2-Butanone, 1,3,4-trihydroxy-

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:

From July 20, 2022 to September 15, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 476 and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-800-9

EC Name:

-

Cas Number:

40031-31-0

Molecular formula:

C4H8O4

IUPAC Name:

(3S)-1,3,4-trihydroxybutan-2-one

impurity 1

Test material form:

liquid: viscous

Details on test material:

NA

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Labcorp were stored as frozen stocks in liquid nitrogen. Full details of the supplier are documented in central records. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and placed in an incubator set to 37ºC. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with β-Naphthoflavone/Phenobarbital. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-10°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).

Treatments were carried out both in the absence and presence of S-9 by addition of either 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test system was 1% (v/v).

Test concentrations with justification for top dose:

In the cytotoxicity Range-Finder Experiment, seven concentrations were tested in the absence and presence of S-9, ranging from 18.77 to 1201 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration tested, 1201 µg/mL in the absence and presence of S-9, gave 47% and 99% Relative Survival (RS), respectively.

Vehicle / solvent:

Test article stock solutions were prepared by formulating Erythrulose under subdued lighting in DMSO, with the aid of vortex mixing, warming at 37°C and ultrasonication (for approximately 3 minutes), where required, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within 1.5 hours of initial formulation.

Preliminary solubility data indicated that Erythrulose was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations up to at least 130.52 mg/mL. The solubility limit in culture medium was in excess of 1305.2 µg/mL, as indicated by a lack of any visible precipitation at this concentration over a period of approximately 3 hours after test article addition with warming at 37°C. A maximum concentration of 1201 µg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to 10 mM (based on test article molecular weight of 120.1 g/mol) (OECD, 2016). Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Negative (vehicle) controls comprised treatments with the vehicle DMSO diluted 100-fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

in the presence of S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Negative (vehicle) controls comprised treatments with the vehicle DMSO diluted 100-fold in the treatment medium.

True negative controls:

no

Positive controls:

yes

Remarks:

100-fold dilution

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

in the absence of S9-mix

Details on test system and experimental conditions:

Test System:
The test system was suitably labelled to clearly identify the study number, test article concentration (if applicable), positive and vehicle control, presence and absence of S 9.

Cytotoxicity Range-Finder Experiment:
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment culture volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g) for 5 minutes, washed with tissue culture medium, centrifuged again (200 g) for 5 minutes and resuspended in 20 mL RPMI 10.
Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were placed in a humidified incubator set to 37ºC, gassed with 5% v/v CO2 in air for 8 days. Wells containing viable clones were identified by eye using background illumination and counted.

Mutation Experiment
Treatment of Cell Cultures:
At least 107 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test article or positive control solution was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours in an incubator set to 37°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (see section 7.5) and resuspended in 20 mL RPMI 10 medium.
Cell densities were determined using a Coulter counter and the concentrations adjusted to 2 x 105 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies (Brusick, 1986; Scott et al., 1991). Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity Range-Finder Experiment.

Plating for Survival:
Following adjustment of the cultures to 2 x 105 cells/mL after treatment, samples from these were diluted to 8 cells/mL.

Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were placed in a humidified incubator set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (7 days). Wells containing viable clones were identified by eye using background illumination and counted.

Expression Period:
Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. From observations on recovery and growth of the cultures during the expression period, the following cultures were selected to be plated for viability and 6TG resistance.

Plating for Viability – Mutation Experiment:
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL.

Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (13 days). Wells containing viable clones were identified by eye using background illumination and counted.

Plating for 6TG Resistance – Mutation Experiment:
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 µg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were placed in a humidified incubator, set to 37ºC and gassed with 5% v/v CO2 in air, until scoreable (14 days). Wells containing viable clones were identified by eye using background illumination and counted.

Rationale for test conditions:

A maximum concentration of 1201 µg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to 10 mM (based on test article molecular weight of 120.1 g/mol) (OECD, 2016). Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Evaluation criteria:

The assay was considered valid if all of the following criteria were met:
1. The MF in the concurrent vehicle control cultures was considered acceptable for addition to the laboratory historical vehicle control database
2. The MF in the concurrent positive controls induced responses that were comparable with those generated in the historical positive control database and gave a clear, unequivocal increase in MF over the concurrent vehicle control
3. The test was performed with and without metabolic activation
4. Adequate numbers of cells and concentrations were analysable.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the cytotoxicity Range-Finder Experiment, seven concentrations were tested in the absence and presence of S-9, ranging from 18.77 to 1201 µg/mL (equivalent to 10 mM at the highest concentration tested). Upon addition of the test article to the cultures, precipitate was observed at the highest concentration tested in the presence of S-9 (1201 µg/mL). Following the 3 hour treatment incubation period, no precipitation was observed in the absence and presence of S-9. The highest concentration tested, 1201 µg/mL in the absence and presence of S-9, gave 47% and 99% RS, respectively.

