2-[[3-(dimethylamino)propyl]methylamino]ethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay / Ames test (OECD 471; GLP) with registration substance:

negative with and without activation in Salmonella typhimurium strains (TA 98, 100, 1535, 1537) and Escherichia coli WP2uvrA

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2013 - 13 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

His (-), Trp (-)

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: The tester strains contain 2 additional mutations: rfa wall mutation and a deletion of the uvrB gene. Strains TA 98 and TA 100 also contain the R-factor plasmid, pKM101, which increases the sensitivity of these strains to mutagens.

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA' DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Mutagenicity Assay - (experiment 2) - Pre incubation method: 50, 150, 500, 1500 and 5000 µg/plate. Concentration selection based on:
Dose Range-Finding Study - (Experiment 1) - Plate incorporation method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Sterile distilled water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

yes

Positive controls:

yes

Remarks:

Without S9-mix metabolic activation

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Further substances: 9-Aminoacridine (9AA) and 4-Nitroquinoline-l-oxide (4NQO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

yes

Positive controls:

yes

Remarks:

with S9-mix metabolic activation

Positive control substance:

benzo(a)pyrene

Remarks:

Further substance: 2-Aminoanthracene (2 A A)

Details on test system and experimental conditions:

Test for Mutagenicity (Experiment 1) - Plate Incorporation Method

Dose selection
The test item was tested using the following method. The maximum concentration was 5000 pg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, vehicle or appropriate positive control was added to 2 mL of trace amino-acid supplemented media (at approximately 45 °C) containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A single plate was manually counted for accuracy.


Test for Mutagenicity (Experiment 2) - Pre-incubation Method

As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 50 to 5000 µg/plate.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 °C± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 °Ci: 3 °C for 20 minutes (with shaking) and addition of amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).


NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.9 to 9 x 109 bacteria per mL
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours

Rationale for test conditions:

As indicated in the guidelines. In addition, precipitation/ solubility and cytotoxicity were tested.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon et al, 1989). However, statistical analysis was not needed due to the absence of an increase in the number of revertant colonies at any dose level beyond the positive control.

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

TA1537, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

test substance was considered to be non-mutagenic under the conditions of this test

Executive summary:

A bacterial reverse mutation assay / Ames test (OECD 471; GLP) was conducted with the substance 2-[[3-(dimethylamino)propyl]methylamino]ethanol.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 pg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 50 to 5000 µg/plate.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. In the second experiment (pre-incubation method) the test item induced a slight weakening of the bacterial background lawns of TA100, TA98, TA1537 and WP2uvrA in the absence of metabolic activation only at 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, in the presence of metabolic activation. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 2 (pre-incubation method).

Based on the study results, the test substance is considered not mutagenic under the study test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available in vitro data, the substance 2-[[3-(dimethylamino)propyl]methylamino]ethanol is not genotoxic and does not require classification according to Regulation 1272/2008/EC.