2-dimethylaminoethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All valid studies for genetic toxicity in vitro as well as in vivo showed negative results.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 2

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.

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 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

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 / 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

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.

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.

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.

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.

Reference 3

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").

Reference 4

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").

Reference 5

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:

September 1984 - October 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was performed according to following guidelines: OECD 471 and in compliance with GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction of rat liver homogenate obtained from Arcolor 1254-treated Sprague Dawley rats

Test concentrations with justification for top dose:

50, 166, 500, 1666 and 5000 µg/plate

Vehicle / solvent:

-solvent(s) used: distilled/deionized water

Negative solvent / vehicle controls:

yes

Remarks:

with and without metabolic activation

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 and TA100= 10 µg/plate - without metabolic activation

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA1538 and TA98= 5 µg/plate - without metabolic activation

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537=150 µg/plate - without metabolic activation

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

in all strains=5 µg/plate - with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 48h
- Exposure duration: 48-72h

SELECTION AGENT (mutation assays): histidine and biotin

NUMBER OF REPLICATIONS:
negative, positive controls and compound-treated plates: in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition is tested at following concentrations: 50, 166, 500, 1666 and 5000 µg/plate with strains TA1538 and TA100 (in duplicate)

Evaluation criteria:

revertant colonies are counted (Artek Counter)
- positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies
- negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies

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 determined

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 determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: sreening showed no cytoxicity at any of the doses
COMPARISON WITH HISTORICAL CONTROL DATA: all solvent and positive controls were within acceptable limits of mean historical data

Conclusions:

Interpretation of results: negative with and without metabolic activation
The results for test article, 5601-40-1, Lot #J-214, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166, 500, 1666 and 5000 µg/plate.

Executive summary:

According to guideline OECD 471 different strains of S. typhimurium were investigated in context of gene mutation for 2 -dimethylaminoethanol. The experiment was a bacterial reverse mutation assay (Ames test) and was performed with certificated good laboratory praxis (GLP). S-9 fractions of rat liver homonate were used as metabolic activation system. Destilled / deionized water was used as vehicle, whereby it was incoorparated in agar. The test concentrations were 50, 166, 500, and 5000 µg/plate. S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 showed negative results for genotoxicity and the performed screening showed no cytoxicity at any of the dosis.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08-Oct-2019 - 21-Oct-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 2-Dimethylaminoethanol (abbreviated DMAE), Lot No. A190703581, supplied by Taminco US LLC (Kingsport, TN), was received at ambient conditions and stored at room temperature (19 °C to 25 °C) and purged with nitrogen after use.
- Expiration date of the lot/batch: Sep 2020 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 2-Dimethylaminoethanol was prepared as a stock solution in sterile water at a stock concentration of 200 mg/mL for the dose range-finding assay and all definitive mutagenicity assays (Trials 1-3). The density of the test article (0.886 g/mol) was used as the test article concentration (without any correction factor) from which dilutions were made in sterile water to produce the final experimental formulations.
- Final dilution of a dissolved solid, stock liquid or gel: from the above mentioned stock solution dilutions were made in sterile water to produce the final experimental formulations
- Final preparation of a solid: not applicable

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: All cultures were examined visually for pH change (as indicated by change in the color of the media) and precipitation at the time of dosing and at the end of the 4-hour treatment.

Target gene:

the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (Hprt)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1-BH4 cell line

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO-K1-BH4 cell line
- Suitability of cells: The CHO-K1-BH4 cell line is a proline auxotroph with a modal chromosome number of 20, a population doubling time of 12-14 hours, and a cloning efficiency generally greater than 80%.
- Normal cell cycle time (negative control): the CHO-K1-BH4 cell line has a population doubling time of 12-14 hours,

For cell lines:
- Absence of Mycoplasma contamination: yes, the cells have been tested to ensure that they are free of mycoplasma contamination.
- Number of passages if applicable: Cells can be used up to 20 passages
- Cell cycle length, doubling time or proliferation index: These cells have a population doubling time of 12-14 hours
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: yes, the cells have been tested to ensure that they contain a modal chromosome number of 20
- Periodically ‘cleansed’ of spontaneous mutants: yes, to reduce the frequency of spontaneous HPRT- mutants prior to use in an assay, cells were cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT), prior to cell banking or after thawing.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Stock cultures were maintained in Ham's F12 medium supplemented with 10 % (v/v) heat-inactivated fetal bovine serum (FBS), 2mM L-glutamine, 100 U/mL penicillin and 10 μg/mL streptomycin (F12FBS10) under standard conditions (36 °C to 38 °C in a humidified atmosphere of 4 % to 6 % CO2 in air). Routine culture of cells during the study was done in Ham's F12 medium supplemented with 5 % (v/v) heat-inactivated and dialyzed FBS, 2mM L-glutamine, 100 U/mL penicillin and 10 μg/mL streptomycin (F12FBM5). Treatment medium was Ham's F12 medium supplemented with 2mM L-glutamine, 100 U/mL penicillin and 10 μg/mL streptomycin (serum free). Hypoxanthine-free F12FBM5 (HX-F12FBM5) was used for mutant selection and to determine cloning efficiency at the time of selection. The medium for selection of mutants also contained 10 µM TG.

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Molecular Toxicology, Inc. (Lot Nos. 3923, 3932, 4105, and 4188)
- method of preparation of S9 mix : It was reconstituted as per the manufacturer’s instructions (to yield the same recipe as described by Ames et al.), except that it also contained 10mM CaCl2, which has been demonstrated to enhance the mutagenicity of some promutagens.
- concentration or volume of S9 mix and S9 in the final culture medium: 1 mL S9 mix and 4 mL of treatment medium were used for the cultures

The exogenous metabolic activation system (MUTAZYME™; S9) was obtained from Molecular Toxicology, Inc. (Lot Nos. 3923, 3932, 4105, and 4188), and consisted of Aroclor™ 1254-induced male rat liver S9 lyophilized with NADPH-regenerating system cofactors and phosphate buffer. It was stored and reconstituted as per the manufacturer’s instructions (to yield the same recipe as described by Ames et al.), except that it also contained 10mM CaCl2, which has been demonstrated to enhance the mutagenicity of some promutagens. Reconstituted S9 mix was prepared fresh on the day of testing and was maintained refrigerated (2 °C to 8 °C) or on ice.

