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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted at a reputable laboratory with expertise in these types of studies. Not conducted under GLP

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
2013
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
no guideline followed
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: liquefied gas
Details on test material:
- Name of test material (as cited in study report): trans-1-Chloro-3,3,3-trifluoropropene (trans-HCFO-1233zd)
- Purity: 99.99%
Radiolabelling:
no

Test animals

Species:
other: Rat and rabbit in vivo and in vitro; human in vitro
Strain:
other: Sprague Dawley, New Zealand, and human liver microsomes
Sex:
male/female
Details on test animals and environmental conditions:
Male Sprague Dawley rats (Harlan, Venray, Netherlands) and female albino New Zealand rabbits (Charles River, Sulzfeld, Germany) were used for all inhalation exposures. Animal weights ranged from 212 g to 244 g for male rats and from 2.4 kg to 2.8 kg for female rabbits. All animals were stabilized for at least 7 days and examined daily (general conditions, skin and fur, eyes, respiration, nose, oral cavity and external genitalia) during the stabilization period to confirm suitability for the study. Signs of toxic effects were recorded during treatment and post treatment period.

Administration / exposure

Route of administration:
inhalation
Details on exposure:
Inhalation exposure of rats and rabbits to trans-HCFO-1233zd: Male rats (n=5/concentration) and female rabbits (n=3/concentration) were exposed to target concentrations of 0, 2,000, 5,000 and 10,000 ppm of trans-HCFO-1233zd for 6 h in open dynamic exposure chambers (Bayer et al., 2002; Schuster et al., 2008, 2010). trans-HCFO-1233zd chamber concentrations were determined by GC/MS (HP-Plot Q column). Chamber air samples (100 μL) were injected (split 5:1). The oven temperature was increased from 60 to 180°C within 7 min and a mass fragment characteristic for trans-HCFO-1233zd was recorded (trans-HCFO-1233zd, 95 [m/z], Rt=7.7 min; internal standard—oxygen, 32 [m/z], Rt=1.8 min). Quantification of trans-HCFO-1233zd was based on calibration curves of air samples containing known amounts of trans-HCFO-1233zd.

