Ammonium undecafluorohexanoate

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

- Principle of test: Binding and activation of the test item to the human peroxisome proliferator-activated receptor gamma (hPPARg)
1. Determination of the binding affinity of the test item to the hPPARg ligand binding domain (LBD)
2. Activation of the hPPARg by the test item
3. Molecular docking of the test item with the hPPARg-LBD

- Short description of test conditions:
1. Determination of the binding affinity of the test item to the hPPARg ligand binding domain (LBD)
The binding affinity of the test item to the nuclear receptor was assessed using fluorescence polarization based displacement assay. The PPARg-LBD, a fluorescent probe (C8-BODIPY) and different concentrations of the test item were incubated for 10 min at room temperature. Afterwards, fluorescence polarization was determined at 510 nm wave length using a Horiba Fluoromax-4 spectrofluorimeter.

2. Activation of the hPPARg by the test item
Co-transfected HepG2 cells expressing full length hPPARg and a firefly luciferase reporter PPAR response element (PPRE) were exposed to non-cytotoxic concentrations of the test item for 24 h to study the agonistic activity of the test item on the hPPARg pathway. In addition, the cells were co-transfected with a renilla luciferase encoding plasmid as internal control to correct for difference in transfection and harvest efficiencies. After 24 h of exposure, the effect of the tests item on PPRE binding and subsequent PPARg activation was evaluated by quantification of renilla and firefly luciferase activity.

3. Characterisation of the binding interaction of test item with the hPPARg-LBD
Molecular docking was performed to understand and characterise the interactions between the test item and the hPPARg-LBD. Hydrogen bond interactions of the test item with the molecular structure of the receptor and the orientation of the test item in the amino acids of the protein folding structure were simulated.

- Parameters analysed:
Dissociation constant Kd and IC50 of the test item and the receptor ligand binding domain
Cytotoxicity
Luciferase activity as reporter for test item-induced receptor activity
Molecular interactions of the test item with the receptor

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: structure-dependent binding and activation of the human peroxisome proliferator activated receptor gamma

Details on study design:

1. Determination of the binding affinity of the test item to the hPPARg ligand binding domain (LBD):
hPPARg was titrated to 40 nM C8-BODIPY in Tris-HCl buffer and the fluorescence polarisation was measured at 510 nm using a Horiba Fluoromax-4-spectrofluorimeter equipped with a fluorescence polarisation optics module. Fluorescence polarisation values were plotted against the hPPARg-LBD concentration and a curve fit was performed to extract the dissociation constant Kd.
The binding affinity of the test item was determined in a competitive binding assay. 800 nM hPPARg-LBD in phosphate buffered saline (PBS), 40 nM C8-BODIPY and different concentrations of the test item were incubated for 10 min at room temperature. The fluorescence polarisation values were recorded and the displacement to bound fluorescent probe was calculated. The curves were fitted with a sigmoidal model to derive an IC50 value.
The dissociation constant Kd of the test item and hPPARg-LBD were determined according to the following equation:

IC50 (ligand)/C8-BODIPY (total) = Kd (ligand)/Kd (C8-BODIPY)

2. Activation of the hPPARg by the test item:
HepG2 cells were cultured in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum, 100U/mL penicillin and 100 µg/mL streptomycin at 37 °C and 5% CO2. Cytotoxicity of the test item was determined in a MTT assay to identify non-cytotoxic concentrations of the test item after 24 h of exposure.
The cells were further transfected with two plasmids, (i) a firefly luciferase reporter PPAR response element (PPRE) for determination of hPPARg activation and (ii) a renilla luciferase encoding plasmid as internal control to correct for difference in transfection and harvest efficiencies. After 24 h of exposure to different concentrations of the test item, the effect of the tests item on PPRE binding and subsequent PPARg activation was evaluated by quantification of renilla and firefly luciferase activity.


3. Molecular docking of the test item with the hPPARg-LBD:
The test item was docked into the 3D crystal structure of hPPARg-LBD using Lamarckian genetic algorithm provided by Autodock 4.2 software.

Details on results:

1. Determination of the binding affinity of the test item to the hPPARg ligand binding domain (LBD):
The IC50 and the dissociation constant of the test item were not detectable.

2. Activation of the hPPARg by the test item:
There was an apparent dose-dependent increase in activation of hPPARg compared to vehicle controls starting at concentrations of about 75 µM.

3. Molecular docking of the test item with the hPPARg-LBD:
The test item formed hydrogen bonds with the following amino acids of hPPARg-LBD: Ser-289, His-449 and Tyr-473. The test item structure was shown to bind to the hPPARg binding cavity and successfully docked into the molecular structure of the receptor.

Conclusions:

The test item was shown to dose-dependently activate the human PPARg receptor. Activation was shown at concentrations ≥ 75 µM. In addition, molecular docking was performed and showed that the test item binds to the hPPARg binding cavity and forms hydrogen bonds to hPPARg amino acids : Ser-289, His-449 and Tyr-473.