No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentrations tested (1201 µg/mL) as compared to the concurrent vehicle controls (measured data not reported).
In the Mutation Experiment eight concentrations, ranging from 50 to 1201 µg/mL, were tested in the absence and presence of S-9. No precipitation was observed in the absence or presence of S-9, either at the time of treatment or at the end of the treatment incubation period. The highest concentration analysed was 1201 µg/mL in the absence presence of S-9, which gave 54% and 95% RS, respectively

A summary of the results for the Mutation Experiment is shown in Text Table 2. The individual plate counts observed and the data analysis are shown in Table 8.1 to Table 8.16. The historical vehicle control ranges, based on the last 20 experiments performed in this laboratory, are presented in Section 7.4. The acceptance criteria were met and the study was accepted as valid.
No evidence of concentration-related toxicity or post-treatment precipitate were observed in the absence and presence of S-9. No statistically significant increases in MF, compared to concurrent vehicle controls, were observed at any concentration analysed in the absence and presence of S-9 and there were no positive, statistically significant linear trends. The vehicle control MF values in this study were acceptable for addition to the laboratory historical vehicle control database and were within the historical vehicle control ranges (mean ± 2 standard deviations), based on the last 20 experiments performed in this laboratory prior to this one.

Any other information on results incl. tables

Test article stock solutions were prepared by formulating Erythrulose under subdued lighting in DMSO, with the aid of vortex mixing, warming at 37°C and ultrasonication (for approximately 3 minutes), where required, to give the maximum required concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within 1.5 hours of initial formulation. The following concentration ranges were tested:

Experiment	Treatment Period (hours)	Concentration Range (mg/mL)			Final Concentration Range (µg/mL)
Range-Finder	3 hours, -S-9	1.877	to	120.1	18.77	to	1201
	3 hours, +S-9	1.877	to	120.1	18.77	to	1201
Mutation Experiment	3 hours, -S-9	5.000	to	120.1	50.00	to	1201
	3 hours, +S-9	5.000	to	120.1	50.00	to	1201

 

Text Table 1: RS Values - Range-Finder Experiment – 3 Hour Treatments in the Absence and Presence of S-9

Concentration	-S-9	+S-9
µg/mL	%RS	%RS
0	100	100
18.77	99	102
37.53	101	103
75.06	79	106
150.1	84	124
300.3	89	91
600.5	59	81
1201	47	99 P

%RS                      Percent relative survival
P                             Precipitate noted at time of treatment

 

Text Table 2: Summary of Mutation Data - 3 Hour Treatments in the Absence and Presence of S-9

3 Hour Treatment –S-9			3 Hour Treatment +S-9
Concentration	%RS	MF §	Concentration	%RS	MF §
µg/mL			µg/mL
0	100	3.51	0	100	4.96
50	109	3.52 NS	50	81	4.96 NS
100	115	3.08 NS	100	97	3.25 NS
200	97	2.90 NS	200	99	2.83 NS
400	96	4.29 NS	400	104	1.69 NS
600	78	2.15 NS	600	93	5.68 NS
800	70	1.17 NS	800	107	2.53 NS
1000	52	3.45 NS	1000	101	3.23 NS
1201	54	5.48 NS	1201	95	4.63 NS
NQO 0.15	46	30.78	B[a]P 4	72	36.32
NQO 0.2	51	29.34	B[a]P 6	55	39.88

 

Test for Linear trend

-S-9		+S-9
Slope	-9.27E-10	Slope	7.64E-12
Variance	6.94E-19	Variance	9.17E-19
b² / Sb	1.240	b² / Sb	0.000

-S-9 Linear trend test on mutant frequency: Not significant (negative trend)
+S-9 Linear trend test on mutant frequency: Not significant

 

• 6‑TG resistant mutants/10⁶ viable cells 7 days after treatment

%RS                      Percent relative survival adjusted by post treatment cell counts

NS                          Not significant

Applicant's summary and conclusion

Conclusions:

It is concluded that (S)-1,3,4-trihydroxybutan-2-one did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to a maximum concentration of 1201 µg/mL (equivalent to 10 mM, as required by current regulatory test guidance) for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Executive summary:

(S)-1,3,4-trihydroxybutan-2-one was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by a β‑Naphthoflavone/Phenobarbital-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, seven concentrations were tested in the absence and presence of S-9, ranging from 18.77 to 1201 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration tested, 1201 µg/mL in the absence and presence of S-9, gave 47% and 99% Relative Survival (RS), respectively.

In the Mutation Experiment eight concentrations, ranging from 50 to 1201 µg/mL, were tested in the absence and presence of S-9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance was 1201 µg/mL in the absence and presence of S-9, which gave 54% and 95% RS, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore, the study was accepted as valid.

No evidence of concentration-related toxicity or post-treatment precipitate were observed in the absence and presence of S-9. No statistically significant increases in MF, compared to concurrent vehicle controls, were observed at any concentration analysed in the absence and presence of S-9 and there were no positive, statistically significant linear trends. The vehicle control MF values in this study were acceptable for addition to the laboratory historical vehicle control database and were within the historical vehicle control ranges (mean ± 2 standard deviations), based on the last 20 experiments performed in this laboratory prior to this one.

It is concluded that (S)-1,3,4-trihydroxybutan-2-one did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to a maximum concentration of 1201 µg/mL (equivalent to 10 mM, as required by current regulatory test guidance) for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.