Duration and frequency of treatment / exposure:
6 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 2,000, 5,000 and 10,000 ppm
No. of animals per sex per dose:
5 male rats and 3 female rabbits
Details on study design:
- Chemicals: trans-1-Chloro-3,3,3-trifluoropropene (trans-HCFO-1233zd, purity of 99,99%) was supplied by Honeywell (Morristown, New Jersey). 3,3,3-Trifluorolactic acid (96%) was purchased from Alfa Aesar (Karlsruhe, Germany) and ammonia (purity≥99.98%) was purchased fromLinde (Pullach, Germany). All other chemicalswere obtained from Sigma-Aldrich (Taufkirchen, Germany) in the highest available purity. Synthesis and characterization of S-(1,2-dichlorovinyl)-glutathione (Vamvakas et al., 1988) and S-(3,3,3-trifluoro-trans-propenyl)- mercaptolactic acid were reported previously (Schuster et al., 2009). N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine, N-acetyl-d3-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine, and S-(3,3,3- trifluoro-trans-propenyl)-glutathione were synthesized.
- Animal treatment: To induce cytochrome P-450 2E1, Sprague Dawley rats were treated with pyridine (100 mg/kg ip, dissolved in isotonic chloride solution) once daily for 5 days before euthanized (Urban et al.,1994). Liver microsomes were prepared according to standard protocols (Herbst et al., 1994; Koster et al., 1994; Urban et al., 1994). Cytochrome P-450 2E1 activity in the liver microsomes was determined as 3.33±0.05 [nmol/min×mg (protein)] by analysis of the oxidation of p-nitrophenol (Koop, 1986). Urine was collected for approximately 12 h before initiation of the inhalation exposure and for approximately 48 h after exposure, at 6 and 12 h intervals. Urine was collected at 4°C and volume was measured for each collection interval. Blood samples were taken from rats approximately 12 h before initiation of the inhalation exposure and 0, 24 and 48 h after end of inhalation exposure. All samples were stored at −20°C until analysis.
- In vitro incubations: To study biotransformation, pooled human liver microsomes (Becton Dickinson, Heidelberg, Germany), rabbit liver S9 (Becton Dickinson, Heidelberg, Germany), or rat liver microsomes from pyridine-induced male rats (3 mg protein/mL) were incubated in sealed GC vials with either 10 mM glutathione or a NADPH regenerating system (10 mM NADP, 10 mM glucose-6-phosphate and 0.25 units/mL glucose-6-phosphate dehydrogenase) or 10 mM glutathione and a NADPH regenerating system in potassium phosphate buffer pH 7.4 containing 1 mM EDTA (final volume of 1 mL). Liquid trans-HCFO-1233zd (20 μL) was added through a septum with a microsyringe and the incubations were performed for up to 12 h at 37°C. Reactions were stopped by placing the opened GC vials on ice. For in vitro kinetic experiments either pooled human, male rat, or female rabbit S9 liver fractions (4 mg protein/mL) were pre-incubated with 10 mM glutathione in 100 mM potassium phosphate buffer pH 7.4 containing 1 mM EDTA (end volume 0.5 mL) in sealed GC vials for 5 min at 37 °C. Reaction mixtures were incubated for another 30 min, after adding gaseous trans-HCFO-1233zd (100, 250, 500, 750, 1,000 or 2,000 μL) through the septum with a gastight microliter syringe (equal amount of air was removed). Reactions were stopped by placing the opened GC vials on ice and adding 50 μL of acetonitrile.
- 19F NMR analyses: 19F NMR spectra were recorded with a Bruker DRX 300 NMR spectrometer with a 5 mm fluoride probe operating at 282.4 MHz. 19F chemical shifts were referenced to external CFCl3. Spectra were recorded with a 30° pulse, a pulse length of 13.6 μs and a cycle delay of 6 μs. The acquisition time was 3 s and 10,240 scans were recorded to obtain a good signal to noise ratio (S/N). The 19F spectra were acquired with proton coupling and a spectral width of 200 ppm (+10 to−190 ppm). Before integration of peak areas, an accurate baseline correction and spectra phasing were performed between chemical shifts of −60 and −85 ppm.
