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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Heptanoic anhydride
EC Number:
210-940-5
EC Name:
Heptanoic anhydride
Cas Number:
626-27-7
Molecular formula:
C14H26O3
IUPAC Name:
heptanoic anhydride
Constituent 2
Reference substance name:
Heptanoic acid anhydride
IUPAC Name:
Heptanoic acid anhydride

Method

Target gene:
histidine gene locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1 (Plate incorporation test): 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate. Since only moderate toxic effects were observed 5000 μg/plate were chosen as maximal concentration.

Experiment 2 (pre-incubation test):

TA 100 (without S9 Mix): 1; 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
TA 1535, TA 1537, TA 98 & TA 102 (without S9 mix): 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
All strains with S9 mix: 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
DMSO
The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene, 4-nitro-o-phenylene-diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION:

Experiment 1: in agar (plate incorporation);
Experiment 2: preincubation;

DURATION Experiment 1:
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours

Each concentration and the controls were tested in triplicate.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test item precipitated in the overlay agar in the test tube in experiment I from 1000 to 5000 μg/plate and in experiment II from 2500 to 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 1000 to 5000 μg/plate and in experiment II from 2500 to 5000 μg/plate. The undissolved particles had no influence on the data recording.

Reduced background growth or a reduction in the number of revertants were observed at the following concentrations:
TA1535 - from 1000 µg/plate on without S9 mix in Experiment I and from 2500 µg/plate on without S9 mix in Experiment II; none with S9 mix
TA 1537 - from 100 µg/plate on without S9 mix and at 5000 µg/plate with S9 mix in Experiment I; from 2500 µg/plate on without S9 mix
TA 98 - from 333 µg/plate on without S9 mix and at 5000 µg/plate with S9 mix in Experiment I; from 2500 µg/plate on without S9 mix
TA 100 - from 100 µg/plate on without S9 mix in Experiment I and from 333 µg/plate on without S9 mix in Experiment II; none with S9 mix
TA 102 - from 1000 µg/plate on without S9 mix in Experiment I and from 2500 µg/plate on without S9 mix in Experiment II; none with S9 mix

Any other information on results incl. tables

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Enanthsäureanhydrid at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Applicant's summary and conclusion

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations (according to OECD guideline 471) according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation in concentrations up to 5000 µg/plate. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Enanthsäureanhydrid is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

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