1-Propene, 1,3,3,3-tetrafluoro-

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Reason / purpose for cross-reference:

reference to same study

Objective of study:

metabolism

Principles of method if other than guideline:

The biotransformation of HFO-1234ze following inhalation exposure was evaluated by determining urinary metabolites excreted for up to 48 hours following exposure.

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann, Borchen Germany
- Weight at study initiation: 220 - 250 g

Route of administration:

inhalation: gas

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

Male rats (n = 5) were exposed to targeted concentrations of 2000, 10000, and 50000 ppm HFO-1234ze in a dynamic exposure chamber consisting of a 20.6-L desiccator, a stirrer and connections to compressed air and a cylinder of HFO-1234ze fitted with flow meters. Metered amounts of HFO-1234ze were mixed with air and introduced into the exposure chamber. Chamber concentrations of HFO-1234ze were monitored at 15-min intervals by taking samples (100 µL) of the chamber atmosphere with a gastight syringe. The content of HFO-1234ze in these samples was determined by GC/MS.

Duration and frequency of treatment / exposure:

acute 6 hour exposure

Dose / conc.:

2 000 ppm

Dose / conc.:

10 000 ppm

Dose / conc.:

50 000 ppm

No. of animals per sex per dose / concentration:

5

Control animals:

no

Details on study design:

- Dose selection rationale: doses used were the same as those used for a study on a comparable fluorocarbon (HFO-1234yf)

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: every 6 hours for 48 hours
- From how many animals: not pooled
- Method type(s) for identification: NMR, LC/MS, GC/MS
- Limits of detection and quantification: 1.2 pmol/mL N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine, 2.9 pmol/mL S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid, 250 pmol/mL of 3,3,3-trifluoropropionic acid

- Tissues and body fluids sampled : urine
- Time and frequency of sampling: every 6 - 12 hours for 48 hours
- From how many animals: 5
- Method type(s) for identification: NMR, LC/MS, GC/MS

Statistics:

Not described

Metabolites identified:

yes

Details on metabolites:

S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid, S-(3,3,3-Trifluoro-trans-propenyl)-l-cysteine, N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-l-cysteine and 3,3,3-trifluoropropionic acid

In urine samples of rats exposed to 50,000 ppm HFO-1234ze, the predominant metabolite was S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid and accounted for 66% of all integrated (19)F-NMR signals in urines. No (19)F-NMR signals were found in spectra of rat urine samples collected after inhalation exposure to 2000 or 10,000 ppm HFO-1234ze likely due to insufficient sensitivity.

S-(3,3,3-Trifluoro-trans-propenyl)-l-cysteine, N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-l-cysteine and 3,3,3-trifluoropropionic acid were also present as metabolites in urine samples of rats.

Quantification of three metabolites in urines of rats was performed, using LC/MS-MS and GC/MS. The quantified amounts of the metabolites excreted with urine in both mice and rats, suggest only a low extent (<1% of dose received) of biotransformation of HFO-1234ze and 95% of all metabolites were excreted within 18 h after the end of the exposures (t(1/2) app. 6 h). The obtained results suggest that HFO-1234ze is likely subjected to an addition-elimination reaction with glutathione and to a CYP 450 mediated epoxidation at low rates.

The very low extent of biotransformation following inhalation to high exposures of HFO-1234ze indicates covalent binding resulting in potential liver toxicity is likely prevented by effecient detoxification by glutathione. These results are consistent with the lack of hepatotoxic in rats following 90 day inhalation exposure to HFO-1234ze (see section 7.5 for details of this study).

Conclusions:

In urine samples of rats exposed to 50000 ppm HFO-1234ze, the predominant metabolite was S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid and accounted for 66 % of all integrated (19)F-NMR signals in urines. S-(3,3,3-Trifluoro-trans-propenyl)-l-cysteine, N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-l-cysteine and 3,3,3-trifluoropropionic acid were also present as metabolites in urine samples of rats. Quantification of three metabolites in urines of rats was performed, using LC/MS-MS and GC/MS. This quantification suggest only a low extent (<1% of dose received) of biotransformation of HFO-1234ze and 95 % of all metabolites were excreted within 18 h after the end of the exposures (t(1/2) app. 6 h). The main difference between the rat and mice metabolites is the fact the concentration of S-(3,3,3-Trifluoro-trans-propenyl)-mercaptolactic acid attributes to 66 % of the metabolites in rat in comparison to only 8 % in mice. The obtained results suggest that HFO-1234ze is likely subjected to an addition-elimination reaction with glutathione and to a CYP 450 mediated epoxidation at low rates. The very low extent of biotransformation following inhalation to high exposures of HFO-1234ze indicates covalent binding resulting in potential liver toxicity is likely prevented by efficient detoxification by glutathione. These results are consistent with the lack of hepatotoxic in rats following 90 day inhalation exposure to HFO-1234ze (see section 7.5 for details of this study).