- Sample preparation for 19F NMR analysis: After thawing at 4 °C, urine samples (1 mL) were vortexed and then centrifuged for 15 min at 14,000 rpm and at 4 °C. Aliquots (720 μL) of the obtained supernatants were added to 80 μL of deuterium oxide. NMR spectra were analyzed with TopSpin 3.0 (Bruker) and trans-HCFO-1233zd metabolites were identified by comparison of their chemical shifts,multiplicities and coupling constants with spectra of authentic standards (Schuster, 2009; Schuster et al., 2008, 2010).
- LC-MS/MS analyses were performed with an API 3000 mass spectrometer (Applied Biosystems, Darmstadt, Germany) or an API/Q-Trap 2000 (Applied Biosystems-MDS Sciex, Darmstadt, Germany) both coupled to an Agilent 1100 HPLC-pump equipped with an Agilent 1100 autosampler (Agilent, Waldbronn, Germany).
- Sample preparation for LC-MS/MS analysis: Before analysis, frozen urine samples were thawed at 4 °C. Aliquots (1 mL) of urine or incubation mixtures were vortexed and then centrifuged for 15 min at 14,000 rpm and 4°C. Plasma (20 μL) was added to 40 μL of methanol. After keeping samples on ice for 30 min followed by centrifugation (14,000 rpm, 4°C, 10 min), obtained supernatants were mixed with 40 μL of acetonitrile and subjected to a second centrifugation at 14,000 rpm (4°C, 10 min). Obtained urine and plasma supernatants were diluted with water (1:10). If analyte responses were outside the linear range of the calibration curve, an additional dilution by 10 or 100 fold was performed. GC/MS analysis was performed with an Agilent 5973 mass spectrometer and electron impact ionization. The MS was coupled to an Agilent 6890 GC (Agilent, Santa Clara, United States). Analytical columns were either a HP-Plot Q column (30 m∗0.32 mm i.d., 20 μm film thickness; Agilent, Santa Clara, United States) or an Agilent DB-WAX column (30 m∗0.25 mm i.d., 25 μm film thickness; Agilent, Santa Clara, United States). Helium served as carrier gas at a flow rate of 1 mL/min.
- Quantification of N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine: Diluted samples (10 μL) were added to 90 μL of a 90 pg/μL internal standard solution (N-acetyl-d3-S-(3,3,3-trifluoro-trans-propenyl)-Lcysteine), vortexed, transferred into glass autosampler vials and sealed. Aliquots (10 μL) were injected into the LC. Separation was achieved using a Reprosil Pur C18 AQ column (2∗150 mm, 5 μm; Maisch, Ammerbuch, Germany) with a gradient elution using water containing 0.1% formic acid (solvent A) and methanol containing 0.1% formic acid (solvent B). The elution conditions were: flow-rate of 300 μL/min; 2% solvent B for 1 min followed by a linear increase to 90% solvent B within 5 min, then 90% solvent B for another 3 min. R2-values of the calibration curves were >0.996 and deviations between repeatedly measured samples were 5.4±5.0%. The LOQ for N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine was between
0.21 and 0.26 μg/L (0.81–1.00 nmol/L) in rat and rabbit urine, with LODs between 0.05 and 0.07 μg/L (0.19–0.26 nmol/L). In plasma, the quantitation method for N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine achieved a LOQ of 0.62 μg/L (2.38 nmol/L) and a LOD of 0.17 μg/L (0.65 nmol/L). R2-values of the calibration curves in plasma were >0.998 and deviations between repeatedly measured samples were 6.6±4.5%.