Test concentrations with justification for top dose:

The test concentrations were based on a preliminary Dose-Range-finding assay.
In the definitive mutagenicity assay concentrations of 250, 500, 750, 1000, 1333, 1666, and 2000 μg/mL were used for experiments for 4-hours with and without S9-Mix. Furthermore, 125, 250, 400, 500, 600, 700, 800, 900, 1000, and 2000 μg/mL were employed in additional tests with 4-hour without S9 treatment and 250, 500, 700, 1000, 1200, 1400, 1600, 1800, and 2000 μg/mL were employed in the tests with 4-hour treatment with S9 in Trial 2. However, due to the cytotoxicity in the without S9 treatment and equivocal results of the with S9 treatment, the assay was repeated.
In Trial 3, 125, 250, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, and 2000 μg/mL were employed for the 4-hour treatments with and without S9.
The concentrations of the dose formulations were not determined. Concentrations were limited by cytotoxicity of the test article, demonstrating the maximum feasible dose was achieved in the test system. Therefore, this exception has no impact on the quality or integrity of the study.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqueous solvents (water), in detail sterile water (Mediatech, Lot Nos. 21718004 and 12219013) was used

Untreated negative controls:

not specified

Remarks:

however, historical control data were available, with which the current data has been compared as well

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle control for all assays was sterile water, Mediatech, Lot Nos. 21718004 and 12219013.

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethylnitrosamine (DMN)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of replicates: duplicate cultures
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Cells for treatment were seeded (on Day -1) in 75-cm2 flasks at a density of approximately 2.0 × 106 to 2.5 × 106 cells in 10 mL F12FBM5. Following an overnight incubation (on Day 0), the cultures were washed twice with 10 mL DPBS and re-fed with 10 mL treatment medium, or 8 mL treatment medium and 2 mL S9 mix, as appropriate. Following addition of the test or control articles, the cultures were incubated for 3.5 to 4.5 hours
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3.5 to 4.5 hours
- Harvest time after the end of treatment (sampling/recovery times): After the 4-hour treatment, the treatment media was removed, the cultures were washed twice with 10 mL CMF-DPBS, washed once with 5 mL trypsin, and incubated at 36 °C to 38 °C for 3 - 5 minutes (or until the cells detached). Four mL F12FBM5 was added and the cells were resuspended and counted using a Coulter counter. All cultures were examined visually for pH change (as indicated by change in the color of the media) and precipitation at the time of dosing and at the end of the 4-hour treatment.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Approximately 2.4 × 10E6 cells (or all available) from each resuspended day 0 culture were also added to a 225-cm² flask in 30 mL F12FBM5 for phenotypic expression and incubated under standard conditions. The cultures were subcultured on Days 2 and 5 (Trial 1) or Days 3 and 6 (Trials 2 and 3) to maintain logarithmic growth and permit expression of the mutant phenotype. At each subculture, the cultures were washed twice with 15 mL CMF-DPBS, washed once with 5 mL trypsin and incubated at 36 °C to 38 °C for 3 - 5 minutes (or until the cells detached). Five mL F12FBM5 were added and the cells were resuspended and counted using a Coulter counter. However, if visual observation indicated that < 2.4 × 10E6 cells are available, the culture was re-fed with fresh media and incubated under standard conditions until the next subculture.
At the end of the phenotypic expression period (on Day 7 for Trial 1 or Day 8 for Trials 2 and 3), cultures were trypsinized as above (except using 5 mL HX-F12FBM5 to inactivate the trypsin) and 2.4 × 10E6 cells from each culture were seeded at a density of 6 × 10E5 cells/150-mm plate (4 plates total) in 30 mL HX-F12FBM5 containing 10μM TG. An additional aliquot of cells from each culture was diluted in DPBS (1:100) and seeded at a density of 200 cells/60-mm plate in 5 mL HX-F12FBM5 in triplicate to determine the cloning efficiency at the time of selection. All plates were incubated under standard conditions for 7 to 10 days.
- Selection time (if incubation with a selective agent): 7- to 10-days
- Fixation time (start of exposure up to fixation or harvest of cells): After the 7- to 10-day incubation period, the colonies were fixed, stained and counted as above. Mutant frequencies are expressed as the number of TGr mutants/10E6 clonable cells. The number of clonable cells was determined from the triplicate 60-mm plates.
- Criteria for small (slow growing) and large (fast growing) colonies: not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative survival (RS)
- Any supplementary information relevant to cytotoxicity: Cytotoxicity of the test article to the test system was determined in order to allow the selection of appropriate dose levels to be tested in the definitive mutagenicity assays.
Cells were treated with 2-Dimethylaminoethanol at concentrations of 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μg/mL, as well as the vehicle control, in the presence and absence of S9 using single cultures. The highest concentration evaluated approximated the limit dose for this assay (2000 μg/mL).
An aliquot of the resuspended cells from each day 0 culture was diluted in DPBS (1:100) and seeded at a density of 200 cells/60-mm plate in 5 mL F12FBM5 in triplicate for determination of initial survival. The 60-mm plates were incubated under standard conditions for 7 - 10 days, and the resulting colonies were fixed in absolute ethanol, stained with crystal violet, rinsed in running tap water, air dried and counted using an automated colony counter.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: cloning efficiency; relative survival (RS)

Evaluation criteria:

CRITERIA FOR A VALID ASSAY
Each treatment condition (i.e., with or without S9) is considered independent with regard to assay acceptance and is repeated independently as necessary to satisfy the acceptance criteria.

Vehicle Control Cultures
The average absolute cloning efficiency of vehicle control article must be ≥ 50 % (at initial survival and selection). In addition, the average spontaneous mutant frequency of the vehicle control article must be comparable to the 95 % control interval of the historical negative controls.
Spontaneous mutant frequencies are calculated separately for cultures with and without S9.

Positive Control Cultures
The positive control articles must induce a statistically significant increase compared with the concurrent vehicle controls (p ≤ 0.01). A significant increase in the absence of S9 indicates the test system can identify a mutagen, while a significant increase in the presence of S9 will be considered to have demonstrated the efficacy of the S9 mix as well as the ability of the test system to detect a mutagen.

Maximum Concentration Evaluated
The highest test article concentrations evaluated must be the limit dose for this assay (2000 μg/mL or 10mM), or be the highest that is able to be prepared and administered as a uniform fine suspension, or be minimally insoluble in treatment medium, or induce 10 - 20 % relative survival (the cytotoxicity limit for this assay), whichever is lowest. Lower concentrations may be justified for test articles which clearly show mutagenic activity.

Number of Concentrations
A minimum of four concentrations with acceptable relative survival is required for a valid assay. Fewer concentrations may be justified for test articles which clearly show mutagenic activity.

Statistics:

A one-tailed Fisher’s Exact test was performed to compare the mutant frequencies observed in the treated cultures with those of the concurrent vehicle control article cultures. The Cochran-Armitage test was used to assess dose dependency.

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1-BH4

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test article was found to be freely soluble at all concentrations evaluated

RANGE-FINDING/SCREENING STUDIES (if applicable):
2-Dimethylaminoethanol was prepared in sterile water and evaluated in a preliminary dose range-finding assay at concentrations of 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μg/mL with and without S9 (Table 1). The highest concentration evaluated approximated the limit dose for this assay (2000 μg/mL). Sterile water was evaluated concurrently as the vehicle control. Relative survivals at concentrations of 2000 μg/mL with S9 and 2000 μg/mL without S9 were 22.93 and 1.07 %, respectively. In addition, the test article was found to be freely soluble at all concentrations evaluated.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
In all valid trials the DMN and EMS positive controls induced significant increases in mutant frequencies (p < 0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : No dose-dependent increases observed
- Statistical analysis; p-value, if any: No statistically significant or dose-dependent increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p > 0.05)

- Genotoxicity results:
please refer to 'any other information on results'

EVALUATION CRITERIA

Criteria for a Positive Response

The test article will be considered to be positive for inducing mutations if it induces a statistically significant and dose-dependent increase in mutant frequency (p ≤ 0.05) that exceeds the 95 % control limit of the historical negative/vehicle control articles.

Criteria for a Negative Response

The test article will be considered negative for inducing mutations if no statistically significant or dose-dependent increases in mutant frequency are observed (p > 0.05), and none of the observed mutant frequencies exceed the 95 % control limit of the historical negative/vehicle control articles.

Criteria for an Equivocal Response

Responses that do not clearly fit into the positive or negative category may be judged equivocal (e.g., a response that meets only one or two of the three criteria above). In these cases, the data should be evaluated by expert judgement and/or further investigations. In rare cases, even after further investigations, the data set may preclude making a definitive assessment and the result may be concluded to be equivocal.

RESULTS AND DISCUSSION

Dose Range-Finding Assay

Table 1
Dose Range-Finding Assay
Treatment	Dose Level (µg/mL)	S9	Initial Survival
			Cells/mL	Cloning Efficiency				Relative Survival(adj.,%)
			(x 10^6)	(Colonies/Plate)			(%)
di-H2O	10.0^a	-	1.072	119	117	115	58.50	100.00
DMAE	3.91	-	0.837	90	88	92	45.00	60.10
DMAE	7.81	-	1.093	120	120	119	59.83	104.35
DMAE	15.6	-	0.938	76	74	81	38.50	57.61
DMAE	31.3	-	0.869	122	120	120	60.33	83.60
DMAE	62.5	-	1.022	144	122	138	67.33	109.71
DMAE	125	-	1.017	78	81	86	40.83	66.22
DMAE	250	-	1.003	101	98	111	51.67	82.61
DMAE	500	-	0.832	126	120	132	63.00	83.55
DMAE	1000	-	0.338	85	83	84	42.00	22.63
DMAE	2000	-	0.041	26	38	34	16.33	1.07
di-H2O	10.0^a	+	0.974	122	125	146	65.50	100.00
DMAE	3.91	+	0.986	142	154	100	66.00	102.00
DMAE	7.81	+	0.848	138	141	139	69.67	92.58
DMAE	15.6	+	0.928	126	141	133	66.67	96.95
DMAE	31.3	+	0.937	152	124	137	68.83	101.06
DMAE	62.5	+	0.957	146	144	150	73.33	109.92
DMAE	125	+	0.859	142	152	150	74.00	99.61
DMAE	250	+	0.987	127	142	146	69.17	107.00
DMAE	500	+	0.705	177	188	169	89.00	98.36
DMAE	1000	+	0.633	228	191	194	102.17	101.28
DMAE	2000	+	0.194	153	142	158	75.50	22.93
^aµL/mL

Definitive Mutagenicity Assay

Trial 1

2-Dimethylaminoethanol was evaluated in the definitive mutagenicity assay at concentrations of 250, 500, 750, 1000, 1333, 1666, and 2000 μg/mL for 4-hours with and without S9 (Table 2).

All test article concentrations, as well as the concurrent positive and vehicle controls, were evaluated in duplicate cultures. 2-Dimethylaminoethanol again was found to be freely soluble at all concentrations evaluated. Those cultures treated at concentrations of ≥ 1000 μg/mL without S9 were discarded prior to selection or excluded from evaluation of mutagenicity due to excessive cytotoxicity. Therefore, only 3 analyzable concentrations were available for the treatment without metabolic activation. The average relative survivals at the highest concentrations remaining and evaluated for mutagenicity were approximately 15.1 and 17.2 % with and without S9, respectively, and met the acceptance criteria of 10-20 % relative survival. No statistically significant or dose-dependent increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p > 0.05), and all observed values were within historical control limits. Statistically significant increases were noted in the treatment with S9 at multiple concentrations, however all increases noted were within historical range of the vehicle control.

