4-hydroxybutan-2-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 May 2015 - 22 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP / OECD guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxybutan-2-one

EC Number:

209-693-6

EC Name:

4-hydroxybutan-2-one

Cas Number:

590-90-9

Molecular formula:

C4H8O2

IUPAC Name:

4-hydroxybutan-2-one

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report: 4-Hydroxybutan-2-one
- Test substance No.: 14/0671-1
- Batch Identification: 10781727
- Physical state: liquid, colorless, clear
- Analytical purity: 96,2 area-%
- Stability under test conditions: guaranteed until 04 Nov 2016 as indicated by the sponsor
- Storage condition of test material: Room temperature

Method

Target gene:

Salmonella typhimurium: histidine
Escherichia coli WP2 uvrA: tryptophan

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/b-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Standard plate test and Preincubation test: 33; 100; 333; 1000; 2600; and 5200 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, water was used as vehicle

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S9 mix: 2-aminoanthracene (2-AA). Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 4-nitro-o-phenylenediamine (NOPD); 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO).

Details on test system and experimental conditions:

TEST SYSTEM
For testing, deep-frozen (-70°C to -80°C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) werethawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth
+ 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria cultures was
determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 109 cells per mL).
These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

1. Standard plate test +/- S9 mix:
METHOD OF APPLICATION: in agar (plate incorporation) according to Ames et al. 1975, Maron et al. 1983
NUMBER OF REPLICATIONS: 3 test plates /dose or control
2. Preincubation Test +/- S9 mix (because no mutagenicity was observed in the Standard plate test)
METHOD OF APPLICATION: in medium, according to Yahagi et al. 1977 und Matsushima et al. 1980
NUMBER OF REPLICATIONS: 3 test plates /dose or control

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10exp9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT: only in one tester strain at a concentration of 5200 µg/plate. PIT: E.coli without S9 mix at a concentration of 2600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No test substance precipitation was found with and without S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionallyobserved in the standard plate test depending on the
strain and test conditions at a concentration of 5200 μg/plate.
In the preincubation assay weak bacteriotoxicity (slight decrease in the number of trp+ revertants) was observed only in the tester strain
E. coli WP2 uvrA without S9 mix at a concentration of 2600 μg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions chosen here, it is concluded that 4-Hydroxybutan-2-one is not a mutagenic test substance in the bacterial
reverse mutation test in the absence and the presence of metabolic activation

Executive summary:

The test substance 4 -Hydroxybutan-2-one was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5200 μg/plate (SPT) ; 33 μg - 5200 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions at 2600 and 5200 μg/plate

MUTAGENICITY:

A biologically relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

CONCLUSION:

Thus, under the experimental conditions of this study, the test substance 4 -Hydroxybutan-2-one is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

According to the results of the present study, the test substance did not lead to a biologically relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.