- Quantification of 3,3,3-trifluorolactic acid. An ion pair chromatography combined with mass spectrometry was developed based on a published procedure (Loos and Barcelo, 2001). Samples (7.5 μL) were added to 92.5 μL of a 150 pg/μL internal standard solution (dichloroacetic acid), vortexed and transferred into glass autosampler vials. Aliquots (10 μL) were analyzed. Quantification was performed using a Reprosil Pur C18 column (2∗150 mm, 5 μm; Maisch, Ammerbuch, Germany) with an isocratic elution by water containing 5 mM triethylamine and 5% acetonitrile adjusted to pH 6.2 with acetic acid at a flow-rate of 200 μL/min for 10 min. R2-values of the calibration curves were >0.997 and deviations between repeatedly measured samples were 0.5±11.4%. The limit of quantification (LOQ) for 3,3,3-trifluorolactic acid was 0.43 μg/L (2.99 nmol/L) with a limit of detection (LOD) of 0.14 μg/L (0.97 nmol/L) in urine.
- Quantification of S-(3,3,3-trifluoro-trans-propenyl)-glutathione. Incubation supernatants (50 μL) were added to 50 μL of a 500 pg/μL solution of the internal standard S-(1,2-dichlorovinyl)-glutathione, vortexed and transferred into glass autosampler vials. Aliquots (10 μL) were injected. Analytes were separated on a Reprosil Pur C18 column (2∗150 mm, 5 μm; Maisch, Ammerbuch, Germany) by gradient elution with water containing 0.1% formic acid (solvent A) and acetonitrile containing 0.1% formic acid (solvent B) at a flow-rate of 200 μL/min. Conditions were set to 10% solvent B for 1 min, increasing to 90% solvent Bwithin 9 min followed by solvent B at 90% for 3 min. R2-values of the calibration curves were >0.999 and deviations between repeatedly measured samples were 7.7±3.7%. The limit of quantification (LOQ) for S-(3,3,3-trifluoro-trans-propenyl)-glutathione was 5.5 μg/L (13.7 nmol/L) with a limit of detection (LOD) of 1.7 μg/L (4.2 nmol/L) in matrix.
- Identification of minor metabolites. Trifluoroacetic acid, 3,3,3-trifluoropropionic acid. and 3,3,3-trifluoro-1,2-propanediol were identified by GC/MS (Schuster et al., 2008). 3,3,3-Trifluoropropionic acid was identified by its characteristic mass fragments m/z 142, 111, 83 and 69 (Rt=7.5 min), trifluoroacetic acid by m/z 59 and 69 (Rt=4.6 min) and 3,3,3-trifluoro-1,2-propanediol by m/z 80, 69 and 31 (Rt=9.6 min). Analysis of 3,3,3-trifluoro-1-propanol involved heating of 200 μL urine at 85 °C for 30 min. A 1 mL headspace sample was injected splitless on a HP-Plot Q column with a carrier gas flow rate of 2 mL/min. Oven temperature was kept at 40 °C for 1 min, followed by an increase to 160 °C within 8 min holding the final temperature for 1 min. The intensities of m/z 95 and 69 were recorded with dwell times of 100 ms during analysis. 3,3,3-Trifluoro-1-propanol was eluted at 9.1 min.
- Determination of S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid, S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine and screening for the presence of other conceivable urinary metabolites was performed by LC-MS/MS analysis on a Reprosil Pur C18 AQ column (2∗150 mm, 5 μm; Maisch, Ammerbuch, Germany) using gradient elution with 0.1% formic acid in ACN or methanol and 0.1% formic acid in water at a flow rate of 200 μL/min. Organic mobile phase content was increased from10% to 90% within 30 min and held for further 2 min. Scans to detect conceivable metabolites were performed with negative ionization either in the sensitive multiple reaction monitoring (MRM)/enhanced product ion (EPI), enhanced mass spectrometry (EMS)/EPI or constant neutral loss (CNL)/EPI modes monitoring loss of 20 Da (hydrogen fluoride), 73 Da (glycine), 80 Da (sulfate), 86 Da (mercaptopyruvic acid), 87 Da (cysteine), 88 Da (mercaptolactic acid), 129 Da (mercapturic acid or glutathione), and 176 Da (glucuronide).
Statistics:
All data were analyzed by Prism 5.01 (GraphPad Software, Inc, La Jolla, CA 92037 USA).

