N-[2-[2-(2-methoxyethoxy)ethoxy]ethyl]-2-methyl-propan-2-amine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: negative (+/- S9), (BASF, 2018)

- in-vitro CA : negative (+/- S9), (Exxon, 1983)

- in vitro ML : negative (+/- S9), Exxon (1988)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according 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, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from induced rats

Test concentrations with justification for top dose:

DOSE RANGE:
33 μg - 5000 μg/plate (SPT)
33 μg - 5000 μg/plate (PIT)

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other:

Details on test system and experimental conditions:

DURATION
- Exposure duration: 48 - 72 hours at 37 °C

Evaluation criteria:

Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups

Statistics:

Not applicable

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Conclusions:

According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria.

Executive summary:

Under the experimental conditions chosen here, it is concluded that 2-Propanamine, N-[2-[2 - (2-methoxyethoxy)ethoxy]ethyl]-2-methyl- is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 14, 1983 to December 30, 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary, line K1, ATCC CEL G1

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver (S9 mix)

Test concentrations with justification for top dose:

1.82 μg/ml, 18.2 μg/ml, 182 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 24 hours
- Exposure duration: 7.5 hours and 24 hours.
- Expression time (cells in growth medium): 24 hours
- Selection time (if incubation with a selection agent): Not applicable.
- Fixation time (start of exposure up to fixation or harvest of cells): 2 hours prior to harvest


SELECTION AGENT (mutation assays): Not applicable.
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa stain


NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: Fifty metaphase spreads from each flask were evaluated.


DETERMINATION OF CYTOTOXICITY
- Method: Metaphase spreads were evaluated for the presence of any and all structural abnormalities.

Evaluation criteria:

Levels of damage that were above the accepted spontaneous damage level for this system would be considered a positive result.

Statistics:

For each treatment and control group the total and mean values for each type of aberration were calculated. Mean values, with standard deviation, were calculated for numbers of abnormal cells and numbers of abnormalities observed.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A toxicity test was performed with and without S9 activation, to determine which doses to use. An LD50 was observed at 250 μg/ml, and an LD0 was observed at 50 μg/ml. The LD30 was then calculated. A high dose of 182 μg/ml was used with and without activation. The intermediate and low doses were one tenth and one hundredth serial dilutions of the top dose (18.2 and 1.82 μg/ml)




COMPARISON WITH HISTORICAL CONTROL DATA: Historically, the spontaneous damage levels for non-treated or vehicle controls are 0.0% - 8.0%. All vehicle controls for this study fall within accepted values (2.00%-5.34%).


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Test substance: All three dose levels, at both time periods, with and without metabolic activation showed types of damage and damage levels comparable to the vehicle and non-treated controls (2.00%-6.00%). These values fall well within historical spontaneous damage levels, observed in this laboratory.

Cyclophosphamide: The positive controls showed damage levels of 21.00%.

Nontreated control plus activation: The nontreated controls showed damage levels of 5.00%.

Nontreated control w/o activation: The nontreated controls showed damage levels of 2.00%

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative

The test material, was examined for its ability to cause chromosomal mutations in CHinese Hamster Ovary cells when dosed at 182, 18.2 and 1.82 μg/ml with and without metabolic activation for 7.7 and 24 hours. The levels of damage observed were comparable to the controls and are well within the historical control data, or no effect, values.

Within the framework of the test system, the test substance appears to be incapable of cause cytogenetic damage to CHO cells.

Executive summary:

The cytogenetic activity of the test substance was evaluated in Chinese Hamster Ovary (CHO) cells, grown in vitro. The test material was administered in the culture medium at 182, 18.2 and 1.82  μg/ml with and without S9 activation. Test and control flasks were divided into two groups, harvested at about 7.5 and 24 hours following a one hour dosing period. Approximately two hours prior to harvest all flasks received 1  μg/ml of colcemid, to induce metaphase arrest in the cells. The metaphase cells were harvested and prepared for cytogenetic evaluation. Fifty metaphase spreads from each flask were evaluated, under code to avoid bias, for the presence of any and all structural abnormalities.

The cytogenetic evaulation revealed no levels of damage that were above the accepted spontaneous damage level for the system (less than 8.00%). The positive controls, however, showed a marked increase in chromosomal abnormalities (21.00%).

Within the framework of this test system the test substance was not found to cause increased chromosomal damage.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 88 to 06 June 88

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK +/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Culture preparation: L5178Y TK +/- Mouse Lymphoma Cells were treated with methotrexate, thymidine, glycine and hypoxanthine prior to freezing to eliminate possible spontaneous TK -/- mutants. As needed, cells were then removed from liquid nitrogen storage, cultured and maintained in logarithmic phase for five days before use in the assay system.

Culture medium - F10p
Cloning medium - F20p

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver (S9 mix)

Test concentrations with justification for top dose:

Toxicity assay: Doses ranged from 1 to 1000 μg/ml.
Mutagenesis assay: Based on the results of the toxicity assay, six doses were used in the mutagenesis assay; 500, 700, 900, 1100, 1300 and 1500 μg/ml, but only five doses were plated. Because all doses had greater than 10% relative suspension growth, the lowest dose was eliminated; and 700, 900, 1100, 1300 and 1500 μg/ml were treated with and without activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle:Considered under the conditions of the assay to be stable for the duration of the assay.

The test material and positive controls were diluted and dosed prior to administration.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hour and 20 minute exposure
- Expression time (cells in growth medium): 2-day expression period


SELECTION AGENT (mutation assays): TFT = Trifluorothymidine Deoxyriboside


NUMBER OF REPLICATIONS: 3 plates per dose group.