Table 2
Definitive Mutagenicity Assay (Trial 1)
Treatment	Dose Level (µg/mL)	S9			Cells/mL (x 10^6)	Initial Survival								Relative Survival (%, adj.)		Selection
						Cloning Efficiency										TG^rMutants/Plate								Total Mutant Colonies		Cloning Efficiency								Mutant Frequency (x 10^-6)
						(Colonies/Plate)						(%)														(Colonies/Plate)						(%)		Individual		Average
di-H2O	10.0^a			0.901		128	136		109		62.17		99.49		2		2		1		0		5		125		122		101		58.00		3.59
di-H2O	10.0^a			0.959		110	140		104		59.00		100.51		3		1		1		3		8		b		b		b		b				3.59
DMAE	250			0.907		132	103		101		56.00		90.29		2		1		2		1		6		99		110		97		51.00		4.90
DMAE	250			0.914		118	116		118		58.67		95.27		1		0		1		1		3		115		89		68		45.33		2.76		3.83
DMAE	500			0.636		112	106		104		53.67		60.65		1		3		0		1		5		16		22		34		12.00		17.36
DMAE	500			0.645		119	113		106		56.33		64.59		0		2		1		0		3		89		107		82		46.33		2.70		10.03
DMAE	750			0.215		98	76		86		43.33		16.55		1		0		8		2		11		31		34		43		18.00		25.46
DMAE	750			0.241		85	84		82		41.83		17.90		0		1		1		1		3		53		82		60		32.50		3.85		14.65
DMAE	1000			0.086		69	74		71		35.67		5.46		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND
DMAE	1000			0.103		51	43		56		25.00		4.59		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND
DMAE	1333			0.063		46	66		61		28.83		3.23		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND
DMAE	1333			0.058		50	54		40		24.00		2.45		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND
DMAE	1666				0.026	30		33		34		16.17		0.75		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND
DMAE	1666				0.021	37		30		31		16.33		0.60		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND
DMAE	2000				0.013	7		9		7		3.83		0.09		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND
DMAE	2000				0.011	4		3		4		1.83		0.04		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND		ND
EMS	200				0.936	70		84		65		36.50		60.69		63		84		79		72		298		54		58		55		27.83		446.11
EMS	200				0.925	58		90		57		34.17		56.16		68		66		83		71		288		94		66		88		41.33		290.32		368.22
di-H2O	10.0^a	+			0.998	128		101		118		57.83		90.03		4		1		2		0		7		140		124		127		65.17		4.48
di-H2O	10.0^a	+			1.110	116		136		129		63.50		109.97		0		1		2		0		3		134		102		119		59.17		2.11		3.29
DMAE	250	+			0.927	123		133		117		62.17		89.90		4		2		2		1		9		130		100		127		59.50		6.30
DMAE	250	+			1.031	110		113		124		57.83		93.04		0		2		6		1		9		178		91		137		67.67		5.54		5.92
DMAE	500	+			1.102	99		100		85		47.33		81.41		1		0		1		0		2		107		123		111		56.83		1.47
DMAE	500	+			1.121	110		89		103		50.33		88.07		8		1		3		2		14		100		115		102		52.83		11.04		6.25
DMAE	750	+			1.028	94		80		97		45.17		72.42		2		6		10		1		19		116		141		124		63.50		12.47
DMAE	750	+			1.075	80		82		93		42.50		71.28		4		3		1		1		9		130		140		118		64.67		5.80		9.13
DMAE	1000	+			0.893	73		100		75		41.33		57.58		5		9		4		8		26		82		104		81		44.50		24.34
DMAE	1000	+			0.914	85		90		97		45.33		64.64		6		2		3		1		12		86		88		60		39.00		12.82		18.58
DMAE	1333	+			0.881	90		123		97		51.67		70.99		2		1		3		0		6		89		82		86		42.83		5.84
DMAE	1333	+			0.868	98		89		58		40.83		55.28		4		2		8		3		17		74		84		75		38.83		18.24		12.04
DMAE	1666	+			0.597	96		75		92		43.83		40.83		2		2		2		2		8		94		83		76		42.17		7.91
DMAE	1666	+			0.510	97		87		107		48.50		38.60		2		0		1		2		5		67		98		93		43.00		4.84		6.38
DMAE	2000	+			0.238	65		73		59		32.83		12.20		5		3		5		10		23		75		80		98		42.17		22.73
DMAE	2000	+			0.319	66		73		79		36.33		18.08		7		6		0		3		16		80		97		86		43.83		15.21		18.97
DMN	100	+			0.951	16		27		15		9.67		14.34		31		21		19		17		88		55		58		77		31.67		115.79
DMN	100	+			1.038	22		18		24		10.67		17.28		14		31		22		C		67		55		71		58		30.67		121.38		118.58
^aµL/mL
--^bData not reported due to plating error
C = Contaminated plate
ND = Not determined or reported due to excessive cytotoxicity
*= Fisher's Exact p< 0.05
**= Fisher's Exact p< 0.01
Note: 4-hour without S9 Statistics were not run on test article concentrations due to lack of analyzable concentrations, additionally only VC duplicate 1 was included in statistical analysis of the positive control response
statistical analysis of the positive control response

Trial 2 and Trial 3

Due to the cytotoxicity in the without S9 treatment and equivocal results of the with S9 treatment, the assay was repeated. Trial 2 of the assay did not meet acceptance criteria for a valid assay and will not be reported. Trial 3 was conducted at test article concentrations of 125, 250, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, and 2000 μg/mL with and without S9 (Table 3). All test article concentrations, as well as the concurrent positive and vehicle controls, were evaluated in duplicate cultures. 2-Dimethylaminoethanol was found to be freely soluble at all concentrations evaluated. Those cultures treated at concentrations of ≥ 1400 μg/mL without S9 were discarded prior to selection or excluded from evaluation of mutagenicity due to excessive cytotoxicity. The average relative survivals at the highest concentrations remaining and evaluated for mutagenicity with S9 was approximately 29.9 (limit dose of 2000 μg/mL). The treatment without S9 met the protocol acceptance criteria of 10 - 20 % relative survival with a value of 10.2 % (at 1200 μg/mL) relative survival compared to the vehicle control.

