1H-Pyrrole-1,3-dicarboxylic acid, 4-ethyl-2,5-dihydro-, 1-(phenylmethyl) ester

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:

23 May - 21 July, 2017

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Sterility checks were conducted on both the top concentration of the formulated test article and onthe metabolic activation mix (S9), even though there was no directive in the study plan or test method to do so. This deviation has no impact to the study as the sterility checks were used solely to ensure that there was no contamination of the reagents used in this study.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-[(benzyloxy)carbonyl]-4-ethyl-2,5-dihydro-1H-pyrrole-3-carboxylic acid

EC Number:

813-192-3

Cas Number:

1869118-25-1

Molecular formula:

C15H17NO4

IUPAC Name:

1-[(benzyloxy)carbonyl]-4-ethyl-2,5-dihydro-1H-pyrrole-3-carboxylic acid

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Aroclor™ 1254-induced exogenous rat liver metabolic activation system (S9)

Test concentrations with justification for top dose:

The test article was formulated in dimethyl sulfoxide. The test material formd a colorless slution at hte highest concentration tested of 50 mg/mL. The test substance was evaluated in the mutagenicity assay using the plate incorporation treatment method at concentrations of 0.3, 0.8, 2, 7, 22, 67, 200, 600 and 1000 μg/well both with and without S9.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was the vehicle control

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: N-methyl-N'-nitro-N-nitrosoguanidin and 2-aminoanthracene

Details on test system and experimental conditions:

Preparation of Overnight Cultures: Inoculation:
Overnight cultures were inoculated into flasks containing culturing broth and the flasks were placed in a shaker/incubator programmed to begin operation (shaking, 130± 25rpm; incubation, 37 ±2°C) so that overnight cultures were in late log phase when optical density (OD) monitoring began.To ensure cultures were harvested at the appropriate phase, the growth of each culture was monitored using a spectrophotometer. An aliquot of each culture was used to determine the OD at 660nm. Cultures were analyzed by spectroscopy so that an absorbance reading of 1.5 to 2.3 was reached. This suggested that the bacterial growth was in log phase growth and bacteria were at a concentration of ≥ 1 x 10^9 cells/mL. Overgrown (stationary) cultures may have exhibited decreased sensitivity to some mutagens. Once the incubation period ended, the cultures were removed and were used the same day after the target OD was reached.

Tester Strain Media:
Culturing Broth: Nutrient broth was used to grow overnight cultures of the tester strains.
Minimal Bottom Agar Plates: Bottom agar (4-5 mL per well of a 6-well plate) was 1.5% (w/v) agar and 2.0% (w/v) glucose in Vogel-Bonner minimal medium.
Nutrient Bottom Agar Plates: Nutrient bottom agar (4-5 mL per well of a 6-well plate) was 7.5% (w/v) agar and 12.5% (w/v) nutrient broth No. 2 with Vogel-Bonner salts and glucose added.
Top Agar for Selection of Revertants: Top agar contained 0.5% (w/v) agar and 0.5% (w/v) NaCl, supplemented with 0.5 mM histidine, 0.5 mM biotin, and 0.5 mM tryptophan.

Controls:
Spontaneous Controls: Spontaneous controls (bacterial samples plated without test item or vehicle) were plated (n = 3 per bacterial strain, with and without S9) for each assay run. Spontaneous controls were not used in the evaluation of mutagenicity, but rather added to the characterization of the bacterial strains used for testing in each assay.

Vehicle Controls: The vehicle was plated concurrently in all strains, in the presence and absence of S9 at the same volume as used to administer the test articles.

Positive Controls: The combinations of positive controls, activation conditions and tester strains evaluated.

Metabolic Activation System (S9):
Liver Homogenate and S9 MixAroclor™ 1254-induced rat liver homogenate (S9) was purchased commercially and used with co-factors (S9 mix). S9 mix was prepared by standard procedures on the day of use and kept on ice until needed.

