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Basic toxicokinetics

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basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Partial information, only interim results are available, without a formal report

Data source

Reference Type:
other: interim results
Report date:

Materials and methods

Objective of study:
Principles of method if other than guideline:
- Principle of test: assess the in vitro metabolism of hexafluorobutadiene and the presence of glutathione conjugates in rat microsomes and cytosol
- Short description of test conditions: rat or human liver microsomes (1 mg/ml protein) were incubated with 10 mM reduced glutathione, then the gas was bubbled through the mixture. Samples taken at various timepoints and analysed by ESI-LC-MS.
- Parameters analysed / observed: analysis of glutathione conjugates (additiona and/or substitution.
GLP compliance:

Test material

Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Details on test material:
No details available

Results and discussion

Metabolite characterisation studies

Metabolites identified:
the analysis was not technically feasible
Details on metabolites:
Results with hexafluorobutadiene:
3 Peaks were identified on the mass spectra, but full identification of the conjugates was not possible due to impossibility to synthesise the relevant standards.

Any other information on results incl. tables

Theoritical reactions:

- Addition product resulting from reaction of reduced glutathione with hexafluorobutadiene would result in a peak in positive ion spectrum with a mass of 470.

- substitution product resulting from reaction of reduced glutathione with hexafluorobutadiene would result in a peak in positive ion spectrum with a mass of 450.

Results obtained with TFE:

- only 1 addition product was detected with TFE experiment.

- slight glutathione depletion (<10%)

Results obtained with HCBD:

- only 1 main substitution product was detected with HCBD experiment.

- little or no GHS depletion (<5%)

Results obtained with hexafluorobutadiene (HFBD):

- two peaks were detected for the addition reaction. (mono-glutathione conjugates)

- three peaks were detected for the substitution reaction.

Those results were obtained with rat and human liver microsomes, and cytosol.

The nature of the conjugates could not be identified due to technical feasibility (the corresponding standards could not be successfully synthesised and the study was ended).

With regard to formation of glutathione addition and substitution reaction products, there was a greater reactivity with rat liver microsomes compared to rat cytosol and human liver microsomes or cytosol.

There was a greater glutathione depletion with time in mixture containing rat liver microsomes incubated with hexafluorobutadiene (approx 50% decrease after 30 min incubation), compared to the two other chemicals TFE and HCBD (<5% decrease).

Comparatively, the glutathione depletion was more limited in human liver microsomes (approx 20% decrease after 30 min incubation with HFBD).

Applicant's summary and conclusion

These preliminary in vitro data supported the formation of a mixture of addition and substitution products resulting from reactions of hexafluorobutadiene (HFBD) with reduced glutathione in rat liver microsomes. A greater efficiency was found with the rat microsomes compared to cytosolic fraction indicating that the reactions are catalysed mainly by the microsomal glutathione S-transferase.
This was further supported by the time-dependent depletion of reduced glutathione observed in the incubation mixtures, especially with rat liver microsomes incubated in the presence of HFBD.
These results support the hypothesis that hexafluorobutadiene is metabolised in the liver by a conjugation reaction with glutathione (catalysed by hepatic glutathione S-transferase), which is the first step of a complex metabolic pathway previously identified for other fluoroalkenes which ultimately may lead to specific renal toxicity.