Table 3
Definitive Mutagenicity Assay (Trial 3)
Treatment	Dose Level (µg/mL)	S9	Cells/mL (x 10^6)	Initial Survival				Relative Survival (%, adj.)	Selection
				Cloning Efficiency					TG^rMutants/Plate				Total Mutant Colonies					Mutant
															Cloning Efficiency			Frequency (x 10^-6)
				(Colonies/Plate)			(%)							(Colonies/Plate)			(%)	Individual	Average
di-H2O	108^a		0.807	108	93	114	52.50	91.41	5	7	5	5	22	116	121	108	57.50	15.94
di-H2O	10.0^a		0.896	119	126	92	56.17	108.59	4	1	8	8	21	146	124	119	64.83	13.50	14.72
DMAE	125		0.684	104	106	121	55.17	81.42	6	7	3	7	23	115	123	108	57.67	16.62
DMAE	125		0.984	128	119	118	60.83	129.18	11	8	15	10	44	131	114	113	59.67	30.73	23.67 **
DMAE	250		0.964	94	126	81	50.17	104.38	5	6	9	7	27	106	108	114	54.67	20.58
DMAE	250		0.924	118	113	89	53.33	106.35	7	9	10	5	31	100	138	115	58.83	21.95	21.27 *
DMAE	400		0832	127	152	122	66.83	91.14	9	9	5	8	31	105	115	102	53.67	24.07
DM AE	400		0.707	114	134	107	59.17	90.32	12	16	11	11	50	139	105	106	58.33	35.71	29.89 **
DM AE	500		0.789	113	116	102	55.17	93.90	5	3	7	3	18	116	132	112	60.00	12.50
DM AE	500		0.866	81	123	103	51.17	95.60	9	7	7	8	31	107	88	116	51.83	24.92	18.71
DM AE	600		0.680	112	135	112	59.83	87.83	5	10	6	7	28	128	111	110	58.17	20.06
DM AE	600		0.727	132	126	128	64.33	100.88	3	4	4	2	13	111	105	116	55.33	9.79	14.92
DM AE	700		0.658	134	104	95	55.50	78.79	8	2	5	6	21	124	84	113	53.50	16.36
DM AE	700		0.674	120	121	103	57.33	83.37	12	14	8	20	54	90	87	106	47.17	47.70	32.03 **
DM AE	800		0.412	118	151	107	62.67	55.79	7	5	2	3	17	108	121	126	59.17	11.97
DM AE	800		0.555	140	73	119	55.33	66.23	8	7	4	9	28	110	111	119	56.67	20.59	16.28
DM AE	900		0.314	110	118	91	53.17	36.01	8	10	10	5	33	119	117	115	58.50	23.50
DM AE	900		0.347	60	133	84	46.17	34.53	9	1	3	5	18	132	127	93	58.67	12.78	18.14
DM AE	1000		0.228	142	160	120	70.33	34.66	2	5	1	6	14	125	117	99	56.83	10.26
DMAE	1000	-	0.263	83	118	97	49.67	28.15	3	1	3	4	11	102	85	96	47.17	9.72	9.99
DM AE	1200		0.123	90	107	52	41.50	11.01	10	5	9	9	33	112	100	102	52.33	26.27
DM AE	1200		0.100	87	81	96	44.00	9.48	0	1	3	0	4	86	95	90	45.17	3.69	14.98
DM AE	1400		0.142	101	110	104	52.50	16.07	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
DM AE	1400		0.096	105	69	108	47.00	9.78	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
DM AE	1600		0.076	104	85	80	44.83	7.36	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
DM AE	1600		0.093	97	96	92	47.50	9.56	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
DM AE	1800		0.071	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
DMAE	1800		0.057	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
DMAE	2000		0.036	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
DMAE	2000		0.023	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
EMS	200		1.150	99	90	93	47.00	116.68	98	76	129	102	405	89	99	93	46.83	360.32
EMS	200		1.108	75	64	96	39.17	93.63	70	76	68	66	280	104	96	103	50.50	231.02	295.67 **
di-H2O	10.0^a	+	1.073	117	154	73	57.33	102.10	7	9	3	4	23	111	113	150	62.33	15.37
di-H2O	10.0^a	+	1.237	76	124	86	47.67	97.90	10	7	7	9	33	104	107	92	50.50	27.23	21.30
DMAE	125	+	1.252	76	144	90	51.67	107.39	10	5	5	11	31	161	101	104	61.00	21.17
DMAE	125	+	1.114	92	125	114	55.17	102.00	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	21.17
DM AE	250	+	1.105	95	92	100	47.83	87.76	4	2	8	4	18	162	150	145	76.17	9.85
DM AE	250	+	1.054	94	118	127	56.50	98.87	10	9	7	11	37	118	99	106	53.83	28.64	19.24
DM AE	400	+	0.970	130	165	128	70.50	113.58	1	4	3	1	9	114	124	120	59.67	6.28
DM AE	400	+	0.918	110	100	87	49.50	75.46	6	7	8	10	31	0	0	0	0.00		6.28
DM AE	500	+	1.078	138	134	111	63.83	114.27	8	6	11	4	29	254	245	227	121.00	9.99
DM AE	500	+	1.168	103	113	89	50.83	98.60	11	6	5	8	30	113	127	131	61.83	20.22	15.10 *
DM AE	600	+	1.279	95	89	105	48.17	102.31	6	6	3	5	20	106	108	96	51.67	16.13
DM AE	600	+	1.131	94	151	125	61.67	115.76	5	12	10	10	37	81	92	95	44.67	34.51	25.32
DM AE	700	+	1.086	107	145	79	55.17	99.49	4	6	5	4	19	102	150	155	67.83	11.67
DM AE	700	+	1.099	101	101	114	52.67	96.08	3	15	12	5	35	117	100	127	57.33	25.44	18.55
DM AE	800	+	0.920	94	148	107	58.17	88.81	7	7	4	8	26	102	103	97	50.33	21.52
DM AE	800	+	1.018	107	129	132	61.33	103.65	10	9	12	6	37	146	152	137	72.50	21.26	21.39
DM AE	900	+	0.958	117	121	107	57.50	91.45	7	9	6	7	29	106	108	114	54.67	22.10
DM AE	900	+	1.016	126	110	121	59.50	100.39	6	8	12	7	33	133	115	150	66.33	20.73	21.42
DM AE	1000	+	0.948	100	109	103	52.00	81.83	4	7	4	5	20	122	100	125	57.83	14.41
DMAE	1000	+	0.996	136	95	123	59.00	97.58	4	6	8	5	23	110	94	79	47.17	20.32	17.36
DMAE	1200	+	0.888	99	159	132	65.00	95.77	6	7	12	6	31	141	134	114	64.83	19.92
DMAE	1200	+	1.043	107	123	100	55.00	95.24	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	19.92
DMAE	1400	+	1.048	117	81	89	47.83	83.19	7	1	3	4	15	113	111	102	54.33	11.50
DMAE	1400	+	0.762	177	179	131	81.17	102.64	13	5	6	7	31	123	143	93	59.83	21.59	16.55
DMAE	1600	+	0.701	117	133	138	64.67	75.27	13	6	18	8	45	130	121	89	56.67	33.09
DMAE	1600	+	0.989	100	114	90	50.67	83.15	4	3	3	1	11	123	133	125	63.50	7.22	20.15
DMAE	1800	+	0.634	89	95	89	45.50	47.91	8	6	10	8	32	128	127	103	59.67	22.35
DMAE	1800	+	0.758	100	115	99	52.33	65.83	14	7	22	25	68	113	103	115	55.17	51.36	36.85 **
DMAE	2000	+	0.599	49	40	76	27.50	27.33	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
DMAE	2000	+	0.558	60	84	66	35.00	32.43	11	5	7	9	32	129	108	100	56.17	23.74	23.74
DMN	100	+	0.988	35	37	52	20.67	33.91	45	39	25	45	154	136	71	68	45.83	140.00
DMN	100	+	1.072	53	41	71	27.50	48.92	33	30	34	32	129	115	87	102	50.67	106.09	123.04 **
^aµL/mL
C = Contaminated plate
ND = Not determined or not reported due to excessive cytotoxicity
*= Fisher's Exact p< 0.05
**= Fisher's Exact p< 0.01

A statistically significant increase in mutant colonies as compared to the concurrent vehicle control, and outside the historical control range was observed at 400 and 700 μg/mL without S9.