NUMBER OF CELLS EVALUATED:
Approximately 9XE5 cells were plated in 30ml F20p with 2.5 μg/ml TFT. Concurrently, approximately 180 cells were plated in 30 ml F20p without a selective agent, to determine viable colony counts (VC).


DETERMINATION OF CYTOTOXICITY
- Method: Relative suspension growths, relative cloning growths, relative total growths, mutant frequencies amd induced mutant frequencies were calculated.

Plates were labelled with study number, compound identification number, the dose administered, VC or TFT, +S9 or -S9. Colonies were enumerated after eleven days incubation at 36-38 deg C with 4.5-5% CO2. Plate counts were made with a Biotran III colony counter and recorded.

Evaluation criteria:

A test material is considered positive if it yields a 2-fold increase over the spontaneous mutant frequency of vehicle controls at one or more dose levels, if a dose related increase in response is observed and if the relative total growth is greater than 10%. Should the relative total growth, (survival) be less than 10% and a two-fold increase in mutant frequency observed, the test material will be considered negative.

Statistics:

The following equations were used in the data analysis of the forward mutation assay:
Relative Suspension Growth, % =
Total suspension growth of test culture / mean total suspension growth of vehicle controls x 100%

Relative cloning growth, % =
Mean number of VC Colonies on “X” treated plates / Average of mean of VC colonies on vehicle control plates x 100%

Relative total growth, % =
(Relative suspension growth, %) (Relative cloning growth, %) / 100%

Mutant Frequency (mutants / 10E4) =
Mean number of TFT colonies for “X” / Mean number of VC colonies for “X” x 2

Induced Mutant Frequency (IMF) = Mutant frequency of treated cultures - mean mutant frequency of vehicle control cultures

VC = Viable colonies
"X" = Dose being evaluated

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Dose selection for the repeat mutation assay* was based on a toxicity (dose determination) assay. The doses selected were 700, 900, 1100, 1300 and 1500 μg/ml. These were tested with and without metabolic activation.

*The initial mutation assay was repeated because plate count values were out of range.

Please see attached background material for Table 1 - Summary of mouse lymphoma mutagenicity data, and Table 2 - Mouse growth results with and without metabloic activation.

A mutant frequency greater than 2(X) the mean control mutant frequency was not observed at any dose. The positive (DMBA and EMS) and negative (Controls 1 and 2, + and - S9) controls responded in a manner consistant with data from previos analysis.

Remarks on result:

other: strain/cell type: L5178Y cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information): negative

A mutant frequency that was greater than two times the mean vehicle control frequency was not observed at any dose level, nor was there any evidence of a dose related response.
For this reason, it can be concluded that, under the conditions of this assay, the test substance does not induce gene mutations in L5178Y mouse lymphoma cells.

Executive summary:

The mutagenic activity of the test substance was tested in the L5178Y mouse lymphoma assay. The positive controls were dimethylbenzanthracene (DMBA) and ethylmethanesulphonate (EMS). The substance was tested at 700, 900, 1100, 1300, 1500 μg/ml with and without metabolic activation. Following a three hour and 20 minute exposure period, test and control cultures were allowed a two day expression period. Cultures were cloned in selective medium, then incubated for eleven days. Surviving and mutant colonies were then counted and the mutant frequencies calculated.

There was no evidence of a dose related increase in mutagenic frequency, and the mutant frequencies were not significantly different from control at any dose. For this reason, it can be concluded that, under the conditions of this assay, the test substance does not induce gene mutations in L5178Y mouse lymphoma cells.

The positive (DMBA, EMS) and negative (DMSO) controls responded in a manner consistant with data from previous studies.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test:

- Under the experimental conditions of this study, the test substance 2-Propanamine, N-[2-[2 - (2-methoxyethoxy)ethoxy]ethyl]-2-methyl- is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Read-across:

Chromosome abberation

The cytogenetic activity of the read across substance was evaluated in Chinese Hamster Ovary (CHO) cells, grown in vitro. The test material was administered in the culture medium at 182, 18.2 and 1.82  μg/ml with and without S9 activation. Test and control flasks were divided into two groups, harvested at about 7.5 and 24 hours following a one hour dosing period. Approximately two hours prior to harvest all flasks received 1  μg/ml of colcemid, to induce metaphase arrest in the cells. The metaphase cells were harvested and prepared for cytogenetic evaluation. Fifty metaphase spreads from each flask were evaluated, under code to avoid bias, for the presence of any and all structural abnormalities.

The cytogenetic evaulation revealed no levels of damage that were above the accepted spontaneous damage level for the system (less than 8.00%). The positive controls, however, showed a marked increase in chromosomal abnormalities (21.00%).

Within the framework of this test system the read across substance was not found to cause increased chromosal damage.

Mouse lymphoma

The mutagenic activity of the read across substance was tested in the L5178Y mouse lymphoma assay. The positive controls were dimethylbenzanthracene (DMBA) and ethylmethanesulphonate (EMS). The substance was tested at 700, 900, 1100, 1300, 1500 μg/ml with and without metabolic activation. Following a three hour and 20 minute exposure period, test and control cultures were allowed a two day expression period. Cultures were cloned in selective medium, then incubated for eleven days. Surviving and mutant colonies were then counted and the mutant frequencies calculated.

There was no evidence of a dose related increase in mutagenic frequency, and the mutant frequencies were not significantly different from control at any dose. For this reason, it can be concluded that, under the conditions of this assay, the test substance does not induce gene mutations in L5178Y mouse lymphoma cells.

The positive (DMBA, EMS) and negative (DMSO) controls responded in a manner consistant with data from previous studies.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Negative results were obtained with an Ames test of the registered substance as well as with an chromosome abberation and mouse lymphoma test with the read-across substance.

Therefore no classification for mutagenicity is required according to Regulation (EC) No. 1272/2008.