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Objective of study:

metabolism

Principles of method if other than guideline:

The biotransformation of HFO-1234ze following inhalation exposure was evaluated by determining urinary metabolites excreted for up to 48 hours following exposure.

GLP compliance:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan-Winkelmann, Borchen Germany
- Weight at study initiation: 27 - 30 g

Route of administration:

inhalation: gas

Details on exposure:

TYPE OF INHALATION EXPOSURE: nose only

Inhalation exposure of male mice (n=5) was performed using only a single concentration of 50,000 ppm HFO-1234ze in a dynamic exposure chamber consisting of a 20.6-L desiccator, a stirrer and connections to compressed air and a cylinder of HFO-1234ze fitted with flow meters. Metered amounts of HFO-1234ze were mixed with air and introduced into the exposure chamber. Chamber concentrations of HFO-1234ze were monitored at 15-min intervals by taking samples (100 µL) of the chamber atmosphere with a gastight syringe. The content of HFO-1234ze in these samples was determined by GC/MS.

Duration and frequency of treatment / exposure:

acute 6 hour exposure

Dose / conc.:

50 000 ppm

No. of animals per sex per dose / concentration:

5

Control animals:

no

Details on study design:

- Dose selection rationale: doses used were the same as those used for a study on a comparable fluorocarbon (HFO-1234yf)

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: every 6 hours for 48 hours
- From how many animals: not pooled
- Method type(s) for identification: NMR, LC/MS, GC/MS
- Limits of detection and quantification: 1.2 pmol/mL N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine, 2.9 pmol/mL S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid, 250 pmol/mL of 3,3,3-trifluoropropionic acid

- Tissues and body fluids sampled : urine
- Time and frequency of sampling: every 6 - 12 hours for 48 hours
- From how many animals: 5
- Method type(s) for identification: NMR, LC/MS, GC/MS

Statistics:

Not described

Metabolites identified:

yes

Details on metabolites:

S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid, S-(3,3,3-Trifluoro-trans-propenyl) -l-cysteine, N-acetyl-S-(3,3,3 -trifluoro-trans-propenyl) -l-cysteine and 3,3,3-trifluoropropionic acid

S-(3,3,3-Trifluoro-trans-propenyl)-mercaptolactic acid, S-(3,3,3 -Trifluoro-trans-propenyl)-l-cysteine, N-acetyl-S-(3,3,3 -trifluoro-trans-propenyl)-l-cysteine and 3,3,3 -trifluoropropionic acid were present as metabolites in urine samples of mice. A presumed amino acid conjugate of 3,3,3-trifluoropropionic acid was the major metabolite of HFO-1234ze in urine samples of mice exposed to 50000 ppm and related to 18 % of total integrated (19)F-NMR signals.

 

Quantification of three metabolites in urines of mice was performed using LC/MS-MS and GC/MS ( S-(3,3,3-Trifluoro-trans-propenyl)- mercaptolactic acid, N-Acetyl-S-(3,3,3-trifluoro-trans-propenyl)-l–L-cysteine and 3,3,3 -Trifluoroprprionic acid). The quantified amounts of the metabolites excreted with urine in both mice and rats, suggest only a low extent (<1% of dose received) of biotransformation of HFO-1234ze and 95% of all metabolites were excreted within 18 h after the end of the exposures (t(1/2) app. 6 h).

 

The obtained results suggest that HFO-1234ze is likely subjected to an addition-elimination reaction with glutathione and to a CYP 450 mediated epoxidation at low rates.

The very low extent of biotransformation following inhalation to high exposures of HFO-1234ze indicates covalent binding resulting in potential liver toxicity is likely prevented by effecient detoxification by glutathione. These results are consistent with the lack of hepatotoxic in rats following 90 day inhalation exposure to HFO-1234ze (see section 7.5 for details of this study).