However these increases were not dose-dependent, and at least 4 concentrations evaluated above 700 μg/mL were not significantly increased and observed values were within historical control limits. Additionally, the increase was not reproducible across the replicat treatments and is not considered biologically relevant. Taken together with the first trial, 2-Dimethylaminoethanol is considered to be negative for mutagenicity in the 4-hour treatment without S9.

In the 4-hour treatment with S9, statistically significant increases were noted in mutant colonies as compared to the concurrent vehicle control and outside the historical control range at 1800 μg/mL. However this increase was not reproducible across the replicate treatments and is not considered biologically relevant. Additionally, the 2000 μg/mL dose level is not significantly increased. Therefore, 2-Dimethylaminoethanol is considered to be negative for mutagenicity in the 4-hour treatment with S9.

In all valid trials the DMN and EMS positive controls induced significant increases in mutant frequencies (p < 0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

Conclusions:

2-Dimethylaminoethanol (DMAE) was found to be negative in the In Vitro Mamalian Cell Gene Mutation Test (CHO-HPRT Assay) under the conditions, and according to the criteria, of the test protocol. In all valid trials the DMN and EMS positive controls induced significant increases in mutant frequencies (p < 0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

Executive summary:

2-Dimethylaminoethanol (Lot No. A190703581) was evaluated for the potential to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (Hprt) in Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr) according to OECD 476 in compliance with GLP standards.

2-Dimethylaminoethanol was prepared in sterile water and evaluated in a preliminary dose range-finding assay at concentrations of 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μg/mL for 4-hours with and without S9. The highest concentration evaluated approximated the limit dose for this assay (2000 μg/mL). Sterile water was evaluated concurrently as the vehicle control. Relative survivals at concentrations of 2000 μg/mL with S9 and 2000 μg/mL without S9 were 22.93 % and 1.07 %, respectively. In addition, the test article was found to be freely soluble at all concentrations evaluated.

Based upon these results, 2-Dimethylaminoethanol was evaluated in the definitive mutagenicity assay at concentrations of 250, 500, 750, 1000, 1333, 1666, and 2000 μg/mL for 4-hours with and without S9. All test article concentrations, as well as the concurrent positive and vehicle controls, were evaluated in duplicate cultures. 2-Dimethylaminoethanol again was found to be freely soluble at all concentrations evaluated. Those cultures treated at concentrations of ≥ 1000 μg/mL without S9 were discarded prior to selection or excluded from evaluation of mutagenicity due to excessive cytotoxicity. Therefore, only 3 analyzable concentrations were available for the treatment without metabolic activation. The average relative survivals at the highest concentrations remaining and evaluated for mutagenicity were approximately 15.1 and 17.2 % with and without S9, respectively, and met the acceptance criteria of 10 - 20 % relative survival. No statistically significant or dose-dependent increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p > 0.05), and all observed values were within historical control limits. Statistically significant increases were noted in the treatment with S9 at multiple concentrations, however all increases noted were within historical range of the vehicle control.

Due to the cytotoxicity in the without S9 treatment and equivocal results of the with S9 treatment, the assay was repeated. Trial 2 of the assay did not meet acceptance criteria for a valid assay and will not be reported. Trial 3 was conducted at test article concentrations of 125, 250, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, and 2000 μg/mL with and without S9. All test article concentrations, as well as the concurrent positive and vehicle controls, were evaluated in duplicate cultures. 2-Dimethylaminoethanol was found to be freely soluble at all concentrations evaluated. Those cultures treated at concentrations of ≥ 1400 μg/mL without S9 were discarded prior to selection or excluded from evaluation of mutagenicity due to excessive cytotoxicity. The average relative survivals at the highest concentrations remaining and evaluated for mutagenicity with S9 was approximately 29.9 (limit dose of 2000 μg/mL). The treatment without S9 met the protocol acceptance criteria of 10-20 % relative survival with a value of 10.2 % (at 1200 μg/mL) relative survival compared to the vehicle control.

A statistically significant increase in mutant colonies as compared to the concurrent vehicle control, and outside the historical control range was observed at 400 and 700 μg/mL without S9. However these increases were not dose-dependent and at least four concentrations evaluated above 700 μg/mL were not significantly increased and observed values were within historical control limits. Additionally, the increase was not reproducible across the replicat treatments and is not considered biologically relevant. Taken together with the first trial, 2-Dimethylaminoethanol is considered to be negative for mutagenicity in the 4-hour treatment without S9. In the 4-hour treatment with S9, statistically significant increases were noted in mutant colonies as compared to the concurrent vehicle control and outside the historical control range at 1800 μg/mL.

However this increase was not reproducible across the replicate treatments and is not considered biologically relevant. Additionally, the 2000 μg/mL dose level is not significantly increased. Therefore, 2-Dimethylaminoethanol is considered to be negative for mutagenicity in the 4-hour treatment with S9. In all valid trials the DMN and EMS positive controls induced significant increases in mutant frequencies (p < 0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

These results indicate 2-Dimethylaminoethanol was negative in the In Vitro Mammalian Cell Gene Mutation Test (CHO/HPRT Assay) under the conditions, and according to the criteria, of the test protocol.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

All valid studies for genetic toxicity in vitro as well as in vivo showed negative results.

Link to relevant study records

Reference

Reference 1

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.

Reference 2

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:

3 May 1988 - 15 June 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study followed OECD guideline 474. In compliance with GLP Regulation. The OECD Guideline 474 states:that samples of bone marrow should be taken at least twice, starting not earlier than 24h after treatment, but not extending beyond 48h after treatment with appropriate interval(s) between samples. In this assay, bone marrow samples are taken at 30, 48 and 72h after treatment. A significant increase in PCE/NCE ratios is observed for the 72h treated group as compared to the vehicle control. However, it's biological significance, if any, is unknown.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

The OECD Guideline 474 states: that samples of bone marrow should be taken at least twice, starting not earlier than 24h after treatment, but not extending beyond 48h after treatment with appropriate interval(s) between samples. In this assay, bone marrow samples are taken at 30, 48 and 72h after treatment.
Also, the likelihood that the test substance reach the general circulation or the target tissue has not been discussed.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, Massachusetts
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 34-38g, females: 25-29g
- Assigned to test groups randomly: yes, randomized by bodyweight, assigned to groups by use of a random number table
- Fasting period before study: no
- Housing: 5 per cage (according to sex and dose group)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 3
- Humidity (%): 30-70% humidity
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark, 12 hrs light

IN-LIFE DATES: From: May 1988 To: June 1988

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water

Details on exposure:

one single intraperitoneal dose per mouse

Duration of treatment / exposure:

1 single dose

Frequency of treatment:

once

Post exposure period:

test substance: 30, 48 and 72h
positive control: 30h
negative control: 48h

Dose / conc.:

75 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Route of administration: intraperitoneal
- Doses / concentrations: 0.5 mg/kg

Tissues and cell types examined:

bone marrow of femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
based on dose range finding study with following doses: 10, 50, 75, 100, 250 mg/kg bw.
All mice died at 250 mg/kg bw and 1 of 4 died at 100 mg/kg bw. Du to the mortality observed at 100 mg/kg bw, 75 mg/kg bw was selected as an estimate of the maximum tolerated dose for the micronucleus test.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
All mice were sacrified by cervical dislocation.

