2-dimethylaminoethanol

Genetic toxicity: in vitro

Administrative data

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-reference

Cross-reference 1

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").

Cross-reference 2

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 10^5)
	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 x10^5cells/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 x10^5cells inoculated in each of 5 plates (1 x10^6total 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/10^6viable cells, but a 95 percentile range of 0-25 mutants/10^6viables 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").

Cross-reference 3

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 ± 3.6^b	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 ± 8.7^b	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

Test material information

Constituent 1

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

Controls

Controls 1

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

Controls 2

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 10^5)
	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 x10^5cells/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 ± 0.37^d
DMN (0.3 mg/mL)		2.38 ± 0.48^d
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.