Conclusions:

The quantified amounts of the metabolites excreted with urine in both mice and rats, suggest only a low extent (<1 % of dose received) of biotransformation of HFO-1234ze and 95 % of all metabolites were excreted within 18 h after the end of the exposures (t(1/2) app. 6 h). The obtained results suggest that HFO-1234ze is likely subjected to an addition-elimination reaction with glutathione and to a CYP 450 mediated epoxidation at low rates. The major metabolite of HFO-1234ze detected in urine samples of mice exposed to 50,000 ppm and related to 18 % of the total (19) F-NMR signal is a presumed to be an amino acid conjugate of 3,3,3-trifluoropropionic acid. Additional identified identified metabolites are S-(3,3,3-Trifluoro-trans-propenyl)- mercaptolactic acid S-(3,3,3-Trifluoro-trans-propenyl) -l-cysteine, N-acetyl-S-(3,3,3 -trifluoro-trans-propenyl) -l-cysteine and 3,3,3-trifluoropropionic acid. The main difference between the rat and mice metabolites is the fact the concentration of S-(3,3,3-Trifluoro-trans-propenyl)-mercaptolactic acid attributes to 66% of the metabolites in rat in comparison to only 8 % in mice. Overall, metabolite recovery from mouse urine is lower than that of rat, likely due to lower CYP 2E1 activity in the mouse liver.

Reference 3

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Objective of study:

metabolism

Principles of method if other than guideline:

The biotransformation of HFO-1234ze was evaluated in rat hepatocytes +/- rat or human S9.

GLP compliance:

yes

Remarks:

conducted under GLP conditions without certificate

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Test animals
- Source: Harlan-Winkelmann, Borchen Germany
- Weight at study initiation: 220 - 250 g

For the in vitro enzymatic reactions:

Liver subcellular fractions and microsomes of rats were prepared as described previously (Schuster et al., 2008). Human S9 material was obtained from BD Biosciences (Woburn, MA). Incubations with HFO-1234ze contained microsomes or S9 (2–4 mg/mL) and a NADPH-generating system and/or glutathione (Werner et al., 1995). The supernatants were used to record 1H-coupled and 1H-decoupled 19F-NMR spectra. Hydroxylation of p-nitrophenol was determined as described (Koop, 1986; Harris et al., 1991). The absorbance of 4-nitrocatechol was measured spectrophotometrically at 510 nm (a = 14.6 mmol− 1 cm− 1).

Route of administration:

other: in vitro microsomes

Duration and frequency of treatment / exposure:

Reaction mixtures containing microsomes/9,000xg supernatants, cofactors, and buffer were placed in sealed 2-mL GC vials with gaseous HFO-1234ze for 40 minutes

Remarks:

10 - 100 μL

Control animals:

no

Details on study design:

To induce CYP2E1, rats were given pyridine (100 mg/kg ip, dissolved in isotonic sodium chloride solution) once daily for 5 days . All animals were fasted 18 h before sacrifice and preparation of microsomes .

Statistics:

Not described

Metabolites identified:

yes

Details on metabolites:

2-S-(1-carboxy-3,3,3-trifloropropyl)-glutathione and S-(3,3,3-trifluoro-trans-propenyl)-glutathione

To characterize biotransformation by subcellular fractions, rat liver microsomes and rat liver cytosol and human S9 fractions were incubated with HFO-1234yf and appropriate cofactors. In incubations of HFO-1234ze with cytosol or microsomes with and without glutathione, but without NADPH, metabolite formation was not detected by 19F-NMR.

The absence of any signal in¹⁹F-NMR spectra of incubations with subcellular fractions is indicative for a very low biotransformation of HFO-1234ze in vitro and consistent with the observation of a very low extent of biotransformation of HFO-1234ze in vivo, even after exposures to high concentrations of up to 50,000 ppm. More sensitive LC/MS-MS analysis of microsomal incubations showed small signals indicative of formation of 2-S-(1-carboxy-3,3,3-trifloropropyl)-glutathione and S-(3,3,3 -trifluoro-trans-propenyl)-glutathione. However, the in vitro findings support the proposed pathway of biotransformation of HFO-1234ze in rats by both, epoxidation and addition-elimination reaction with glutathione. The low reactivity of HFO-1234ze with glutathione may be due to steric and electronic factors reducing the reactivity of HFO-1234ze with soft nucleophiles such as the thiolate ion of glutathione.

Conclusions:

Little if any metabolism was observed in vitro using rat liver microsomes with rat or human S9. Consequently none of the metabolites found in vivo could be confirmed in vitro. The absence of any signal in 19F-NMR spectra of incubations with subcellular fractions is indicative for very low biotransformation of HFO-1234ze in vitro and consistent with the observation of very low extent of biotransformation of HFO-1234ze in vivo, even after very high exposure concentrations.
The in vitro findings do support the proposed pathway of biotransformation of HFO-1234ze in rats by both epoxidation and addition-elimination reaction with glutathione.