DETAILS OF SLIDE PREPARATION:
Femurs were opened carefully at the proximal end with a scissors until a small opening to the marrow canal became visible. A 1 mL tuberculin syringe filled with approx. 0.2 mL fetal bovine serum was inserted into the marrow cavity and the marrow was gently flushed (to assure maximum dispersion) into 1.0 mL of fetal bovine serum in a 3 mL conical centrifuge tube. The femora were flushed with fetal bovine serum until all the marrow was out and the bone appeared almost translucent.
The suspension was centrifuged at 1000 rpm for 5 minutes. The supernatant was removed leaving a small amount of fetal bovine serum with the remaining cell button. The button was mixed with a pasteur pipette to assure a homogenous mixture. The marrow smears were made by placing immediately a small drop of the cell suspension near the frosted end of a glass slide pre-cleaned in absolute ethanol and smeared by pulling the cell suspension behind with another pre-cleaned slide at a 45° angle. The slides were quickly dried on a slide warmer set at approx. 56 °C, dipped in absolute methanol and air dried.
Stained with Giemsa.

METHOD OF ANALYSIS:
Slides were screened for good preparation, i.e. well spread, undamaged, perfectly stained.
1000 polychromatic erythrocytes (PCE)(immature) were counted for the presence of micronuclei per animal. Also the number of normochromatic erythrocytes (NCE) (mature) present in these 1000 PCE were recorded.
Data are expressed as the number of micronucleated PCE versus total normal PCE in 1000 PCE per animal.
A total of 100 PCE and NCE was also counted per animal. These data were expressed as the ratio PCE/NCE.

OTHER: Slides were coded randomly by study number and number designation. The code was kept on a separate sheet until the slides were evaluated. Following evaluation, slides were decoded. Coding of the slides was carried out by an individual not involved in the actual scoring.

Evaluation criteria:

If the spontaneous rate of micronuclei in the PCE is less than 0.2 % and the positive control is statistically significantly greater (p<0.05) than the spontaneous rate and at least seven animals per group survived the treatment, the results are deemed acceptable.

Statistics:

- one-tailed t-tests were used to make pairwise comparisons between each treatment group and its concurrent vehicle control for statistically significant increases in the number of micronucleated PCE.
- the proportion of PCE per 1000 erytrocytes per animal were evaluated by pairwise two-tailed t-test after an arcsin transformation was performed.
All comparisons were made for each sacrifice time separately comparing treated groups versus the vehicle control group.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

writhing, abnormal gait, piloerection, decreased body tone, no mortality observed

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 10-250 mg/kg
- Solubility: ok
- Clinical signs of toxicity in test animals:
all mice died at 250 mg/kg and 1 of 4 died at 100 mg/kg.
most mice (10-250 mg/kg): writhing, piloerection and/or decreased body tone.
also observed: abnormal gait, decreased activity (not in lowest dose group)
250mg/kg: tremors and ptosis at 24h and 48h.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 6/10000 (neg. control), 422/10000 (pos. control), 4/10000 (30h), 8/10000 (48h), 4/10000 (72h)
- Ratio of PCE/NCE (for Micronucleus assay): 1.173 (neg control), 0.905 (pos control), 1.433 (30h), 1.424 (48h), 1.755 (72h)
- Statistical evaluation: OK
Test article did not produce any statistically significant increase in the incidence of micronucleated PCEs at any of the sacrifice intervals.
There was a significant increase in PCE/NCE ratios for the 72h treated group as compared to the vehicle control.
The biological significance of this positive result, if any, is unknown at the present time.
Animals of the positive control group showed statistically significant increase in the incidence of micronucleated PCE in comparison with the negative control.

Conclusions:

Interpretation of results: negative
The test article, administered in single intraperitoneal dose at 100 mg/kg bw, was considered negative in the micronucleus test at the time intervals evaluated under the experimental condition of this assay.
The findings are based on the inability of the test article to produce a statistically significant increase in the incidence of micronuclei per 1000 PCE per animal in the treated group versus the negative control group under the conditions of this assay.

Executive summary:

The chromosome aberration as micronucleus assay was performed equivalent to OECD guideline 474 with certificated good laboratory praxis (GLP). As test species 5 male and 5 female mouse were used, strain CD-1. Destilled water was the vehicle and one single intraperitoneal dose per mouse was injected. The post exposure period is reported as 30, 48, and 72 hours, the nominal concentration was 75 mg/kg. The bone marrow of femur was examined. The genotoxicity is stated with a negative result. Toxicity is stated to be positive, due to different abnormalities: writhing, abnormal gait, piloerection, and decreased body tone. No mortality was observed during the experiment.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

According to the guideline OECD 471 different strains of S.typhimurium were investigated in context of gene mutation for 2 -dimethylaminoethanol. The experiment was a bacterial reverse mutation assay (Ames test) and was performed with certificated good laboratory praxis (GLP). S-9 fractions of rat liver homogenates were used as metabolic activation system. Distilled / deionized water was used as vehicle, whereby it was incorporated in agar. The test concentrations were 50, 166, 500, and 5000 µg/plate. S.typhimurium strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538 showed negative results for genotoxicity and the performed screening showed no cytoxicity at any of the doses.(Pharmakon Research International, 1984).

