(3R,6S)-1-benzyl-6-methylpiperidin-3-amine acetyl-L-leucinate [1:1]

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:

31st January 2020 to 5th March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Guideline No. 440/2008. Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.13/14: "Mutagenicity: Reverse Mutation Test using Bacteria”. Official Journal of the European Union No. L142, 31 May 2008.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(3R,6S)-1-benzyl-6-methylpiperidin-3-amine acetyl-L-leucinate [1:1]

EC Number:

840-203-9

Cas Number:

2295746-51-7

Molecular formula:

C21H35N3O3

IUPAC Name:

(3R,6S)-1-benzyl-6-methylpiperidin-3-amine acetyl-L-leucinate [1:1]

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 homogenate: 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).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized.
- concentration or volume of S9 mix and S9 in the final culture medium:
To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.

Test concentrations with justification for top dose:

Selection of an adequate range of doses was based on a dose-range finding test with the
strains TA100 and WP2uvrA, both with and without 5% (v/v) S9-mix. Eight concentrations,
1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate. The highest
concentration of the test item used in the subsequent mutation assays was 5000 µg/plate.

Based on the results of the dose-range finding test, the following dose-range was selected for the
first mutation experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 µg/plate.

Vehicle / solvent:

The vehicle of the test item was ethanol (Extra pure, Merck, Darmstadt, Germany).

Solvents for Positive Control Items:
Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands)
DMSO = dimethyl sulfoxide (Merck, Darmstadt, Germany)

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10 9 cells/mL
- Test substance added in top agar tubes
In the second part of this experiment, the test item was tested both in the absence and presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test item was tested both in the absence and presence of 10% (v/v) S9-mix in all tester strains.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10 9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in ethanol or control solution and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4h.After this period revertant colonies(histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that PF-07202371-S7 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli
reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of PF-07202371-S7 and/or its metabolites to induce reverse mutations at the histidinelocus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).
The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 19-AN-00524 of the test item was a white powder with a purity of 99.9%. The vehicle of the test item was ethanol.
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 absence and presence of 5% (v/v) S9-mix. In the first mutation experiment, the test item was again tested up to concentrations of 5000 µg/plate in the strains TA1535, A1537 and TA98 in the absence and presence of 5% (v/v) S9-mix. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 492 to 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. In all three experiments the test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed, except in tester strain TA100 in the absence of S9-mix in the first experiment and in the absence and presence of S9-mix in the second experiment, where toxicity, as evidenced by a decrease in the number of revertants, was observed.
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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.
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.
In conclusion, based on the results of this study it is concluded that PF-07202371-S7 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichiacoli reverse mutation assay.