Results and discussion

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
- Incubation of trans-HCFO-1233zd with liver microsomes: These incubations were performed both in the presence or absence of glutathione and/or NADPH regenerating system and the incubation mixtures were analyzed by 19F NMR and LC-MS/MS. The major signal in the 19F NMR spectra observed in rat, rabbit, and human was allocated to S-(3,3,3-trifluoro-trans-propenyl)-glutathione (δ=−62.0 ppm; dd, 3JHF=6.5 Hz, 4JHF=2.1 Hz). The structure of S-(3,3,3-trifluoro-trans-propenyl)-glutathione was confirmed by LC-MS/MS analysis. Minor metabolites observed in 19F NMR spectra of incubations of trans-HCFO-1233zd in rat liver microsomes were identified as 3,3,3-trifluorolactic acid (δ=−75.4 ppm; d, 3JHF=8.2 Hz), 3,3,3-trifluoro-1,2-propanediol (δ=−77.2 ppm; d, 3JHF=7.3 Hz) and 3,3,3-trifluoro-1-hydroxyacetone (δ=−83.4 ppm; s). 19F NMR spectra of rabbit microsomal incubations also showed several minor metabolites and two signals were attributed to 3,3,3-trifluoro-1,2-propanediol (δ=−77.2 ppm; d, 3JHF=7.3 Hz) and 3,3,3-trifluoro-1-propanol (δ=−64.3 ppm; t, 3JHF=11.2 Hz). The minor biotransformation products were present in both incubations containing a NADPH regenerating system and glutathione or only a NADPH regenerating system. In order to identify the additional signals in the 19F NMR spectra, LC-MS/MS experiments were performed in the CNL/EPI, EMS/EPI and MRM/EPI scan mode with negative ionization. Four minor metabolites were thus characterized in incubations in the presence of glutathione. All EPI spectra showed the characteristic fragmentation pattern of glutathione adducts (m/z 272, m/z 254 and m/z 179) (Farkas et al., 2007). Two of the EPI spectra suggested the presence of 2-S-(3,3,3-trifluoro-1, 1-propanediol)-glutathione, and 1-S-(1-chloro-3,3,3-trifluoropropyl)-glutathione or 2-S-(1-chloro-3,3,3-trifluoropropyl)-glutathione. EPI spectra indicative of 2-S-(3,3,3-trifluoro-1-propanol)-glutathione and 2-S-(3,3,3-trifluoropropionic acid)-glutathione were only obtained in supernatants of rat liver microsomes. The latter metabolites will give doublets in 19F NMR spectra and the unidentified signals observed in the 19F NMR spectra of rat microsomal incubations at −73.5 ppm (d, 3JHF=4.9 Hz) and at −84.8 ppm (d, 3JHF=4.0 Hz) may be assigned to these structures. Kinetics of the formation of S-(3,3,3-trifluoro-trans-propenyl)-glutathione were characterized in rabbit, rat and human subcellular liver fractions. Kinetic parameters were determined by nonlinear regression analysis as Km (trans-HCFO-1233zd headspace gas concentration) — 249,000 ppm (rabbit), 213,400 ppm (rat) and 163,400 ppm (human) and Vmax as 21.8 [pmol/min ∗mg protein] (rabbit), 13.1 [pmol/min ∗mg protein] (rat), and 5.0 [pmol/min∗mg protein] (human). Kinetic analyses of the oxidative biotransformation of trans-HCFO-1233zd in liver subcellular fractions could not be performed due to the low sensitivity of the quantitation procedures available for the highly polar products formed.
- Inhalation exposures of rats and rabbits to trans-HCFO-1233zd: Mean chamber concentrations of trans-HCFO-1233zd were 1,961±261, 5,084±549 and 10,089±818 ppm for rabbit and 1,977±256, 5,220±1147 and 10,128±714 ppm for rat exposures. Predominant metabolites in 19F NMR spectra of rat urine were 3,3,3-trifluorolactic acid (δ=−75.3 ppm; d, 3JHF=8.2 Hz) and N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine (δ=−61.8 ppm; dd, 3JHF=6.6 Hz, 4JHF=2.1 Hz), representing 32% and 40%, of the total fluorine signal area (0–6 h post inhalation urine fraction), respectively. Additional identified metabolites were S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid (δ=−61.7 ppm; dd, 3JHF=6.6 Hz, 4JHF=2.1 Hz), trifluoroacetic acid (δ=−75.4 ppm; s), 3,3,3-trifuoro-1,2-dihydroxypropane (δ=−77.2 ppm; d, 3JHF=7.1 Hz), and 3,3,3-trifluoropropionic acid (δ=−63.4 ppm; t, 3JHF=11.2 Hz). The structures of the chemical entities giving the doublets at −71.3 and −77.1 ppm as well as the doublet of doublets at −61.5 ppm could not be elucidated. In 19F NMR spectra of rabbit urine, the major metabolite was N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine (δ=−61.8 ppm; dd, 3JHF=6.6 Hz, 4JHF=2.1 Hz) representing 46% of total 19F NMR signal area (0–6 h post inhalation urine fraction). S-(3,3,3-trifluorotrans-propenyl)-mercaptolactic acid (δ=−62.1 ppm; dd, 3JHF=6.5 Hz, 4JHF=2.1 Hz), trifluoroacetic acid (δ=−75.4 ppm; s) and 3,3,3-trifuoro-1,2-dihydroxypropane (δ=−77.2 ppm; d, 3JHF=7.1 Hz) were also present in rabbit urine. Besides, additional signals were observed at −72.3 (d, 3JHF=6.8), −72.4 (d, 3JHF=7.0 Hz) and −62.1 ppm dd, 3JHF=6.5 Hz, 4JHF=2.1 Hz), the latter was assigned to S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine. A weak signal for 3,3,3-trifluoro-1-propanol was seen at a chemical shift of −64.3 ppm (t, 3JHF=11.2 Hz). Again, the chemical entity giving the doublets appearing at −72.3, −72.4 and −77.1 ppm could not be identified, signals indicative of the presence of 3,3,3-trifluorolactic acid were not present. The structures of the major metabolites N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine, 3,3,3-trifluorolactic acid, S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid, and S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine were confirmed by LC-MS/MS in the sensitiveMRM/EPI scanmode. Metabolite retention times and fragmentation patterns in the negative scan mode were compared with synthesized or purchased standards. More detailed analysis by LC-MS/MS revealed an EPI mass spectrum assigned to the S-oxide of N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine. The fragmentation showed the characteristic pattern of trans-HCFO-1233zd conjugates (loss of two molecules of hydrogen fluoride). This signal was only observed in rabbit urine. Minor volatile metabolites in urine were detected by GC/MS analysis. Trifluoroacetic acid, 3,3,3-trifluoropropionic acid and 3,3,3-trifluoro-1,2-propanediol were confirmed in rat and rabbit urine. 3,3,3-Trifluoro-1-propanol was only present in rabbit urine. While the presence of trifluoroacetic acid was verified by 19F NMR and GC/MS analysis, no reasonable mechanisms of formation can be developed and the detection of trifluoroacetic acid may be an artifact (Bayer et al., 2002; Schuster et al., 2008). Excretion kinetics of metabolites were determined by quantitation of N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine in rat/rabbit urine and in rat plasma and of 3,3,3-trifluorolactic acid in rat urine. Due to low amounts of rat blood, 3,3,3-trifluorolactic acid kinetics could not be determined in plasma. The urinary excretion profiles of N-acetyl-S-(3,3,3-trifluoro-transpropenyl)-L-cysteine showed the highest concentrations in urine fractions collected between 0 and 6 h after the end of the inhalation exposure in both rats and rabbits. In contrast, 3,3,3-trifluorolactic acid peaked in urine samples collected between 6 and 12 h after the end of the inhalation exposures. Within the first 24 h after termination of the inhalation exposures,>98.2±3.0% (rats) and >97.1±3.5% (rabbits) of the N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine and >87.2±4.4% of 3,3,3-trifluorolactic acid were recovered. Elimination half-life times were determined as 5.6±3.8 h for 3,3,3-trifluorolactic acid in rats and 4.3±4.4 h and 2.9±2.1 h for N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine in rats and rabbits, respectively. Mean peak plasma levels of N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine were determined in all blood samples taken before and after the inhalation exposure in all three exposure groups. In general, concentration levels of N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine were higher in the 10,000 ppm exposure group compared to 5,000 and 2,000 ppm, but large standard deviations, in part due to the low concentrations of the analyte present, did not permit meaningful analysis.