2 -dimethylaminoethanol was investigated for DNA damage & repair by unscheduled DNA synthesis in mammalian cell in vitro. This experiment was performed in equivalence to OECD guideline 482 (with a few discrepancies). No metabolic activation was done, WME with 10 % calf serum and 50 µg/mL gentamycin was used as media and hepatocytes, which were isolated from adult male F344 rats were used as test material. The test concentrations were 5E-6, 1E-5, 5E-5, 1E-4, 5E-4, 1E-3, 5E-3, 1E-2, 5E-2, 1E-1, 5E-1, and 1 % v/v, DMSO was used as vehicle, the application was done in the medium. In context of genotoxicity the result was reported negative. When the slides exposed to the highest concentrations of the test substance were examined under the microscope, after they had been processed for autoradiography, cytotoxicity was observed in the slides exposed to the four highest concentrations (1 % to 5E-2 %). Counting of slides thus began with slides exposed at 1E-2 % and lower. The mean net nuclear grain counts of the slides exposed at 1E-2 % and at lower concentrations did not exceed 5. Therefore it was concluded that the test substance was not genotoxic to the hepatocytes in the assy.(Naylor Dana Institute, 1985).

DMAE 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, DMAE was not judged to be positive in the in vitro gene mutation test performed by Leung and Ballantyne (1997).

However, according to the European Chemicals Agency, this "study does not provide the information required by Annex VIII, Section 8.4.3., because the top dose only reaches 80% or 60% survival, while according to the OECD test guideline 476, it should “result in approximately 10-20 % (but not less than 10%) relative survival”. Since the test was conducted, significant changes have been made to the OECD test guideline 476 and this means that the study provided does not meet the current guidelines because the top dose used did not result in 10-20% relative survival as required in the updated OECD TG 476, nor can it be considered as providing equivalent data according to the criteria in Annex XI, 1.1.2. of the REACH Regulation." As such, a new HPRT test (according to CLP and OECD Guideline was requested in the final decision issued on 1st of August 2016 and the adequacy of this study was changed from key to supporting.

As requested by the European chemical agency - DMAE was tested again in an in vitro gene mutation test according to OECD 476. DMAE was evaluated for the potential to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (Hprt) in Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). In the definitive mutagenicity assay concentrations of 250, 500, 750, 1000, 1333, 1666, and 2000 μg DMAE /mL for 4-hours with and without S9 were employed. 2-Dimethylaminoethanol was found to be freely soluble at all concentrations evaluated. Those cultures treated at concentrations of ≥ 1000 μg/mL without S9 were discarded prior to selection or excluded from evaluation of mutagenicity due to excessive cytotoxicity. Therefore, only 3 analyzable concentrations were available for the treatment without metabolic activation. The average relative survivals at the highest concentrations remaining and evaluated for mutagenicity were approximately 15.1 and 17.2 % with and without S9, respectively, and met the acceptance criteria of 10 - 20 % relative survival. No statistically significant or dose-dependent increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p > 0.05), and all observed values were within historical control limits. Statistically significant increases were noted in the treatment with S9 at multiple concentrations, however all increases noted were within historical range of the vehicle control.

Due to the cytotoxicity in the without S9 treatment and equivocal results of the with S9 treatment, the assay was repeated. Trial 2 of the assay did not meet acceptance criteria for a valid assay and will not be reported. Trial 3 was conducted at test article concentrations of 125, 250, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, and 2000 μg/mL with and without S9. All test article concentrations, as well as the concurrent positive and vehicle controls, were evaluated in duplicate cultures. 2-Dimethylaminoethanol was found to be freely soluble at all concentrations evaluated. Those cultures treated at concentrations of ≥ 1400 μg/mL without S9 were discarded prior to selection or excluded from evaluation of mutagenicity due to excessive cytotoxicity. The average relative survivals at the highest concentrations remaining and evaluated for mutagenicity with S9 was approximately 29.9 (limit dose of 2000 μg/mL). The treatment without S9 met the protocol acceptance criteria of 10-20 % relative survival with a value of 10.2 % (at 1200 μg/mL) relative survival compared to the vehicle control.

A statistically significant increase in mutant colonies as compared to the concurrent vehicle control, and outside the historical control range was observed at 400 and 700 μg/mL without S9. However these increases were not dose-dependent and at least four concentrations evaluated above 700 μg/mL were not significantly increased and observed values were within historical control limits. Additionally, the increase was not reproducible across the replicate treatments and is not considered biologically relevant. Taken together with the first trial, 2-Dimethylaminoethanol is considered to be negative for mutagenicity in the 4-hour treatment without S9. In the 4-hour treatment with S9, statistically significant increases were noted in mutant colonies as compared to the concurrent vehicle control and outside the historical control range at 1800 μg/mL.

However this increase was not reproducible across the replicate treatments and is not considered biologically relevant. Additionally, the 2000 μg/mL dose level is not significantly increased. Therefore, 2-Dimethylaminoethanol is considered to be negative for mutagenicity in the 4-hour treatment with S9. In all valid trials the DMN and EMS positive controls induced significant increases in mutant frequencies (p < 0.01). All positive and vehicle control values were within acceptable ranges, and all criteria for a valid assay were met.

These results indicate 2-Dimethylaminoethanol was negative in the In Vitro Mammalian Cell Gene Mutation Test (CHO/HPRT Assay) under the conditions, and according to the criteria, of the test protocol.

Genetic toxicity in vivo:

The chromosome aberration as micronucleus assay was performed equivalent to OECD guideline 474 with certificated good laboratory praxis (GLP). As test species 5 male and 5 female mouse were used, strain CD-1. Distilled water was the vehicle and one single intraperitoneal dose per mouse was injected. The post exposure period is reported as 30, 48, and 72 hours, the nominal concentration was 75 mg/kg. The bone marrow of femur was examined. The genotoxicity is stated with a negative result. Toxicity is stated to be positive, due to different abnormalities: writhing, abnormal gait, piloerection, and decreased body tone. No mortality was observed during the experiment (Pharmakon Research International, 1988).

In the second mouse micronucleus test conducted by Leung and Ballantyne (1997), there were no major gender differences in mortality responses. The LD50 (combined sexes) DMAE 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. Furthermore, no significant increases in the incidence of micronucleated polychromatic erythrocyte were observed at any sampling time. Therefore, DMAE was not considered to be inducer of micronuclei under the condition of this in vivo test.(Leung and Ballantyne, 1997).

Conclusion:

N,N-dimethylethanolamine 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.

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. (Leung and Ballantyne, 1997).

DMAE 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 (Leung and Ballantyne, 1997) nor any statistically significant increases in the incidence of mutations of CHO cells with or without an S9 metabolic activation system (Hurtado, 2020 and Leung and Ballantyne, 1997). There were no increases in the numbers of first division cells which suggests that the dose ranges used were appropriate.

Neither in the first nor in the second mouse micronucleus test genotoxicity was observed and DMAE is not considered to be an induces of micronuclei.

Justification for classification or non-classification

Due to the negative results in the available studies on genetic toxicity in vitro and in vivo, classification is not warranted according to the criteria of EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.