Mutagenicity Assay:
Design: Six- well plates were used and each testing condition was conducted in a well of the 6-well plate. Using such wells, the limit dose of 5000 μg/well, suggested by OECD471, was reduced to 1000 μg/well based on the surface area of the well compared to that of a Petri plate. The assay was performed using the plate incorporation method. The test articles were evaluated in the mutagenicity assay at concentrations 0.3, 0.8, 2, 7, 22, 67, 200, 600 and 1000 μg/well. Positive and vehicle controls were also evaluated. Test and positive control article concentrations were evaluated using triplicate wells; vehicle controls were evaluated using six wells.

Frequency and Route of Administration:
Tester strains were exposed to the test article via the plate incorporation methodologyoriginally described by Maron and Ames. In the plate incorporation methodology, the tester strain, test article, and S9mix (where appropriate) are combined in molten supplemented top agar, which is then overlaid on minimal bottom agar in a well of a 6-well plate. Following incubation, revertant colonies were counted.

Plating Procedures:
Each 6-well plate was labeled with the study number, date, test article, tester strain, activation condition, and concentrations. Treatments in the absence of S9 were performed by adding 20 μL test or control articles, 100 μL of phosphate buffered saline (PBS) and 25 μL tester strain to sterile tubes containing 0.5mL molten supplemented top agar (maintained at 46± 2°C). The mixture was overlaid onto the surface of bottom agar wells and gently spread with swirling of the plate. The plates were kept at room temperature until the top agar solidified. After the top agar solidified, the 6-well plates were inverted and incubated for two days at 37 ± 2°C. When S9 was required, 100μL S9 mix was used instead of PBS.

Bacterial Background Lawn Evaluation:
The condition of the background lawn was evaluated for evidence of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control.

Counting Revertant Colonies:
Revertant colonies were counted by hand or automated colony counter (Accucount, Biologics Inc.). Colony counts were transcribed to a Microsoft Excel spreadsheet.

Evaluation criteria:

Assay Evaluation Criteria:
Once criteria for a valid assay have been met or the Study Director determined that the assayis valid, the responses observed in the assay are evaluated as follows:
Criteria for a Positive Response:
A test article is considered to have produced a positive response if it induces a concentration-dependent increase in revertant frequency that was ≥ 2.0-foldvehicle control values for tester strains TA98, TA100, and E. coli, or ≥ 3.0-fold vehicle control values for tester strains TA1535 and TA1537. Reproducibility of the response may be determined at the Study Director's discretion.
Criteria for a Negative Response:
A test article is considered to have produced a negative response if no concentration-dependent, ≥ 2.0-fold or ≥ 3.0-fold increases are observed in tester strains TA98, TA100, and E. coli, or TA1535 and TA1537, respectively.
Criteria for an Equivocal Response:
Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the concentration-dependency or fold increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response.Other criteria also may be used in reaching a conclusion about the study results (e.g.,comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly report and described anysuch considerations.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxicity observed with all Salmonella strains, without metabolic condition, beginning at 600 μg/well. Toxicity was observed at 1000 μg/well with the TA1537 strain with S9.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxicity observed with all Salmonella strains, without metabolic condition, beginning at 600 μg/well

Vehicle controls validity:

valid

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:

other: Toxicity observed with all Salmonella strains, without metabolic condition, beginning at 600 μg/well

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxicity observed with all Salmonella strains, without metabolic condition, beginning at 600 μg/well

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Toxicity was observed at 1000 μg/well with the E. coli strain without S9.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Ater the incubation period with the test material, there was toxicity observed with all Salmonella strains, without metabolic condition, beginning at 600 μg/well. Toxicity was observed at 1000 μg/well with the E. coli strain without S9 and the TA1537 strain with S9.

The criteria for a positive response were not met for any condition tested.

Any other information on results incl. tables

All vehicle control values were within acceptable ranges and the positive controls demonstrated the tester strains were capable of detecting mutagens. Therefore, the study was considered valid.

Applicant's summary and conclusion

Conclusions:

The results indicate that the test material was negative (non-mutagenic) in the 6-well 5-strain bacterial reverse mutation assay under the conditions, and according to the criteria, of the study plan.