2,2-bis(hydroxymethyl)propionic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Four in vitro studies of genotoxicity are available and a weight of evidence approach is taken for this endpoint. No adverse effects are observed and all the four studies report negative results. Therefore, Bis-MPA (dimethylol propionic acid) was not mutagenic or clastogenic when tested in vitro in a bacterial reverse mutation assay, a mammalian cell gene mutation assay, and a mammalian chromosome aberration test.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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

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

Target gene:

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

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.

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)

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.

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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, moisture % 0.13, ash as sodium oxide % 0.008
- Appearance: free flowing granular solid, off-white
- Expiry date: not provided
- Storage conditions: ambient

Target gene:

Not applicable - chromosome aberration study.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO cells (CHO K-1 line) were obtained from Dr. Natarajan, University of Leiden, The Netherlands. The cell cycle is 12-14 h. The cells were stored as frozen stock cultures in liquid nitrogen. Subcultures were prepared from these stocks (passage 14) for experimental use. Each passage CHO cells in the liquid nitrogen is checked for mycoplasma contamination and karyotype stability, which were absent and stable, respectively.
Culture medium: Ham's F-12 medium (with Glutamax-I), supplemented with heat-inactivated (30 min, 56°C) foetal calf serum (10%), penicillin (100 IU/ml medium) and streptomycin (100 µg/ml medium).

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:

0 (negative control), 1, 5, 10, 25, 50, 100, 200, 300, 400, 500, 750, 1000, 1250 µg/mL.

Vehicle / solvent:

The test substance was dissolved in Ham's F12 medium at 50 mg/ml resulting in a clear solution. Serial dilutions (ranging from 50 to 1.56 mg/ml) were prepared in Ham's F12 medium, and 500 µl of each of these stock dilutions was added to 4.5 ml Ham's F12 medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Used in the presence of S9-mix (3.75 µg/mL)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Used in the absence of S9-mix (0.025 µg/mL)

Details on test system and experimental conditions:

Exponentially growing cells were seeded in sterile, screw-capped tissue culture flasks (surface area 75 cm², 400,000 cells per flask) containing 10 ml culture medium and then incubated at 37°C in humidified air containing 5% CO2. On the next day, the cells were exposed to the test substance, in both the presence and absence of S9. Duplicate cultures were used for test substance concentrations and positive and negative controls. In the absence of S9 the treatment time was 18 h; in the presence of S9 mix the treatment time was 3 h. In both cases the fixation time was 18 h after onset of treatment.

First chromosome aberration assay:
In the absence of S9 mix, 1.0 ml of each dilution of the test substance in Ham's F12 medium was added directly to 9.0 ml freshly added tissue culture medium in the flasks and the culture medium was checked visually. Thereafter, the cultures were incubated at 37°C in humidified air containing 5% CO2 and continuously treated for 18 h. Two hours before the end of the treatment period the cells and culture medium were checked visually.
In the presence of S9 mix, the culture medium was replaced by 8.0 ml Ham's F12 medium with penicillin and streptomycin but without serum. Toe each culture, 1.0 ml of each of the dilutions in Ham's F12 medium was then added to the cell cultures. Thereafter, 1.0 ml of S9 mix was added. The culture medium was checked visually for insoluble test substance. After a 3 h incubation at 37°C in humidified air containing 5% CO2, the cells and culture medium were checked again. Thereafter the medium with the test substance was removed, the cells were washed twice with phosphate buffered saline (pH 7.4) and supplied with 10 ml freshly prepared culture medium. The cells were then incubated for an additional 15 h at 37°C in humidified air containing 5% CO2.

Second chromosome aberration assay:
The second independent assay was carried as above. In the absence of S9 mix the treatment/fixation times were: 18/18 h and 32/32 h. In the presence of S9 mix the treatment/fixation times were: 3/18 h and 3/32 h.

Cell fixation and slide preparation and scoring:
Two hours before the end of the total incubation period, the cells of the remaining cultures were arrested in the metaphase stage of mitosis by the addition of colcemid (final concentration: 0.1 mM medium). At the end of the total incubation period the cells were harvested by trypsinisation, treated for 15 min at 37°C with a hypotonic solution (1% sodium citrate), fixed with a 3:1 mixture of methanol:glacial acetic acid (two refreshments of the fixative), and transferred to clean microscope slides. Two slides were prepared per culture. The slides were stained in 2% Giemsa, rinsed in water, dried and embedded with a Tissue-TEK Coverslipper. The slides were coded by a qualified person not involved in scoring the slides, to enable blind scoring. At least 1000 nuclei in each culture were examined (500/slide) to detemine the mitotic index. After the results of the mitotic index scoring and observations with respect to the quality of the metaphases obtained, a selection for test concentrations was made. At least 3 concentrations of the test substance together with the negative and positive controls were selected for analysis of chromosomal aberrations; the highest concentration should reduce the mitotic index by 50% compared to the negative control, and the lowest concentration should be borderline of mitotic inhibition. The scoring of the slides for endoreduplication was conducted separately to that for structural chromosomal aberrations.
For each selected treatment, 200 well spread metaphases (100/culture) each containing 20-22 centromeres, were analysed by microscopic examination for chromatid-type aberrations (gaps, breaks, fragments, interchanges), chromosome-type aberrations (gaps, breaks, minutes, rings, dicentrics) and other anomalies, such as intersitial deletions, endoreduplication, polyploidy and multiple aberrations (> 10 aberrations per cell, excluding gaps).