Reference 2

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

- Principle of test: Binding and activation of the test item to the human and mouse peroxisome proliferator-activated receptor alpha (hPPARa and mPPARa)

- Short description of test conditions:
Transiently transfected COS-1 cells expressing full length hPPARa or mPPARa were exposed to test item concentrations of 5 – 100 µM for 24 h. After the exposure period, luciferase activity was measured to quantify the test-item induced activation of the corresponding receptors.

- Parameters analysed:
No observed effect concentration (NOEC)
Low observed effect concentration (LOEC)
C20max: Concentration that produces 20% of the maximal response (extrapolated from the regression formula and slope of the dose-response curve)

GLP compliance:

no

Type of method:

in vitro

Details on study design:

COS-1 cells were cultured in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% fetal bovine serum, 200U/mL penicillin and 0.2 mg/mL streptomycin at 37 °C and 5% CO2.
The cells were transiently transfected with plasmids encoding human or mouse PPARa fused with a luciferase reporter gene (1 µg/µL for 3 h). 24 h after transfection, exposure with the test item was performed. Nine different concentrations in the range of 5 – 100 µM were tested in 4 – 8 replicates per concentration. In addition, vehicle controls (water or 0.1% DMSO) and a positive control (10 µM WY-14643) were included. After 24 h of exposure, the cells were washed, lysed and luciferase activity was determined using the Luciferase reporter assay kit (Promega) and a LUMIstar Galaxy luminometer (BMG Labtechnologies, Durham, USA).

Positive control:

The positive control WY-14643 elicited a significant increase in luciferase activity over its vehicle control for every assay (p < 0.0001), indicating proper performance of the assay.

Details on results:

The test item dose-dependently increased luciferase activity, demonstrating the activation of human and mouse PPARa.

NOEC (mouse PPARa): 10 µM
LOEC (mouse hPPARa): 20 µM, (6.28 µg/mL), p < 0.0001

Regression analysis of dose-response activation (mouse):
Slope of regression line: 0.0059
Regression p value: < 0.0001
R2: 0.48

Relative responses of PPARa to the test item in transiently transfected cells
Cmax (mouse) = 38 µM

NOEC (human PPARa): 5 µM
LOEC (human hPPARa): 10 µM (3.14 µg/mL), p < 0.05

Regression analysis of dose-response activation (human):
Slope of regression line: 0.0048
Regression p value: < 0.0001
R2: 0.80

Relative responses of PPARa to the test item in transiently transfected cells
Cmax (human) = 47 µM

Conclusions:

The test item dose-dependently activated human and mouse PPARa. The activation response was higher for mouse PPARa than in human PPARa. Lowest obtained effect concentrations (LOEC) were 20 µM for mouse PPARa and 10 µM for human PPARa.

Description of key information

Binding and activation of the human peroxisome proliferator-activated receptor gamma (hPPARg) in HepG2 cells: positive

Activation of the human and mouse peroxisome proliferator-activated receptor alpha (hPPARa and mPPARa) in COS-1 cells: positive

Additional information

Publications on mechanistical studies in vitro are available for the structurally similar substance undecafluorohexanoic acid (CAS 307-24-4). Thus, a read-across was performed to investigate the mode of action of the target substance ammonium undecafluorohexanoate (CAS 21615-47-4).

 

The first publication investigates the binding of CAS 307-24-4 to the human peroxisome proliferator-activated receptor gamma (hPPARg) and the activation of the receptor (Zhang, 2014). Transfected Hep-G2 cells expressing the hPPARg and the luciferase reporter PPAR response element (PPRE) were exposed to test item concentrations in the range of 0.1 to 100 µM for 24 h. The test item was shown to dose-dependently activate the receptor when compared to vehicle controls.

In addition, molecular docking was performed. The test item (CAS 307-24-4) was found to bind to the binding cavity of the hPPARg ligand binding domain, forming hydrogen bonds to several amino acids in the binding cavity of the nuclear receptor. Inclusion of positive (Rosiglitazone) and vehicle controls confirmed the validity and sensitivity of the test system.

 

The second publication assessed the activation of human and mouse peroxisome proliferator-activated receptor alpha (hPPARa and mPPARa) by source substance CAS 307-24-4 (Wolf, 2008). COS-1 cells were transiently transfected with plasmids encoding the human or mouse PPARa gene and exposed to test item concentrations in the range of 5 – 100 µM, positive (WY-14643) or vehicle (DMSO) controls. After 24 h of exposure, luciferase activity was determined.

The results obtained with the positive and the vehicle controls demonstrated the proper function and sensitivity of the test system. The test item (CAS 307-24-4) showed a dose-dependent activation of the human, as well as the murine PPARa receptor. Lowest observed effect concentrations (LOEC) were higher for mouse (20 µM) compared to human (10 µM) PPARa. In addition, activation responses were higher for the mouse than for the human receptor.

 

Based on the findings obtained with the structurally related substance undecafluorohexanoic acid (CAS 307-24-4), ammonium undecafluorohexanoate is expected to also bind to and activate the nuclear receptors human PPARg, human PPARa and mouse PPARa.