTORICAL CONTROL DATA: There are only data on mutant indices (mentioned in Endpoint 7.6.1.003)

ADDITIONAL INFORMATION ON CYTOTOXICITY: A dose level in the cytotoxicity test (Table 1) which was moderately toxic, but permitted a cell population increase of at least 40-50%, was selected as the maximum dose.
Concentrations which markedly inhibited cell division or SCE differentiation were recorded as cytotoxic and SCEs were not counted.

Any other information on results incl. tables

Table 1

Cytotoxicity determination for the Chinese hamster ovary gene mutation and sister chromatid exchange tests

Chemical (mg/mL)

% Survival relative to control (final cell density X 105)

DMEA

- S9

+ S9

Culture medium

100 (45.8)

100 (44.0)

0.0003

 

 

0.001

 

 

0.003

 

 

0.01

96.2

93.7

0.03

89.1

77.1

0.1

72.4

89.9

0.3

84.2

87.7

0.6

 

 

1

cytotoxic

67.5

3

cytotoxic

cytotoxic

5

 

 

10

cytotoxic

cytotoxic

Cell density was determined approximately 18-24 h after treatment. The initial cell density was 1 x105cells/flask. Cytotoxic: all cells lysed or detached from monolayer

Table 2

Induction of sister chromatid exchange in Chinese hamster ovary cells in vitro by DMEA

Chemical

Number of SCE per chromosome (mean ± SD) *

DMEA

- S9

+ S9

Culture medium

0.52 ± 0.16

0.54 ± 0.15

0.50 ± 0.15

0.52 ± 0.15

EMS (0.1 mg/mL)

1.36 ± 0.37d

 

DMN (0.3 mg/mL)

 

2.38 ± 0.48d

0.1 mg/mL

0.56 ± 0.20

 

0.52 ± 0.15

 

0.2 mg/mL

0.65 ± 0.28

 

0.54 ± 0.19

 

0.3 mg/mL

 

 

 

 

0.4 mg/mL

0.59 ± 0.18

 

0.54 ± 0.15

 

0.6 mg/mL

 

0.58 ± 0.18

 

0.59 ± 0.18

0.8 mg/mL

 

 

 

 

1.0 mg/mL

 

0.57 ± 0.16

 

0.57 ± 0.13

1.5 mg/mL

 

0.51 ± 0.18

 

0.57 ± 0.18

2.0 mg/mL

 

 

 

 

Cells were treated for 5 h without S9; 2 h with S9. EMS, ethylmethane sulfonate; DMN, dimethylnitrosamine.
aDetermined in duplicate culture from the values of the individual cells examined.
Statistical significance above culture medium control:

bp <0.05;cp <0.01;
dp <0.001.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative both with and without metabolic activation
DMEA was not considered to induce reciprocal chromatid exchanges under the condition of this in vitro test.
Executive summary:

N,N-dimethylethanolarnine was evaluated for potential genotoxic activity using the Salmonella/microsome reverse gene mutation test, the CHO/HGPRT forward gene mutation test, a sister chromatid exchange test in cultured CHO cells, and an in vivo peripheral blood micronucleus test in Swiss-Webster mice (see also Endpoints 7.6.1.002, 7.6.1.003 and 7.6.2.001).

DMEA did not produce any statistically significant increase in SCEs above control values in tests both with or without the incorporation of an S9 metabolic activation system (Table 2 in "Remarks on results"). There were no increases in the numbers of first division cells which suggests that the dose ranges used were appropriate.

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