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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Near guideline studies, published in peer-reviewed literature, adequate for assessment

Data source

Reference Type:
A compilation of two decades of mutagenicity test results with the Ames Salmonella typhimurium and L5178Y mouse lymphoma cell mutation assays
Seifried HE, Seifried RM, Clarke JJ, Junghans TB and San RHC.
Bibliographic source:
Chem. Res. Toxicol. 19: 627-644

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
not specified
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetic anhydride
EC Number:
EC Name:
Acetic anhydride
Cas Number:
Molecular formula:
acetic anhydride
Details on test material:
- Name of test material (as cited in study report): acetic anhydride
- No further details reported


Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells grown in Fischer's medium supplemented with 10% horse serum and 0.02% pluronic F-68
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes, at approximately 3 month intervals
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced liver S9 from male Sprague-Dawley rats
Test concentrations with justification for top dose:
0.04-0.3 µg/mL
Controlsopen allclose all
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
Migrated to IUCLID6: 4.7 x 10-6 M (or methyl methanesulfonate at 10-20 µg/mL)
Positive control substance:
Migrated to IUCLID6: 1.86 x 10-5 M (or dimethylbenz[α]-anthracene at 0.5-4 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

- A total of 1.2 x 107 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37± 1°C, washed twice with growth medium, and maintained at 37± 1°C for 48 h in log phase growth to allow recovery and mutant expression.
- Cells in the cultures were adjusted to 3 x 105/mL at 24 h intervals.
- They were then cloned (1 x 106 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0.23% granulated agar.
- Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 µg/mL) to the cloning medium for mutant selection. The 100x stock solution of TFT in saline was stored at -70°C and was thawed immediately before use.
- Plates were incubated at 37±1°C in 5% CO2 in air for 10-12 days and then counted with an automated colony counter. Only colonies larger than -0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells.
Evaluation criteria:
Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
other: Inconclusive. With and without S9
Cytotoxicity / choice of top concentrations:
see remarks on results
Untreated negative controls validity:
not specified
Positive controls validity:
not specified

Applicant's summary and conclusion

Interpretation of results (migrated information):
other: Inconclusive. With and without metabolic activation

Acetic anhydride is considered to have no significant mutagenic activity in mammalian cells.
Executive summary:

Acetic anhydride has been examined for mutagenic activity in mammalian cells in vitro using the mouse lymphoma L5178Y assay. Although the data can be formally regarded as equivocal, they do not indicate any significant genotoxic activity for acetic anhydride in mammalian cells in vitro. This evaluation of the data reports the findings as inconclusive.

Acetic anhydride is considered to have no significant mutagenic activity in mammalian cells.