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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study did not reference OECD guidelines and GLP. One dose level was tested on female rats. No information on age of the animals, housing and environmental conditions. This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Metabolism of hexafluoropropene: Evidence for bioactivation by glutathione conjudate formation in the Kidney
Author:
Koob M, Dekant W
Year:
1990
Bibliographic source:
Drug Metab Dispos. 18(6):911-916

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
yes
Remarks:
one single dose and one sex
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Hexafluoropropene
EC Number:
204-127-4
EC Name:
Hexafluoropropene
Cas Number:
116-15-4
Molecular formula:
C3F6
IUPAC Name:
1,1,2,3,3,3-hexafluoroprop-1-ene
Constituent 2
Reference substance name:
1-Propene, hexafluoro-
IUPAC Name:
1-Propene, hexafluoro-
Details on test material:
Purity: 99%
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Source: Institut fur Versuchstierkunde (Hannover, FRG).
- Age at study initiation: No data.
- Weight at study initiation: 220-260 grams.
- Fasting period before study: No data.
- Housing: No data.
- Individual metabolism cages: Yes.
- Diet (e.g., ad libitum): ad libitum.
- Water (e.g., ad libitum): ad libitum.
- Acclimation period: No data.

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): No data.
- Humidity (%): No data.
- Air changes (per hr): No data.
- Photoperiod (hrs dark/hrs light): No data.

IN-LIFE DATES: No data.

Administration / exposure

Route of administration:
inhalation
Details on exposure:
TYPE OF INHALATION EXPOSURE: Whole body

GENERATION OF TEST ATMOSPHERE/CHAMBER DESCRIPTION:
- Exposure apparatus: Closed exposure system and HFP gas was introduced with an airtight syringe through an airtight septum.
Duration and frequency of treatment / exposure:
1 hr (in vivo study), 1 hr incubation (in vitro).
Doses / concentrations
Remarks:
Doses / Concentrations:
800 ppm (in vivo study); 1mM (in vitro).
No. of animals per sex per dose / concentration:
Two female rats (in vivo study). No information on number of cannulated rats. No information on number of replicates in the in vitro assay.
Control animals:
no
Details on study design:
In vivo study:
Two female rats were transferred into the closed exposure system and hexafluoropropene gas was introduced with an airtight syringe through an airtight septum to give a final concentration of 800 ppm. After 1 hour, the rats were transferred to a metabolic cage and urine was collected for 6 hours, and then extracted. Bile cannulation surgery was performed in rats. Cannulated rats were exposed to hexafluoropropene at 800 ppm for 1 hour and bile and urine were collected over 8 hours. Tap water was supplied ad libidum. Rat liver microsomes and cytosol were analyzed. Urine and bile samples were analyzed.

Enzymatic assay:
Rat liver microsomes and cytosol were prepared. Hexafluoropropene (HFP) gas (1mM) was introduced into the incubation mixture (0.1 M potassium phosphate buffer, pH 7.4, containing 0.1-0.5 mg/mL microsomal or 0.25-1 mg/mL cytosolic protein, 0.1 mM tetrasodium EDTA, and 10 mM GSH in a final volume of 2.5 mL) , with a gastight syringe. Samples were removed with a syringe through a gastight septum. In some experiments, an NADPH-generating system or 1-chloro-2,4-dinitro-benzene were included. The reactions were stopped by addition of 0.1 mL of 30 percent trichloroacetic acid. The precipitated protein was removed by centrifugation. Samples of the supernatant (0.01-0.05 mL) that were fractionated by HPLC. S-conjugates were quantified.

Separation and quantification of S-conjugates from supernatant samples were fractionated by HPLC. Conjugate concentrations from urine and bile samples were separated by HPLC and and identified by GC/MS.
Details on dosing and sampling:
Non-cannulated rats: Urine was collected for 6 hrs.
Cannulated rats: Bile and urine were collected over 8 hrs.
Statistics:
Results were expressed as mean +/- standard deviation.

Results and discussion

Toxicokinetic / pharmacokinetic studies

Details on excretion:
Non-cannulated rats: Urinary excretion of N-acetyl-hexafluoro-propyl-cysteine (N-Ac-HFPC) amounted to 10 +/- 3 percent of the dose of HFP introduced into the exposure system (three replicates). In the time interval from 6 hr to 24 hr, less than 1 percent of the HFP dose could be recovered in urine.

Cannulated rats: Of the administered HFP dose, 8 +/- was recovered as N-AC-HFPC in urine (three replicates).

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
In vitro: Incubations of HFP with liver and kidney subcellular fractions in the presence of GSH resulted in the formation of two metabolites, identified as S-hexafluoropropyl-glutathione (HFPG) and S-pentafluoropropenyl-glutathione (PFPG).

In vivo: a peak with identical retention time and mass spectrum as the synthetic mercapturic acid N-Ac-HFPC methyl ester was identified. The mass spectometric fragmentation of the metabolite definitively identifies N-Ac-HFPC as a urinary metabolite of hexafluoropropene in the rat. The results suggested that hexafluoropropene may be exclusively metabolized by GST in vivo in an addition-reaction to give HFPG.

Hexafluoropropene is metabolized by GST to HFPG and PFPG. Oxidative metabolism could not be demonstrated. The GSH S-conjugates are structural analogues to GSH S-conjugates biosynthesized from other nephrotoxic haloalkenes. The identification of GSH S-conjugates as metabolites of hexafluoropropene thus suggests that these mechanisms also are responsible for hexafluoropropene nephrotoxicity. Collected bile contained PFPG as the only detectable metabolite. Because N-Ac-PFPC or other PFPG metabolites could not be detected in the urine, it is possible that PFPG formed in the liver is not translocated to the kidney to be processed to the corresponding mercapturic acid. The possibility of complete metabolism of PFPG by the enzymes of the mercapturic acid pathway and by b-lyase could not be ruled out. However, the results support the speculation that intrarenal conjugation of hexafluoropropene with GSH may be an important step in the bioactivation of hexafluoropropene.

