Butyraldehyde

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:

2022-09-20 to 2022-10-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD 1997, as corrected in 2020

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:

Butyraldehyde

EC Number:

204-646-6

EC Name:

Butyraldehyde

Cas Number:

123-72-8

Molecular formula:

C4H8O

IUPAC Name:

butanal

Test material form:

liquid: volatile

Specific details on test material used for the study:

- Name of the test material used in the study report: Butyraldehyde
- Appearance: Colourless liquid
- Storage condition of test material: 15°C to 25°C, protected from light. Store under nitrogen.
- Stability under test conditions: no stability analyses undertaken, as fresh preparations of the test article was employed

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: The E. Coli strain was specifically WP2 uvrA pKM101

Metabolic activation:

with and without

Metabolic activation system:

S-9 Fraction obtained from the livers of male Sprague Dawley rats induced with β-Naphthoflavone/Phenobarbital.

Test concentrations with justification for top dose:

Final concentrations: 5, 16, 50, 160, 500, 1600, 5000 μg/plate

Vehicle / solvent:

Water

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

5.0 µg/plate; positive control for strain TA100 and TA1535 in the presence of S9-mix. 10.0 µg/plate; positive control for strain WP2 uvrA pKM101 in the presence of S9-mix.

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

5.0 µg/plate; positive control for strain TA98 and TA1537 in the presence of S9-mix.

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2.0 µg/plate; positive control for strain WP2 uvrA pKM101

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate; positive control for strain TA1537

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

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

Controls 6

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

Details on test system and experimental conditions:

Two independent mutagenicity assays were performed, using triplicate plates without and with S-9 for test article, vehicle and positive controls. A preliminary toxicity test was not conducted, because excessive toxicity was not expected. Therefore, the toxicity test was incorporated into the first mutagenicity assay. The following sequence of additions were supplemented to molten agar plates: 0.1 mL of bacterial culture, 0.1 ml of test article solution/vehicle control/positive control and 0.5 mL of 10% S-9 mix or buffer solution. The ingredients were rapidly mixed and the mix was immediately poured onto agar plates. The plates were inverted and incubated at 34 to 39°C for 3 days. Revertant colonies were then counted electronically using automated colony counter. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test item, S9 mix and sodium phosphate buffer.

Rationale for test conditions:

Standardized test conditions: highest concentration not limited by toxicity or solubility.

Experiment 2 included an incubation in air-tight sealed containers for 3 days following treatment. This was to limit any oxidation of the test article.

Evaluation criteria:

The test article was considered to have provided a mutagenic response if the assay data were valid, and:

1. Treatments with the test article provided a concentration-related increase in revertant numbers at one or more concentrations in at least one strain with or without metabolic activation system

2. An increase in mean revertant colony numbers per plate was observed which was ≥2-fold (in strains TA98, TA100, WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values

3. Any increase in revertant numbers was reproducible, where applicable

The test article was considered positive in this assay if all of the above criteria were met.

The test article was considered negative in this assay if not all the above criteria were met.

Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

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

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A pKM 101

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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 applicable

True 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. From the data it can be seen that vehicle control counts fell within or close to the laboratory’s historical ranges. The study demonstrated correct strain and assay functioning and was accepted as valid. Five analysable concentrations were obtained in each experiment.

Any other information on results incl. tables

It was concluded that n-Butyraldehyde is not mutagenic up to concentrations of 5000 µg/plate.

Applicant's summary and conclusion

Conclusions:

It was concluded that the results obtained with the test substance n-Butyraldehyde in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 as well as E. coli WPS uvrA pKM101 in both the absence and in the presence of the S9-mix indicate that n-Butryaldehyde 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 and E.coli WP2 uvrA pKM101 were exposed to Butyraldehyde in water at concentrations of 5, 16, 50, 160, 500, 1600 and 5000 µg/plate in the presence and absence of mammalian metabolic activation.

Butyraldehyde was tested up to limit concentration of 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable and it satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.