2,2-bis(hydroxymethyl)propionic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 April 1998 to 07 October 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC protocol: "Gene Mutation Test - Mammalian cells In vitro", Directive 87/302/EEC, adopted 18 November 1987

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis(hydroxymethyl)propionic acid

EC Number:

225-306-3

EC Name:

2,2-bis(hydroxymethyl)propionic acid

Cas Number:

4767-03-7

Molecular formula:

C5H10O4

IUPAC Name:

3-hydroxy-2-(hydroxymethyl)-2-methylpropanoic acid

Specific details on test material used for the study:

- Name of the test material used in the study report: 2,2-dimethylol propionic acid (DMPA)
- Batch no.: DO374
- Purity: According to report of analysis, impurities include moisture 0.13%, ash as sodium oxide 0.008%
- Appearance: free flowing granular solid, off-white
- Expiry date: not provided
- Storage conditions: ambient

Method

Target gene:

The TK-locus on chromosome 11 of cultured mouse lymphoma L5178Y cells.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The cells (L5178Y tk+/- 3.2.7.c line) were obtained from Dr. Cole, MRC Cell Mutation Unit, University of Sussex, UK. The chromosome number is 40 (stable aneuploid karyotype, 2n = 40). The cells were stored as frozen stock cultures in liquid nitrogen. Subcultures were prepared from these stocks, each new stock culture was checked for mycoplasma contamination, which was found to be absent.
Type and identity of media: RPMI 1640 medium (with HEPES and L-Glutamine) supplemented with heat-inactivated horse serum (10% v/v for growing in flasks and 20% for growing in microtiter plates), sodium pyruvate and penicillin / streptomycin.

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix obtained from the livers of Aroclor 1254-induced male Wistar rats

Test concentrations with justification for top dose:

First test nominal concentrations: 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 4.2, 5.6, 7.5, and 10 mM.
Second test nominal concentrations: 0.625, 1.25, 2.5, 5, and 10 mM.

Vehicle / solvent:

Just before use, the test substance was dissolved in growth medium (without horse serum). From this stock solution serial dilutions were prepared. The pH was measured of the medium alone, and of test concentrations (in medium) 5.6, 7.5 and 10 mM and found to be 7.45, 7.25, 7.19 and 7.14, respectively. The slight lowering of the pH with increasing concentration was not thought to have influenced the results as the pH did not drop below 7.0.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

growth medium without horse serum

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Methylmethanesulfonate (MMS) used in the absence of S9-mix (0.2 mM)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

3-methylcholanthrene used in the presence of the S9-mix (10 µg/mL)

Details on test system and experimental conditions:

The cells were cultured in a humidified incubator at 37°C in air containing 5% CO2. Five to seven days prior to treatment, the cells were generated from a frozen stock culture by seeding them in sterile, screw-capped tissue culture flasks (about 10,000,000 cells/flask: growth area ± 75 cm²) containing 50 mL growth medium (with 10% horse serum). Fresh cultures of L5178Y cells were harvested from a number of culture flasks and suspended in growth medium (with 10% horse serum), and the number of cells were counted. For the cytotoxicity and gene mutation tests portions of 5,000,000 cells were used per culture. On the day of exposure, the growth rate (doubling time of 9-14 h) and viability (>90%; by tryptophan blue exclusion) of the cells were checked, and found to be within acceptable limits.

Cell treatment: Test substance, negative or positive control, growth medium (without horse serum), and 10% (v/v) S9 mix where appropriate, were added to 5,000,000 cells in 5 mL growth medium (with 10% horse serum) to a final volume of 10 mL. Two cultures treated with the vehicle were used as negative controls. One culture was used for each concentration of the test substance and for the positive control. The cells were exposed for 4 hours at 37 °C and 5% CO2 in a humidified incubator. At the end of treatment, all cell cultures were checked visually and selected cultures were checked for viability.