Any other information on results incl. tables

Discussion

In analogy to published data for trans-1,1,1,3-tetrafluoropropene (Schuster et al., 2009), trans-HCFO-1233zd is metabolized by both glutathione conjugation and by oxidative metabolism by cytochrome P-450 2E1. This is consistent with the information available on the biotransformation of structurally related compounds and the formation of all identified products of the biotransformation of trans-HCFO-1233zd can be readily explained as formed by these two pathways. The identified metabolites in intact animals show that a number of downstream processing reactions result in a complex metabolite pattern.

The biotransformation of trans-HCFO-1233zd mediated by glutathione S-transferase catalyzed addition–elimination reaction of glutathione leads to N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine as final excretory product. Consistent with this observation, S-(3,3,3-trifluoro-trans-propenyl)-glutathione is the major metabolite formed from trans-HCFO-1233zd in liver subcellular fractions. Renal processing of S-(3,3,3-trifluoro-trans-propenyl)-glutathione by γ-glutamyl-transpeptidase and dipeptidases (Anders and Dekant, 1998) results in S-(3,3,3-trifluoro-trans-propenyl)-Lcysteine. This cysteine S-conjugate is further processed by three different pathways. i) Cysteine S-conjugate transaminase to give S-(3,3,3-trifluoro-trans-propenyl)-mercaptopyruvic acid followed by reduction to S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid; ii) renal cysteine S-conjugate β-lyase dependent biotransformation (Anders et al., 1992; Monks et al., 1990) to finally give 3,3,3-trifluoropropionic acid and 3,3,3-trifluoro-1-propanol; and iii) N-acetyl-transferase to form N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine which may be further oxidized to N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine S-oxide.

The second metabolic pathway for trans-HCFO-1233zd is catalyzed by cytochrome P-450, initially resulting in the formation of 1-chloro-3,3,3-trifluoro-1,2-epoxypropane. This epoxide is hydrolyzed to 3,3,3-trifluoro-2-hydroxypropanal which may be oxidized to 3,3,3-trifluorolactic acid or reduced to 3,3,3 -trifluoro-1,2 -propanediol. The latter may be subject to further phase two reactions. Alternatively, ring opening of 1-chloro-3,3,3 -trifluoro-1,2 -epoxypropane may occur by a reaction with glutathione to give 2-S-(3,3,3-trifluoropropanal)-glutathione, which may be oxidized to 2-S-(3,3,3-trifluoropropionic acid)-glutathione or reduced to 2-S-(3,3,3-trifluoro-1-propanol)-glutathione. The glutathione S-conjugates might be subject to further processing by the enzymes of the mercapturic acid pathway. However, the compounds likely are only formed in very low yields and thus could not be detected in urine samples.

The structures of the minor metabolites proposed to be formed in the liver subcellular fractions are consistent with the reaction schemes developed. Tentative minor in vitro metabolites detected by LC-MS/MS in all species were the glutathione conjugate 1-S-(1-chloro-3,3,3-trifluoro-propyl)-glutathione or 2-S-(1-chloro-3,3,3 -trifluoro-propyl)-glutathione and the hydrate of 2-S-(3,3,3-trifluoropropanal)-glutathione. Addition of glutathione to double bonds may not necessarily be followed by elimination of HCl since addition reactions of glutathione to the haloalkene have been described (Dekant, 2003). 2-S-(3,3,3-trifluoropropionic acid)-glutathione and 2-S-(3,3,3-trifluoro-1-propanol)-glutathione, products of the glutathione-mediated ring opening of the epoxide were only detected in supernatants of rat microsomes. Both are formed by oxidation or reduction of the intermediate 2-S-(3,3,3-trifluoropropanal)-glutathione. These products were only detected using highly sensitive LC-MS/MS methods. Related 19F NMR signals were not observed, likely concentrations were below the much higher limit of detection of the 19F NMR method.

