γ-valerolactone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-10-08 to 2021-03-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998-08

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

γ-valerolactone

EC Number:

203-569-5

EC Name:

γ-valerolactone

Cas Number:

108-29-2

Molecular formula:

C5H8O2

IUPAC Name:

5-methyloxolan-2-one

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: Grosss 542
- Expiration date of the batch: 04 June 2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Method

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital and β-naphthoflavone induced Wistar rats
- Method of preparation of S9 mix: The livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (containing 0.05 mL S9) in a total volume of 2.7 mL
- Quality controls of S9: The S9 batch was characterized with benzo(a)pyrene. A sterility control was carried along in both experiments.

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 2500, 5000 µg/plate in both experiments. In agreement with the recommendations of current guidelines, 5000 µg/plate were selected as maximum test dose.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (2-AA) N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) 4-nitro-o-phenylenediamine (NOPD)

Remarks:

+ S9:
2-AA: 2.5 µg/plate (TA1535, TA100, TA1537, TA98), 60 µg/plate (E.coli)
- S9:
MNNG: 5 µg/plate (TA1535, TA100)
NOPD: 10 µg/plate (TA98)
9-AAC: 100 µg/plate (TA1537)
4-NQO: 5 µg/plate (E.coli)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE
- Cell density at seeding: Approximately 10^9 cells per mL
- Test sytem: Plate incorporation (experiment I), preincubation (experiment II)

TREATMENT AND HARVEST SCHEDULE
- Preincubation period: 20 minutes
- Exposure duration/duration of treatment: 48 – 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Background growth inhibition, decrease in the number of revertants (factor ≤ 0.6)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mean number of revertant colonies

Rationale for test conditions:

As long as precipitation does not interfere with the colony scoring, 5000 µg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. No precipitation was observed and only a weak bacteriotoxic effect was observed in experiment I only using tester strain TA 1537 with S9 mix at a concentration of 2500 μg/plate. In experiment II, bacteriotoxicity was observed using tester strain TA 1537 both with and without S9 mix at a concentration of 5000 μg/plate.

Evaluation 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 range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

No statistics are needed

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

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:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Good solubility of the test substance in water, water was used as vehicle
- Precipitation: No precipitation of the test substance was observed with and without S9 mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Ames test:
- Signs of toxicity: A weak bacteriotoxic effect was observed in the standard plate test only using tester strain TA 1537 with S9 mix at a concentration of 2500 μg/plate. In the preincubation assay bacteriotoxicity was observed using tester strain TA 1537 both with and without S9 mix at a concentration of 5000 μg/plate.
- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"