Description of key information

Based on the available toxicokinetics information no quantitative conclusions can be drawn with respect to absorption, metabolism and excretion as no mass balance was determined in the in vivo studies. However, the available in vitro study it can be concluded that the level of biotransformation of the substances is likely to be very low. This was confirmed by the in vivo inhalation studies where less than 1 % of the received dose was recovered from urine in the form of metabolites, thus also indicating that biotransformation of the substance is low. Furthermore, the majority of the absorbed HFO-1234ze compound is likely to have been exhaled as parent compound due to its low boiling point. In the absence of substance-specific absorption data, the default absorption values from the REACH guidance (Chapter 8, R.8.4.2) are used for DNEL derivation, namely: 100% for inhalation, 50% for oral and 50% for dermal absorption.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

50

Additional information

Absorption

Substance-specific absorption data for the oral and dermal route are not available. However, the substance is a gas and therefore dermal and oral exposure is unlikely.

The substance has a vapour pressure of 4271 hPa and therefore inhalation is the major route of exposure. Experiments in rats indicated absorption as systemic effects were observed in a sub-chronic inhalation study at 15000 ppm. Systemic effects were also observed in two supporting repeated dose inhalation studies. Using physiologically based pharmacokinetic (PBPK) modeling techniques, the blood/air (BA) partition coefficient in the human (0.220 ± 0.051, combined mixed and female) was approximately one-half the rat (0.468 ±0.088).

Metabolism

The biotransformation of HFO-1234ze was investigated after inhalation exposure. Male Sprague-Dawley rats were exposed to air containing 2000, 10,000, or 50,000 ppm (n=5/concentration) HFO-1234ze. Male B6C3F1 mice were only exposed to 50,000 ppm HFO-1234ze. All inhalation exposures were conducted for 6 h in a dynamic exposure chamber. After the end of the exposures, animals were individually housed in metabolic cages and urines were collected at 6 or 12 h intervals for 48 h.

For metabolite identification, urine samples were analyzed by (1)H-coupled and (1)H-decoupled (19)F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by (19)F-NMR chemical shifts, signal multiplicity, (1)H-(19)F coupling constants and by comparison with synthetic reference compounds.

In urine samples of rats exposed to 50,000 ppm HFO-1234ze, the predominant metabolite was S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid and accounted for 66% of all integrated (19)F-NMR signals in urines. No (19)F-NMR signals were found in spectra of rat urine samples collected after inhalation exposure to 2000 or 10,000 ppm HFO-1234ze likely due to insufficient sensitivity. S-(3,3,3-Trifluoro-trans-propenyl)-l-cysteine, N-acetyl-S-(3,3,3 -trifluoro-trans-propenyl)-l-cysteine and 3,3,3-trifluoropropionic acid were also present as metabolites in urine samples of rats and mice. A presumed amino acid conjugate of 3,3,3-trifluoropropionic acid was the major metabolite of HFO-1234ze in urine samples of mice exposed to 50,000 ppm and related to 18% of total integrated (19)F-NMR signals. Quantification of three metabolites in urines of rats and mice was performed, using LC/MS-MS and GC/MS.

The quantified amounts of the metabolites excreted with urine in both mice and rats, suggest only a low extent (<1% of dose received) of biotransformation of HFO-1234ze and 95% of all metabolites were 1 excreted within 18 h after the end of the exposures (t(1/2) app. 6 h). The obtained results suggest that HFO-1234ze is likely subjected to an addition-elimination reaction with glutathione and to a CYP 450 mediated epoxidation at low rates. The lack of significant oxidation and glutathione conjugation may explain the lack of hepato or nephrotoxicity with HFO-1234ze.

Conclusion

Based on the presented data, no quantitative conclusions can be drawn with respect to absorption, distribution, metabolism and excretion, as no mass balance was determined in any of the in vivo studies. However, since less than 1% of the administered dose was recovered from urine in the form of metabolites, biotransformation appears to be quite low and most of the absorbed HFO-1234ze will have been exhaled as parent compound. HFO-1234ze total body burden of exposure via the skin (acting as a barrier) is expected to be much lower than via inhalation. In the absence of substance-specific absorption data, the default absorption values from the REACH guidance (Chapter 8, R.8.4.2) are used for DNEL derivation, namely: 100% for inhalation, 50% for oral and 50% for dermal absorption.