Evaluation criteria:

The main criteria for a positive response are a clear dose-related increase in the percentage of cells with structural aberrations over the concurrent control frequencies (in duplicate cultures}, or a reproducible single positive dose (in duplicate cultures).
A test substance is considered to be negative in the chromosome aberration test if it produces neither a dose-related increase in the number of structural chromosomal aberrations nor a reproducible positive response at any of the test points.
Gaps (achromatic lesions) are recorded separately and not included in the final assessment of clastogenic activity.
Both statistical significance and biological relevance are considered together in the evaluation of the results.

Statistics:

Data were analysed statistically by the Fisher's exact probability test (two-sided) to determine significant differences between treated and control
cultures.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1250 µg/ml without S9; and at 1200 µg/ml with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There were no reproducible, biologically relevant and statistically significant increases in the number of cells with chromosome aberrations, both in the absence and presence of metabolic activation, compared to control values. An increase in the number of cells with endoreduplication was observed at 700 µg/mL and higher in tests in the presence of S9-mix at the 18h harvest time. Endoreduplications are not structural aberrations; they are recorded separately and not recorded in the final assessment of clastogenic activity. The positive control substances gave the expected statistically significant increases in the number with aberrations.

The test substance was not clastogenic under the conditions of this study.

Conclusions:

From the findings of two independent chromosome aberration assays it is concluded that 2,2-dimethylol propionic acid (DMPA) was not clastogenic under the conditions used in the study.

Executive summary:

In a mammalian chromosome aberration assay (conducted according to OECD TG 473), CHO cell cultures were exposed to 2,2-dimethylol propionic acid (DMPA) in Ham's F12 medium, at concentrations of 0 (negative control), 1, 5, 10, 25, 50, 100, 200, 300, 400, 500, 750, 1000, 1250 µg/mL with and/or without metabolic activation. Two independent chromosome aberration assays were conducted and in the first assay, the treatment/fixation time was 18/18 h (-S9) and 3/18 h (+S9) and in the second assay were 18/18 and 32/32 h (-S9) and 3/18 and 3/32 h (+S9). Positive controls induced the appropriate response. There were no reproducible, biologically relevant and statistically significant increase in the number of cells with chromosome aberrations, both in the absence and presence of metabolic activation, compared to control values. Therefore, under the conditions used in the test, 2,2 -dimethylol propionic acid (DMPA) was found to be non-clastogenic. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 and EC B10 Test Guidelines.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2015 to 03 December 2015

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of the test material used in the study report: Bis-MPA
- Batch no.: 551124
- Purity: 98.44% (provided in CoA)
- Appearance: Not provided
- Expiry date: 31 July 2018
- Storage conditions: Room temperature

Target gene:

Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by A:T base pair substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other:

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix obtained from the livers of Wistar rats

Test concentrations with justification for top dose:

5000, 2500, 1250, 625 and 313 μg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

10 μg/plate, positive control in the presence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

500 μg/plate, positive control in the absence of S9

Details on test system and experimental conditions:

Two independent mutagenicity assays were performed. A preliminary toxicity test was conducted. Permanent stocks of E. coli WP2 uvr A are kept at -80°C in RTC. Overnight subcultures of this stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
Tryptophan requirement - No Growth on Minimal agar plates. Growth on Minimal plates+Tryptophan.
uvrA - Sensitivity to UV irradiation.
pKM101 - Resistance to Ampicillin.

Evaluation criteria:

The assay was considered valid if the following criteria were met:

(i) Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
(ii) The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
(iii) No more than 5% of the plates should be lost through contamination or other unforeseen event.

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

No statistical analysis was performed; not required for this study type.

