2-ethylaminoethanol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

February to April 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and Guideline compliant study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

May 1983/Revised Draft, March 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Principles of method if other than guideline:

Conducted according to regulatory test guidelines listed

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-ethylethanolamin

IUPAC Name:

N-ethylethanolamin

Constituent 2

Reference substance name:

2-ethylaminoethanol

EC Number:

203-797-5

EC Name:

2-ethylaminoethanol

Cas Number:

110-73-6

Molecular formula:

C4H11NO

IUPAC Name:

2-ethylaminoethanol

Test material form:

other: liquid at room temperature

Details on test material:

- Name of test material (as cited in study report):N-ethylethanolamin

- Physical state:colourless to yellowish liquid at room temperature
- Analytical purity: 99.2%
- Impurities (identity and concentrations): no data

- Purity test date: No data available
- Lot/batch No.: Abl. Nr. 03-9887. Test substance Number 96/412
- Expiration date of the lot/batch: no data

- Stability under test conditions: stability verified by re-analysis throughout the study period
- Storage condition of test material: room temperature
- Other: date of manufacture 20 February 1996. CAS no 110-73-6

Method

Target gene:

reverse mutations of several bacterial mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).
The tester strains TA 1535, TA 1537, TA 98 and TA 100 are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site.
All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity to some mutagens.
All strains also show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.

Strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation, his G 46, and are used to detect base pair substitutions. Strains TA 1537 and TA 98 are used for the detection of frameshift mutagens.
These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98.
The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

E. coli WP2 uvrA has an AT base pair at the primary reversion site and is a derivative of E. coli WP2 with a deficient excision repair. This strain is used to detect substances which induce base pair substitutions. The rate of induced back mutations from tryptophan auxotrophy (trp-) to tryptophan independence (trp+) is determined.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media: minmal agar SAI selective agar
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 fraction from livers of Sprague-Dawley rats receiveing a single intraperitoneal injection of Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1 - plate incorporation - with and without S-9; 0, 20, 100, 500, 2500 and 5000 µg/plate
Experiment 2 - pre-incubation - with and without S-9; 0, 20, 100, 500, 2500 and 5000 µg/plate
Experiment 3 - pre-incubation - with and without S-9; 0, 200, 400, 600, 800 and 1000 µg/plate. Only tester strains TA1535 and TA 100

Vehicle / solvent:

water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

dissolved in DMSO, used in all strains with S-9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine and N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

used without S-9 mix. MNNG with TA1535 and TA100; NOPD with TA98; AAC with TA1537 and ENNG with E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: iin agar (plate incorporation); preincubation for experiments 2 and 3

DURATION
- Preincubation period:20 minutes at 37°C
- Exposure duration: 48 - 72 hours at 37°C
- Expression time (cells in growth medium):48-72 hours

NUMBER OF REPLICATIONS: 3 test plates perconcentration. three experimental replicates
DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants; clearing or diminution of the background lawn; reduction in the titre

Evaluation criteria:

Mutagenicity
Individual plate counts, mean number of revertant colonies per plate and standard deviations were calculated for all dose groups and for the positive and negative controls in all experiments. Typically five doses are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the first experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the first experiment.

Toxicity
Toxicity detected by a:
- decrease in the number of revertants
- clearing or diminution of the background lawn (= reduced his or trp background growth)
- reduction in the titre

Acceptability
Generally, the experiment is to be considered valid if the following criteria are met:
The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.
* The sterility controls revealed no indication of bacterial contamination.
* The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data.
* The titer of viable bacteria was > 10E9/ml.

Evaluation criteria
The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
* The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments
carried out independently of each other.

Statistics:

not required

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A bacteriotoxic effect (slight decrease in the number of revertants, reduction in the titer) was occasionally observed in the standard plate test at 5.000 µg/ plate. In the preincubation assay bacteriotoxicity was found at doses >= 2,500 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No precipitation of the test substance was evident in any group for any of the three experiments. N-ethylethanolamin was soluble under the test conditions. Cytotoxicity was evident with bacteriotoxic effects observed under all test conditions.
An increase in the number of his + or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

ug/plate	Plate incorporation assay
	TA1535		TA100		TA1537		TA98		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
- control	20	21	122	158	12	11	33	45	32	41
20	19	20	159	170	11	11	31	36	32	43
100	19	19	154	212	12	14	22	33	33	40
500	19	21	153	187	11	13	23	40	33	40
2500	20	23	150	147	9	12	25	36	28	37
5000	14	19	149	153	10	9	14	23	28	35
+ control	1285	284	1434	1752	599	152	1075	1065	513	243

 

ug/plate	Pre-incubation assay
	TA1535		TA100		TA1537		TA98		WP2uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
- control	21	22	120	149	11	13	32	44	49	46
20	20	20	134	170	11	14	37	49	45	40
100	16	15	147	158	9	13	28	52	44	41
500	14	15	140	115	8	9	27	41	44	33
2500	-	19	-	140	2	5	18	37	17	17
5000	-	6	-	92	-	3	10	19	12	13
+ control	1848	147	925	978	381	106	712	893	1143	285

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The results of the study, under the experimental conditions chosen indicate 2-ethylethanolamine is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay.

Executive summary:

The substance 2 -ethylaminoethanol (N-Ethylethanolamin) was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay in the standard plate test and in the preincubation test with and without the addition of the S-9 mix metabolizing system obtained from rat liver. The tester strains were the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. In the standard plate incorporation test (experiment 1), there were no increases in his+ or trp+ revertants in any of the tester strains with or without S-9. In the pre-incubation tests (experiments 2 +3), there were no increases in his+ or trp+ revertants in any of the tester strains with or without S-9. A slight decrease in the number of revertants or reduction in the titre (bacteriotoxic effect) was noted in the standard plate test at the highest dose concentration, 5000 µg/plate. bacteriotoxicity was evident in the preincubation test at 2500 µg/plate. No test substance precipitation was observed. The results of the study, under the experimental conditions chosen indicate 2 -ethylaminoethanol is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay.