2,2-bis(hydroxymethyl)butanoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

This study by Caroline Przewalla 2022 was performed to investigate the potential of 2,2-bis(hydroxymethyl)butanoic acid to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79). The study was conducted in accordance with the following guidelines: OECD 476, and EU-Method B.17 of the Commission Regulation (EC) No. 440/2008 in GLP condition.

The assay comprised a pre-test and two independent experiments (experiment I and II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.

Experiment I was performed with and without metabolic activation (liver S9 mix from male rats; Inducing Agent: Phenobarbital-5,6 Benzoflavone (PB/BNF)) and a treatment period of 4 h. Experiment II was performed with a treatment period of 24 hours without metabolic activation.

Ethylmethane sulfonate (EMS) and 7,12-Dimethylbenzanthracene (DMBA) as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

The highest nominal concentration (experiment I +/-S9: 10mM; experiment II -xx mM) applied, was chosen with regard to the solubility of the test item in organic solvents and aqueous media as well as the results of the pre-test.

Precipitation or turbidity of the test item was not visible in all experimental parts at the maximum concentration of the test item.

No dose dependent increase in mutant colony numbers was observed in experiment I. No statistically significant increase in mutant colony number in comparison to the solvent control was detected up to the maximal concentration of the test item. All values remained within the historical control range, except two values exceeded/fall below the historical range of solvent control slightly. However, this effect was judged as irrelevant since it is very minor and the corresponding solvent control remained well within the range of historical controls. Therefore, the result of experiment I is clearly negative.

 

The result of experiment I was clearly negative, however on demand of the sponsor an experiment II was performed for verification of the results of experiment I.

In experiment II again no dose dependent increase in mutant colony numbers was observed. No statistically significant increase in mutant colony number in comparison to the solvent control was detected up to the maximal concentration of the test item. All values remained within the historical control range. Therefore, the result of experiment II is clearly negative.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

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 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

the HPRT locus on chromosome X Chinese Hamster V79 cells.

Species / strain / cell type:

other: Chinese Hamster V79 cells.

Details on mammalian cell type (if applicable):

HPRT locus on chromosome X Chinese Hamster V79 cells.

Metabolic activation:

with and without

Metabolic activation system:

the cells were exposed to the test item with (+S9) and without (-S9) exogenous metabolic activation for 4 hrs (experiment I) resp. 24 hrs (experiments II, only -S9). Following an expression time, the descendants of the treated cell population were monitored for the loss of functional HPRT enzyme by culturing in selective medium.

Test concentrations with justification for top dose:

concentrations in the pre-test (mM): 10; 5; 2.5; 1.25; 0.63; 0.31; 0.16

concentrations in the experiment I (mM): 10; 5; 2.5; 1.25; 0.63; 0.31; -

concentrations in the experiment II (mM): 10; 5; 2.5; 1.25; 0.63; 0.31; -

Vehicle / solvent:

DMEM medium without supplements was used as solvent control for the positive control Ethylmethane sulfonate (EMS) and for the test item in a final concentration of 10 %.
DMSO was used as solvent control for the positive control 7,12-dimethylbenz(a)anthracene (DMBA) in a final concentration of 1 %

Negative solvent / vehicle controls:

yes

Remarks:

DMEM medium - used as solvent contr. for the positive contr. Ethylmethane sulfonate (EMS) and for the test item in a final conc. of 10 %. DMSO was used as solvent control for the positive contr. 7,12-dimethylbenz(a)anthracene (DMBA) in a final conc of 1

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Test vessels: All vessels used were made of glass or sterile plastic. They were sterilized before use in a heating chamber or autoclave.

1. Pre-Test for Detection of Cytotoxicity
cytotoxicity was determined by measuring the survival of the cells (S) after exposure to the test item. Furthermore, the cloning efficiency (CE) of treated cells in comparison to the controls was determined. In the pre-test, 7 concentrations of the test item were used and tested with and without metabolic activation. The exposure time was 4 hours.

2. Dose Selection
According to the results of the pre-test, 6 concentrations were chosen for the main exper-iment and tested with and without metabolic activation

3. Experimental Performance
Experiment I was conducted with and without metabolic activation. Experiment II was conducted for a verification of the results of experiment I.
In experiment II only the approach without metabolic activation was used.
The second experiment is performed in the same manner as Experiment I except for the number of cells seeded (2.25*106_1.8*106) and for the incubation time of the test item without metabolic activation which is 24 h instead of 4 h and is not followed by a washing step. In addition, Passage I is not performed after 2 d and 5 d but after 1 d and 4 d of ex-pression time.
Day 1: First 2.25 * 106 cells per 15 cm culture dish were seeded per tested concentration as well as for the solvent and positive controls and incubated for 47 h + 45 min. The incu-bation conditions during the whole assay were 37.0° 1.5 °C in 5.0 ± 0.5 % CO2.
Again, two control plates (2.25 * 106 cells per 15 cm culture dish) were prepared on Day 1, to generate the exact cell count at the end of treatment on Day 3.
Day 3: At least 20*106 cells the cells were exposed to each concentration of the test item for 4 hours with and without metabolic activation (duplicate cultures per concentration and approach).
Following treatment, the cells were washed with PBS Dulbecco (5 % FBS) twice and once with PBS only (not in experiment II –S9).
To determine the CE, cells were counted and adjusted to 400 cells per 10 cm culture dish and incubated for 6 d at 37.0 ± 1.5 °C under 5.0 ± 0.5 % CO2.
Furthermore, the remaining cells were cultured in 15 cm culture dishes to allow expres-sion of the mutant phenotype for 7 days. During this period, cells were regularly sub-cultured to maintain them in exponential growth.
Therefore, cells were counted and adjusted to 2.25 * 106 cells per 15 cm culture dish and incubated for further 2 d.
Day 5, Passage I: cells were counted and adjusted to 5 * 105 cells per 15 cm culture dish and incubated for further 3 d.
Day 8: Passage II: cells were counted and adjusted to 2.25 * 106 cells per 15 cm culture dish and incubated for further 2 d.
Day 9: For the determination of the CE, after an incubation time of 6 d, the cell colonies of the 10 cm culture dishes, prepared on day 3, were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution and counted for the calculation of cloning efficien-cy CE.
Day 10: After a total expression time of 6 days, the cells were re-plated in medium with and without selective agent (6-thioguanine) for the determination of the number of mu-tants (MF) and cloning efficiency (CE II) at the time of selection.
For the determination of the mutagenicity, the cells were counted and adjusted to 4.2*105 cells per 10 cm culture dish. For all concentrations of the test item and for all controls, 5*10 cm dishes were prepared (duplicate cultures per concentration and approach).
All 5 dishes together, 2.1*106 are seeded for the evaluation of the mutagenicity in selec-tion medium containing 6-TG (final concentration: 2 µg/mL) for further 7 days.
For the determination of the CE II, cells were adjusted to 400 cells per 10 cm culture dish in complete culture medium without selective agent and incubated also for further 6 days.
Day 16: For the determination of the CE II (cloning efficiency in non-selective medium), the cells were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solu-tion after a 6 day-incubation time. The colonies were counted and the cloning efficiency (CE II) was calculated.
Day 17: For the determination of the mutant frequency (MF), after a total expression time of 7 days, the cells were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution, the colonies were counted and the cloning efficiency of mutant colonies in selective medium (CE MUT) was determined.

Evaluation criteria:

7.5 Calculation and Processing of the Data

Cloning Efficiency (CE): Directly after treatment the cells are seeded and incubated for 6 days. The formed colonies are stained and counted. The cytotoxicity of the test item is de-termined by the reduction of the Cloning Efficiency (CE) (how many cells have the potential for further growing after treatment.).


Survival (S): The Survival rate gives information about the cytotoxicity of the test item di-rectly after treatment and is determined by counting the cells directly after treatment.


The Relative Survival (RS): The most exact evaluation of cytotoxicity: The Cloning Effi-ciency (CE) directly after treatment, adjusted by any loss of cells during treatment as com-pared with adjusted Cloning efficiency in solvent controls (assigned a survival of 100 %).


Mutant frequency: is the cloning efficiency in selective medium divided by the cloning efficiency in non-selective medium for the same culture at the same time of selection.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

duration: 24 hours

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

duration: 4 hours

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

duration: 4 hours

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Summary of Results of Experiment I

	Concentration	S9 mix	Treatment time	Culture	RS	MF per 10^6 cells	MF per 10^6 cells
	[mM]		[h]		[%]		Mean
Solvent Control Test Item	-	+	4	A	-	12	11
				B	-	10
Solvent Control DMBA	-	+	4	A	-	12	8
				B	-	4
Positive Control (DMBA)	1.5	+	4	A	91.81%	51	47
				B	76.86%	43
Test item	10	+	4	A	102.52%	2	6
				B	96.61%	10
Test item	5	+	4	A	99.93%	13	10
				B	93.91%	7
Test item	2.5	+	4	A	113.99%	11	12
				B	109.21%	14
Test item	1.25	+	4	A	100.87%	3	6
				B	90.68%	10
Test item	0.63	+	4	A	100.90%	10	11
				B	79.22%	13
Test item	0.31	+	4	A	105.37%	11	10
				B	69.67%	10
Solvent Control Test Item	-	-	4	A	-	13	13
				B	-	12
Solvent Control EMS	-	-	4	A	-	23	19
				B	-	16
Positive Control (EMS)	300	-	4	A	86.05%	461	484
				B	85.87%	508
Test item	10	-	4	A	99.32%	12	13
				B	140.61%	14
Test item	5	-	4	A	103.55%	20	12
				B	140.27%	4
Test item	2.5	-	4	A	98.53%	21	20
				B	117.32%	19
Test item	1.25	-	4	A	99.47%	7	11
				B	133.41%	14
Test item	0.63	-	4	A	107.11%	19	16
				B	125.50%	13
Test item	0.31	-	4	A	101.79%	4	6
				B	138.83%	7

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

 

  Summary of Results of Experiment II

	Concentration	S9 mix	Treatment time	Culture	Relative Survival	mutant frequency per 10^6 cells	mutant frequency per 10^6 cells
	[mM]		[h]		[%]		Mean
Solvent Control Test Item	-	-	24	A	-	11	16
				B	-	21
Solvent Control EMS	-	-	24	A	-	10	10
				B	-	10
Positive Control (EMS)	150	-	24	A	104.61	435	382
				B	90.61	329
Test item	10	-	24	A	94.47	7	5
				B	100.98	4
Test item	5	-	24	A	100.21	13	13
				B	107.29	13
Test item	2.5	-	24	A	111.89	13	17
				B	91.31%	21
Test item	1.25	-	24	A	102.21	7	7
				B	100.43	6
Test item	0.63	-	24	A	100.81	22	14
				B	101.35	7
Test item	0.31	-	24	A	115.11	16	16
				B	93.69	15

 

Conclusions:

It can be stated that under the experimental conditions of this study 2,2-bis(hydroxymethyl)butanoic acid did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.
Therefore, the test item 2,2-bis(hydroxymethyl)butanoic acid is considered to be “non-mutagenic under the conditions of the HPRT assay”.

Executive summary:

This study was performed to investigate the potential of 2,2-bis(hydroxymethyl)butanoic acid to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79). The study was conducted in accordance with the following guidelines: OECD 476, and EU-Method B.17 of the Commission Regulation (EC) No. 440/2008 in GLP condition.

The assay comprised a pre-test and two independent experiments (experiment I and II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the main experiments were determined.

Experiment I was performed with and without metabolic activation (liver S9 mix from male rats; Inducing Agent: Phenobarbital-5,6 Benzoflavone (PB/BNF)) and a treatment period of 4 h. Experiment II was performed with a treatment period of 24 hours without metabolic activation.

Ethylmethane sulfonate (EMS) and 7,12-Dimethylbenzanthracene (DMBA) as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

The highest nominal concentration (experiment I +/-S9: 10mM; experiment II -xx mM) applied, was chosen with regard to the solubility of the test item in organic solvents and aqueous media as well as the results of the pre-test.

Precipitation or turbidity of the test item was not visible in all experimental parts at the maximum concentration of the test item.

No dose dependent increase in mutant colony numbers was observed in experiment I. No statistically significant increase in mutant colony number in comparison to the solvent control was detected up to the maximal concentration of the test item. All values remained within the historical control range, except two values exceeded/fall below the historical range of solvent control slightly. However, this effect was judged as irrelevant since it is very minor and the corresponding solvent control remained well within the range of historical controls. Therefore, the result of experiment I is clearly negative.

 

The result of experiment I was clearly negative, however on demand of the sponsor an experiment II was performed for verification of the results of experiment I.

In experiment II again no dose dependent increase in mutant colony numbers was observed. No statistically significant increase in mutant colony number in comparison to the solvent control was detected up to the maximal concentration of the test item. All values remained within the historical control range. Therefore, the result of experiment II is clearly negative.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Mutation Assay

In bacterial mutation assay by E. Machigaki 1995, the test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrAwere treated with the test item using the Ames plate incorporation method at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 0.5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The experiment was repeated on a separate day using an amended dose range (ranging from 0.5 to 1500 µg/plate), depending on strain type and presence or absence of S9-mix. Fresh cultures of the bacterial strains and fresh test item formulations were employed.

 

Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

 

The vehicle (dimethyl sulphoxide) 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 test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 500 µg/plate both with and without metabolic activation (S9-mix). The test item was tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit depending on bacterial strain type and presence or absence of S9-mix. A test item film (opaque in appearance) was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies.

 

No toxicologically 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 either the range-finding or main tests. Small, statistically significant increases in revertant colony frequency were observed in the range-finding test at 150 µg/plate (TA1537 in the absence of S9-mix) and 1.5, 5, 15 and 150 µg/plate (TA98 in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle controls. 

 The test item was considered to be non-mutagenic under the conditions of this test.

Endpoint Conclusion: No adverse effect observed (negative)

 

The chromosome aberration study in mammalian cells by Sakae Koike 1996 was performed according to The Japanese Guidelines on Industrial Chemicals (1986), Guideline under the Japanese Occupational Safety and Health Law (1987), Annex V (Cytogenetics) in GLP condition.

Concentration range in the main test with and without metabolic activation) was: 370 ... 1480 μg/m.  Chinese hamster lung cell line (CHL/IU) was used. The metabolic activation system was Phenobarbitol and 5,6-Benzoflavone induced rat liver S-9. Exposure period (with metabolic activation): 6 hours. Exposure period (without metabolic activation): 24 hours

Colour change to the culture medium (from red to yellow) was seen upon addition of DMBA at final concentrations of 370 micrograms/ml and higher in the toxicity test and main cytogenetic test.

A statistically significant increase in aberrant cell frequency (27% aberrant cells, compared to 1.5% in controls) was seen in cultures treated with DMBA at 1480 micrograms/ml for 6 hours in the presence of S-9 mix (and harvested 18 hours later) in the main test. In these test cultures, mitotic activity was reduced to 16% of the control value.

It seems likely that the result in the presence of S-9 is a false positive due to decreased pH. The further confirmatory in vitro cytogenetics study was performed.

In a confirmatory test (where cultures were again exposed for 6 hours at 1480 microgrammes/ml in the presence of S-9 mix), a slightly increased aberrant cell frequency was seen when saline was used as the solvent (5.5% aberrant cells, compared to none in controls), but no such effect was seen when HEPES buffer was used as the solvent (no aberrant cells in treated cultures, 1.5% in controls).

Measurement of the pH of the treatment medium showed that there was a reduction in pH (from > 7 to 6 or less) upon addition of DMBA in saline to give a final concentration of 1480 microgrammes/ml. No such effect was seen when HEPES buffer was used as the solvent (reductions were only from CA 7 to 6.5 or more).

No significant increases in aberrant cell frequencies were seen in cultures treated at any other DMBA concentration tested, in the presence or absence of S-9 mix.

This test was carried out specifically to address the positive results seen with S9 in the main study. The cells were treated with notified substance in HEPES-buffered medium (Cytotoxicity / choice of top concentrations: cytotoxicity (740 μg/ml)). When the incubation medium was buffered to pH 7, there was no indication of any genotoxic activity. The results of this test confirm that the aberrations seen in the main study were caused by low pH.

 

Justification for classification or non-classification

The results of bacterial reverse mutation assay, in vitro mammalian chromosome aberration test and In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes are negative. Classification according Regulation (EC) No 1272/2008 is therefore not required.