Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 Dec 2018 - 14 Feb 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(S)-2-(5-Oxo-3-propyl-1,2-dihydropyrrol-1-yl) butyramide
Cas Number:
357338-13-7
Molecular formula:
C11H18N2O2
IUPAC Name:
(S)-2-(5-Oxo-3-propyl-1,2-dihydropyrrol-1-yl) butyramide
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Off-white powder
- Storage condition of test material: In refrigerator protected from light

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
- Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight)
- S9-mix (5% (v/v) S9-fraction) was prepared immediately before use and kept refrigerated.
Test concentrations with justification for top dose:
A correction was made for the purity of the test item. See above for the applied correction factor. Test item concentrations were used within 1.5 hours after preparation;

Experiment 1:
Dose-range finding (TA100 an WP2 uvrA, with and without S9-mix): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Main study (TA1535, TA1537 and TA98, with and without S9-mix): 17, 52, 164, 512, 1600 and 5000 μg/plate

Experiment 2:
TA1535, TA1537, TA98 and TA100, with and without S9-mix: 17, 52, 164, 512, 1000 and 2000 µg/plate
WP2 uvrA, with and without S9-mix: 52, 164, 512, 1000, 2000 and 5000 µg/plate
Vehicle / solvent:
- Solvent used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent: the test item formed a clear solution in DMSO, which is a solvent recommended by international guidelines.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See the section 'any other information on materials and methods'
Details on test system and experimental conditions:
METHOD OF APPLICATION
- Experiment 1: in agar (plate incorporation)
- Experiment 2: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS
Doses of the test substance were tested in triplicate in each strain.
Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED
10E8 per plate

DETERMINATION OF CYTOTOXICITY
Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS
The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
DECISION CRITERIA
- A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
- A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

ACCEPTABILITY CRITERIA
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the test facility.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
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 applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
EXPERIMENT 1
- Precipitation: Not observed
- Cytotoxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix, except in tester strain WP2uvrA where no toxicity was observed at any of the dose levels tested.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test item.

EXPERIMENT 2
- Precipitation: Not observed
- Cytotoxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test item.

COMPARISON WITH HISTORICAL CONTROL DATA
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

- Positive historical control data:

TA1535 TA1537 TA98
S9-mix - + - + - +
Range 125 – 1530 73 – 1481 55 – 1422 54 – 1239 365 – 1995 250 – 1977
Mean 911 245 791 337 1333 902
SD 174 118 357 157 257 354
n 3443 3338 2974 3361 3338 3391

TA100 WP2uvrA
S9-mix - + - +
Range 439 – 1993 408 - 2651 93 – 1999 111 - 1677
Mean 906 1296 1073 436
SD 163 371 527 149
n 3452 3425 3044 3123

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Nov 2015 and Nov 2018.

- Negative (solvent/vehicle) historical control data:

TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 3 – 29 3 – 27 3 – 20 3 – 23 8 - 61 8 – 60 61 – 176 60 - 176 10 – 59 9 - 69
Mean 10 11 6 6 15 22 111 107 25 32
SD 3 3 2 3 5 7 19 20 6 8
n 3496 3467 3416 3369 3476 3507 3520 3463 3144 3115

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Nov 2015 and Nov 2018.

Applicant's summary and conclusion

Conclusions:
In an Ames test, performed according to OECD 471 (1997) and GLP, the substance was found not to be mutagenic with or without metabolic activation.
Executive summary:

The mutagenic potential of the substance and/or its metabolites was assessed in an Ames test, performed according to OECD guideline 471 (1997) and GLP principles. The test item was tested in several strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in one strain of Escherichia coli (WP2uvrA) in the absence and in the presence of a metabolic activation system (S9-mix). The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. The test concentrations were corrected for the purity of the test substance.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction of the bacterial background lawn and/or a decrease in the number of revertants, was observed in tester strain TA100 at dose levels of 1600 and 5000 μg/plate in the absence and presence of S9-mix. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 2000 µg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and up to a concentration of 5000 µg/plate in the tester strain WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates at these top dose levels. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.