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:

not applicable

Positive controls validity:

valid

Table 1: Results of Main assay I (plate incorporation method) and tester strain WP2 uvrA

Concentration [µg/pl]	No. of revertants per plate**	No. of revertants per plate**	Cytotoxicity (yes/no)	Precipitates (yes/no)
	— MA	+ MA	no	no
Untreated	30 ± 2.8	37 ± 1.2	no	no
0.00	27 ± 1.5	34 ± 1.8	no	no
313	31 ± 2.9	33 ± 0.3	no	no
625	30 ± 2.6	41 ± 2.0	no	no
1250	32 ± 3.5	35 ± 1.0	no	no
2500	27 ± 3.8	36 ± 0.9	no	no
5000	28 ± 0.3	40 ± 1.0	no	no
Negative control*	30 ± 2.8	34 ± 1.8	no	no
Positive control#	157 ± 1.2	143 ± 5.3	no	no

*solvent/vehicle control with untreated (-S9) or DMSO (+S9)

# MMS (-S9) or 2-Aminoanthracene (+S9)

** mean of three plates

 

Table 2: Results of Main assay II (pre-incubation method) and tester strain WP2 uvrA

Concentration [µg/pl]	No. of revertants per plate**	No. of revertants per plate**	Cytotoxicity (yes/no)	Precipitates (yes/no)
	— MA	+ MA	no	no
Untreated	29 ± 3.8	33 ± 2.6	no	no
0.00	29 ± 0.9	32 ± 2.3	no	no
313	28 ± 1.2	37 ± 1.0	no	no
625	28 ± 0.7	35 ± 0.3	no	no
1250	27 ± 1.2	33 ± 2.1	no	no
2500	29 ± 1.5	35 ± 1.2	no	no
5000	30 ± 2.6	36 ± 2.1	no	no
Negative control*	29 ± 3.8	32 ± 2.3	no	no
Positive control#	138 ± 3.7	138 ± 1.2	no	no

*solvent/vehicle control with untreated (-S9) or DMSO (+S9)

# MMS (-S9) or 2-Aminoanthracene (+S9)

** mean of three plates

Conclusions:

It is concluded that the test item Bis-MPA does not induce reverse mutation in Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

In a reverse gene mutation assay in bacteria (conducted according to OECD TG 471), strain WP2uvrA of E. coli were exposed to Bis-MPA in DMSO at concentrations of 5000, 2500, 1250, 625 and 313 μg/plate in the presence and absence of mammalian metabolic activation using liver S9 fraction from rats pre-treated with Phenobarbital and 5-6 Benzoflavone (plate incorporation and pre-incubation).
In the toxicity test, the test item was tested up to limit concentration 5000 μg/plate, and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. No toxicity was observed at any dose level, in the absence or presence of S9 metabolism. On the basis of toxicity test results, in main Assay I, using the plate incorporation method, the test item was assayed at 5000, 2500, 1250, 625 and 313 μg/plate. At the end of the incubation period, no precipitation of the test item nor toxicity was noticed at any dose level, in the absence or presence of S9 metabolic activation. In the main Assay II, a pre-incubation step was included and the test item was assayed at 5000, 2500, 1250, 625 and 313 μg/plate. No toxic effect was observed with the tester strain at any dose level, in the absence or presence of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period at any concentration tested. The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in the absence or presence of S9 metabolism. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background and it is concluded that the test item Bis-MPA does not induce reverse mutation in Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. The strains TA1535, TA1537, TA98 and TA100 of S. typhimurium were also investigated in an another study, therefore this study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 April 1998 to 06 August 1998

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

Target gene:

TA98: His D3052, TA100: His G46, TA1535: His G46, TA1537: His C3076.

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix obtained from the livers of male Wistar rats injected i.p. with Arocolor 1254

Test concentrations with justification for top dose:

Nominal concentrations: 0, 62, 185, 556, 1667, 5000 µg/plate

Vehicle / solvent:

Water: Just before use, a stock solution of the test substance was prepared in water at 50000 µg/ml, resulting in a clear solution. Thereafter, the stock solution was sterilised by passage through a micorpore filter and serial dilutions were prepared in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

1.0 µg/plate; positive control for strains TA1535 and TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate; positive control for strain TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

2.0 µg/plate; positive control for strain TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2.0 µg/plate; positive control for strains TA1535, TA98, and TA100 in the presence of S9-mix.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

4.0 µg/plate; positive control for strain TA1537 in the presence of S9-mix.

