2,4,6-tris(dimethylaminomethyl)phenol

Genetic toxicity: in vitro

Administrative data

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:

2010-07-09 to 2010-10-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-tris(dimethylaminomethyl)phenol

EC Number:

202-013-9

EC Name:

2,4,6-tris(dimethylaminomethyl)phenol

Cas Number:

90-72-2

Molecular formula:

C15H27N3O

IUPAC Name:

2,4,6-tris[(dimethylamino)methyl]phenol

Specific details on test material used for the study:

The test material was accurately weighed, formulated in RO medium and serial dilutions prepared.

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr J Cole of the
MRC Cell Mutation Unit at the University of Sussex, Brighton, UK

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Experiment 1: 4 hours exposure, +/- S9: 0, 250, 500, 750, 1000, 1250, 1500, 1750,2000 µg/ml
Experiment 2: 4 hours exposure with S9: 0, 50,100, 200, 300, 400, 500, 600, 700 µg/ml
24 hours without S9: 0, 125, 250, 500, 750, 1000, 1250, 1500, 1750 µg/ml
There was no marked increase in osmolality greater than 50 mOsm when the test material was dosed into media in the solubility test. However, a marked increase in pH greater than 1 pH unit was observed at and above 2500 μg/ml. Therefore, the maximum dose level was reduced to 2000 μg/ml in the subsequent preliminary toxicity test (Scott et al 1991 ).

Vehicle / solvent:

RO medium

Controls

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Mouse lymphoma assay
METHOD OF APPLICATION:
- Metabolic activation system: male Sprague Dawley rats are induced with phenobarbital/ β-naphthoflavone
- Dosing:   
Experiment 1: 4 hours exposure, +/- S9: 0, 250, 500, 750, 1000, 1250, 1500, 1750,2000 µg/mL
Experiment 2: 4 hours exposure with S9: 0, 50,100, 200, 300, 400, 500, 600, 700 µg/mL
24 hours without S9: 0, 125, 250, 500, 750, 1000, 1250, 1500, 1750 µg/mL
- Solvent: RO medium
- Controls
Positive without metabolic activation:
- Ethylmethanesulfonate (EMS) at 400 µg/mL and 150 µg/mL
Positive with metabolic activation:
- Cyclophosphamide (CP) 2 µg/mL

DURATION
- Exposure duration:
Experiment 1: 4 hours exposure, +/- S9
Experiment 2: 4 hours exposure with S9
24 hours without S9
Expression time (cells in growth medium): 2 days in total at 37 °C in 5% CO2/95% humidified air, the cell density was determined each day and adjusted to 2 x 10E5 cells/mL.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. +
Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value.
Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation at 37°C with 5% C02 in air.
The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate).
The numbers of small and large colonies seen in the TFT mutation plates were also recorded.
Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately % to % of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates.
The plates were incubated for approximately two to four hours.
MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

- Cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated
for at least 6 days at 37 °C in a humidified atmosphere with 5% CO2


SELECTION AGENT (mutation assays): selected for 5-trifluoro-thymidine (TFT) resistance

Rationale for test conditions:

The dose range of test material was selected following the results of a preliminary toxicity test and for the first experiment was 250 to 2000 μg/ml in both the absence and presence of metabolic activation. For the second experiment the dose range was 50 to 700 μg/ml in the absence of metabolic activation, and 125 to 1750 μg/ml in the presence of metabolic activation.

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-200 x 10-6 for the TK+/- locus in L5178Y cells at this laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10-6 mutant frequency per survivor are not normally acceptable and will be repeated.

Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance.

Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Statistics:

According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 1 o-6 viable cells.
Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.
The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Applicant's summary and conclusion

Conclusions:

negative with and without metabolic activation. Test item is not mutagenic.

Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-,

locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476) and Method 817 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Methods.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at up to eight dose levels using a 4-hour exposure group in the presence of metabolic activation (1 % S9) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test and for the first experiment was 250 to 2000 μg/ml in both the absence and presence of metabolic activation. For the second experiment the dose range was 50 to 700 μg/ml in the absence of metabolic activation, and 125 to 1750 μg/ml in the presence of metabolic activation. Results. The maximum dose level used in the mutagenicity test was limited by a combination of an excessive increase in pH at dose levels higher than 2000 μg/ml and test material-induced toxicity. Precipitate of test material was not observed at any of the dose levels in the mutagenicity test. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

 

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion:

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.