The results support the speculation that intrarenal conjugation of hexafluoropropene with GSH may be an important step in the bioactivation of hexafluoropropene. HFPG formed in the kidney could be processed by gamma-glutamyltranspeptidase and dipeptidases to the coresponding cysteine S-conjugate, which is metabolized by renal cystein conjudate b-lyase, to give an electrophilic intermediate, most likely a thionoacyl fluoride.
The above hypotheses were supported by analytical results.

Any other information on results incl. tables

Incubations of HFP with rat liver microsomes in the presence of an NADPH-regenerating system and oxygen did not form detectable amounts of pentafluoropropionic acid after 1 hr of incubation. Incubations of hexafluoropropene with liver and kidney subcellular fractions in the presence of GSH resulted in the time and protein concentration-dependent formation of two metabolites. Formation of these two compounds was not observed in incubations with denatured microsomal and cytosolic protein, and could be inhibited completely by the competitive GST inhibitor dinitrochlorobenzene (2mM). These metabolites were identified as S-hexafluoropropyl-glutathione (HFPG) and S-pentafluoropropenyl-glutathione (PFPG) based on GC/MS and NMR analysis. For the in vivo study, the obtained chromatogram showed a peak with identical retention time and mass spectum as the synthetic mercapturic acid N-Ac-HFPC methyl ester. The mass spectometric fragmentation of the metabolite definitively identifies N-Ac-HFPC as a urinary metabolite of hexafluoropropene in the rat. The results suggested that hexafluoropropene may be exclusively metabolized by GST in vivo in an addition-reaction to give HFPG. However, mercapturic acid excretion with urine may not be representative of the total extent of GSH S-conjugate formation in vivo. It is possible that a significant portion of S-conjugates formed in the liver are excreted with feces. GC/MS results for bile indicated that HFPG elimination from the liver with bile was less than 2 percent of the elimination of PFPG. In the urine of cannulated rats, N-Ac-HFPC also was the exclusive hexafluoropropene metabolite identified by GC/MS. Hexafluoropropene is metabolized by GST to HFPG and PFPG. Oxidative metabolism could not be demonstrated. The GSH S-conjugates are structural analogues to GSH S-conjugates biosynthesized from other nephrotoxic haloalkenes. The identification of GSH S-conjugates as metabolites of hexafluoropropene thus suggests that these mechanisms also are responsible for hexafluoropropene nephrotoxicity. The preferential formation of different GSH S-conjugates from hexafluoropropene by membrane-bound and soluble GST may be explained by mechanistic differences in the reaction of GSH with fluoroalkenes. In the first step of the enzymatic reaction between GSH and haloalkenes, the thiolate (GS-) reacts with the alkene to form a carbanion that may either eliminate F- to give a vinylic S-conjugate or react with a proton from water to give fluoroalkyl S-conjugate. The concentration of water near the active site may control the structure of product formed. In cytosol with a high water concentration, the intermediate carbanion reacts with a proton to give HFPG, whereas the water concentration in the lipophilic environment of microsomal membranes likely is lower. Thus, the membrane-bound GST may preferentially catalyze the formation of the vinylic S-conjugate PFPG. In vitro, GST catalyze the formation of two hexafluoropropene-derived GSH S-conjugates. In contrast, in vivo activation only yielded the mercapturic acid N-Ac-HFPC as a urinary metabolite, formed by processing of HFPG by the enzymes of mercapturic acid formation. Collected bile contained PFPG as the only detectable metabolite. Because N-Ac-PFPC or other PFPG metabolites could not be detected in the urine, it is possible that PFPG formed in the liver is not translocated to the kidney to be processed to the corresponding mercapturic acid. The possibility of complete metabolism of PFPG by the enzymes of the mercapturic acid pathway and by b-lyase could not be ruled out. However, the results support the speculation that intrarenal conjugation of hexafluoropropene with GSH may be an important step in the bioactivation of hexafluoropropene. HFPG formed in the kidney could be processed by gamma-glutamyltranspeptidase and dipeptidases to the coresponding cysteine S-conjugate, which is metabolized by renal cystein conjudate b-lyase, to give an electrophilic intermediate, most likely a thionoacyl fluoride. The above hypotheses were supported by analytical results and because 1) the approximately identical proportions of hexafluoropropene introduced into the exposure chamber found as N-Ac-HFPC in the urine of intact and bile duct-cannulated rats, suggesting that S-conjugate excretion with urine, and presumably S-conjugate formation in the kidney, is not affected by cannulation, and 2) hexafluoropropene is toxic to rabbit renal proximal tubule suspension, showing that renal enzymes may bioactivate hexafluoropropene to toxic metabolites, most likely S-conjugates. The different structure of the GSH S-conjugates formed in the liver and kidney may be due to differences in the contribution of membrane-bound and soluble GST to the metabolism of hexafluoropropene in the liver and kidney. In the liver, microsomal GST catalyze the major part of GSH-dependent hexafluoropropene metabolism. Due to the low concentrations of membrane-bound GST present in the kidneys, only HFPG is formed by soluble GST-catalyzed renal biotransformation of hexafluoropropene.

Applicant's summary and conclusion

Conclusions:
The study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

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