The cytotoxicity of the test substance was determined by counting (Coulter counter) the cells (initial cell yield) and by measuring the cloning efficiency. The medium containing the test substance (or controls) was removed and the cells were washed twice with growth medium (with 10% horse serum). Subsequently, the cells were resuspended in growth medium (with 20% horse serum) and the number of cells were counted. A portion of the cells was diluted to 10 cells/mL in growth medium with 20% serum for determining the initial cloning frequency. 200 µL portions of each dilution were transferred to each well of two 96-well microtiter plates, and the plates were incubated for 10-14 days at 37 °C and 5% CO2 in a humidified incubator.

Two assays were used to evaluated the mutagenic potential of the test substance. The remaining cells of the cytotoxicity tests were incubated for about 44 h at 37 °C and 5% CO2 in a humidified incubator to allow near-optimal phenotypic expression of induced mutations. After about 20 h the cells were counted and the cultures were diluted, if required, to 200,000 cells/mL. The frequency of mutants and the final cloning efficiency of the cells were determined 2 days after starting the test. To determine the frequency of mutants, the cell suspensions were diluted to a density of 10,000 cells/mL in growth medium (with 20% horse serum) containing 4 µg TFT/mL. 200 µl portions of each dilution were transferred to each well of two 96-well microtiter plates, and the plates were incubated for 10-14 days at 37 °C and 5% CO2 in a humidified incubator. After this period the number of wells without growth of cells were counted and the cloning efficiency in the plates were calculated. The TK mutant frequency per 1,000,000 cloneable cells were calculated.

The mutant colonies of the negative and positive controls were scored using the criteria of small and large colonies.

Evaluation criteria:

Genotoxicity of the test substance was evaluated using the following criteria (Aaron et al. 1994; Clive et aI., 1995; OECD 1995):
a) a concentration-related increase in mutant frequency,
b) a reproducible positive response for at least one of the test substance concentrations (e.g, the induced mutant frequency [mutant frequency of
test substance minus that of the vehicle negative control] should be more than 100 mutants per 1,000,000 clonable cells),
c) both numerical significance and biological relevance were be considered together in the evaluation.

Statistics:

None reported.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

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:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

At concentrations of 4.2 mM and above, there was a dose-related change of the red colour of the medium to yellow, due to pH lowering. The viability was checked at the end of treatment for the higher concentrations; in test 1 without S9 the cells treated with 7.5 and 10 mM had viabilities of 98% and 95% respectively. In the second test without S9 the cells treated with 10 mM had 98% viability. In the first experiment with S9 the cells treated with 7.5 and 10 mM had viabilities of 98% and 78%, respectively. In the second test with S9 the cells treated with 10 mM had 96% viability.

The test substance was not toxic to the cells in both the absence and presence of S9, up to and including the limit dose of 10 mM.
In both the presene and absence of S9 mix, the test substance did not induce a significant increase in mutant frequency at any dose level.

The positive control substances yielded the expected significant increase in mutant frequency compared to the negative controls, both in the presence and absence of S9.

Applicant's summary and conclusion

Conclusions:

It was concluded that under the conditions used in this study the test substance 2,2-dimethylol propionic acid (DMPA) was not mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

Executive summary:

In a mammalian cell gene mutation assay (conducted according to OECD TG 476), TK-locus of L5178Y cells cultured in vitro were exposed to 2,2-dimethylol propionic acid (DMPA) in growth medium (without horse serum) at concentrations of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 4.2, 5.6, 7.5, and 10 mM (first test nominal concentrations) and 0.625, 1.25, 2.5, 5, and 10 mM (second test nominal concentrations) in the presence and absence of mammalian metabolic activation (S9 mix obtained from the livers of Aroclor 1254-induced male Wistar rats). The test substance was not cytotoxic to the cells in both the absence and presence of S9, up to and including the limit dose of 10 mM.
In both the presene and absence of S9 mix, the test substance did not induce a significant increase in mutant frequency at any dose level. The positive control substances yielded the expected significant increase in mutant frequency compared to the negative controls, both in the presence and absence of S9. The test substance was therefore concluded not to be mutagenic at the TK-locus of mouse lymphoma L5178Y cells. This study is classified as acceptable as it was performed according to GLP and OECD 476 and to EC and US EPA Test Guidelines. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity data.