Details on test system and experimental conditions:

Two independent mutagenicity assays were performed. A preliminary toxicity test was not conducted, because excessive toxicity was not expected. Therefore, the toxicity test was incorporated into the first mutagenicity assay.
Frozen stocks of each strain were checked for histidine or tryptophan requirement and for sensitivity to ampicillin, crystal violet and UV radiation.
To 2 ml molten top agar (containing 0.6% agar, 0.5% NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin), maintained at 46°C, were added subsequently: 0.1 mL of a fully grown culture of the appropriate tester strain, 0.1 mL of the appropriate dilution of the test substance or of the negative control (vehicle: water), of or the positive control substance and 0.5 ml S9 mix or 0.5 ml sodium phosphate 100 m (pH7.4) as appropriate. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5% agar in Vogel and Bonner medium E with 2% glucose). All determinations were made in triplicate. The plates were incubated at 37°C for 3 days. Revertant colonies were then counted.

Evaluation criteria:

A reproducible two-fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed in the vehicle, or a demonstrable concentration-related effect, was taken to indicate a positive response in this assay system.

Statistics:

No statistical analysis was performed; not required for this study type

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The substance was not toxic to the bacteria (evidenced by the absence of a drastic decrease in the mean number of revertant colonies).
In both the absence and presence of S9 mix in all strains tested, the test substance did not cause a reproducible two-fold or greater increase in the mean number of revertant colonies appearing in the test plates compared to the background spontaneous reversion rate observed with the vehicle, and did not give evidence of a dose response. The positive controls gave the expected increase in the mean number of his+ revertants both with and without S9.

It was concluded that 2,2 -dimethylol propionic acid is not mutagenic up to concentrations of 5000 µg/plate.

Conclusions:

It was concluded that the results obtained with the test substance 2,2-dimethylol propionic acid (DPMA) in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, in both the absence and in the presence of the S9-mix indicate that 2,2-dimethylol propionic acid (DMPA) was not mutagenic under the conditions employed in this study.

Executive summary:

In a reverse gene mutation assay in bacteria (conducted according to OECD TG 471) , strains TA1535, TA1537, TA98 and TA100 of S. typhimurium were exposed to 2,2-dimethylol propionic acid in water at concentrations of 0, 62, 185, 556, 1667, 5000 µg/plate in the presence and absence of mammalian metabolic activation. The test substance was tested upto limit concentration of 5000 µg/plate. There was no evidence of toxicity, or mutagenicity up to concentrations of 5000 µg/plate. Therefore it was concluded that 2,2-dimethylol propionic acid was not mutagenic under the conditions of the study. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background. This study is classified as acceptable. The strain E. coli WP2 uvr A is also investigated in a second study, therefore this study satisfies the requirement for Test Guideline OPPTS 870.51001; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro

Bacterial reverse mutation test

The potential of Bis-MPA (Dimethylol propionic acid) to cause genetic damage was evaluated in a GLP study conducted according to OECD TG 471 (van Delft, 1998). Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were tested both in the presence and absence of metabolic activation. There was no evidence of toxicity or mutagenicity up to concentrations of 5000 µg/plate. Therefore it was concluded that Bis-MPA (Dimethylol propionic acid) was not mutagenic under the conditions of the study. In this first study (van Delft, 1998), only four bacterial strains were tested. Therefore, a second GLP study, in accordance with OECD Guideline 471 (RTC, 2015) was conducted by testing exclusively E. coli. In this second bacterial reverse mutation assay, Bis-MPA (Dimethylol propionic acid) was examined for the ability to induce gene mutations in Escherichia coli strain WP2 uvrA. It is concluded that Bis-MPA (Dimethylol propionic acid) did not induce reverse mutation in Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. Therefore these two studies together fulfill the data requirements of gene mutation in bacterial cells and indicate that Bis-MPA (Dimethylol propionic acid) was not genotoxic. 

 

Mammalian cell gene mutation test

A GLP gene mutation study was performed with Bis-MPA (Dimethylol propionic acid), according to OECD TG 476 (van Delft, 1998). The potential of the test substance to induce mutations at the TK-locus of L5178Y cells was determined, both in the presence and absence of metabolic activation. Bis-MPA (Dimethylol propionic acid) was not found to be cytotoxic or genotoxic, either in the presence or absence of metabolic activation, tested up to a concentration of 10 mM.

 

Mammalian chromosome aberration test

A GLP chromosome aberration study was performed with Bis-MPA (Dimethylol propionic acid), according to OECD TG 473 (van Delft, 1998). The study was carried out with Chinese hamster ovary cells, in the presence and absence of metabolic activation, up to a concentration of 1250 µg/ml. Under the conditions of the study, Bis-MPA (Dimethylol propionic acid) was found to be non-clastogenic.

Justification for classification or non-classification

Bis-MPA (Dimethylol propionic acid) was not mutagenic or clastogenic when tested in vitro in a bacterial reverse mutation assay, a mammalian cell gene mutation assay, and a mammalian chromosome aberration test. Therefore, according to Regulation (EC) No 1272/2008, classification is not required.