The 19F NMR spectra of rat and rabbit urine indicated the presence of several unidentified metabolites. The signal (δ=−61.5 ppm) close to the major metabolite N-acetyl-S-(3,3,3-trifluoro-transpropenyl)-L-cysteine (9, Scheme 1) is likely a processing product of S-(3,3,3-trifluoro-trans-propenyl)-glutathione pathway because of its similar multiplicity, 19F chemical shift and coupling constants. The unidentified doublets observed at 19F chemical shifts below −71 ppm possibly have a hydroxyl group in alpha position to the trifluoromethyl group, also indicated by their similar 19F chemical shift, multiplicity and coupling constants compared to 3,3,3-trifluorolactic acid and 3,3,3-trifluoro-1,2-propanediol. Traces of N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine S-oxide, observed by LC-MS/MS analysis in rabbit urine, were below the limit of detection of the 19F NMR method.

The metabolite pattern observed indicates species differences between rats and rabbits and the two pathways of trans-HCFO-1233zd biotransformation may be competitive. Whereas 3,3,3-trifluorolactic acid was the main urinary metabolite of trans-HCFO-1233zd in the rat, N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine was the main urinary biotransformation product in the rabbit. In the rat, urinary metabolite quantification showed an increase of N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine excretion compared to 3,3,3-trifluorolactic acid with increasing trans-HCFO-1233zd exposure concentrations. The ratio of N-acetyl-S-(3,3,3-trifluoro-transpropenyl)- L-cysteine to 3,3,3-trifluorolactic acid was 0.09 at 2,000 ppm, 0.15 at 5,000 ppm and 0.43 at 10,000 ppm. The increased contribution of the glutathione S-transferase catalyzed pathway also explains that the oxidation of trans-HCFO-1233zd in in vitro incubations at very high trans-HCFO-1233zd concentrations is only a minor pathway.

Quantification of the major metabolites 3,3,3 -trifluorolactic acid and N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine revealed rapid excretions, with elimination half-life times of less than 6 h for both substances in both species. Calculations based on total urinary recoveries for N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-L-cysteine or 3,3,3-trifluorolactic acid and estimations of trans-HCFO-1233zd uptake, revealed very low overall biotransformation amounts. The extent of biotransformation of trans-HCFO-1233zd is within the same order of magnitude as trans-1,1,1,3-tetrafluoropropene (Schuster, 2009) with app. 0.007% of the received dose undergoing biotransformation in the rat, the remaining material is likely exhaled due to its high volatility. In vitro enzyme kinetics confirmed the low in vivo biotransformation amount and showed a lower glutathione S-transferase affinity to trans-HCFO-1233zd in humans compared to rodents.

The potential reactive metabolite 1-chloro-3,3,3-trifluoro-1,2 -epoxypropane formed by cytochrome P-450 is formed only in very low amounts due to low overall biotransformation and is apparently rapidly detoxified by epoxide hydrolase. Therefore, covalent binding with tissue nucleophiles may be efficiently prevented by hydrolysis or by glutathione conjugation. Formation of reactive metabolites (thioketenes or thioacylhalodies) by the glutathione S-transferase/renal β-lyase pathway observed with other haloolefins is unlikely for trans-HCFO-1233zd due to the lack of appropriate leaving groups (Dekant et al., 1990; Monks et al., 1990; Vamvakas et al., 1989; Volkel et al., 1998).

In summary, the low biotransformation amount and the rapid excretion of metabolites as well as the absence of potent toxic biotransformation products in both species, support the results of prior studies that trans-HCFO-1233zd has only a low potential for toxicity in mammals. The data on metabolism and toxicokinetics of trans-HCFO-1233zd are in good agreement with previously published data for the related compound trans-1,3,3,3-tetrafluoropropene by Schuster et al. (2009).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
The test substance undergoes both oxidative biotransformation and glutathione conjugation at very low rates. The very low extent of biotransformation and the rapid excretion of metabolites formed are consistent with the very low potential for toxicity of the test substance in mammals.
Executive summary:

The biotransformation of trans-HCFO-1233zd and kinetics of metabolite excretion with urine were assessed in vitro and in animals after inhalation exposures. To characterize biotransformation in vitro, liver microsomes from rats, rabbits and humans were incubated with trans-HCFO-1233zd. Male Sprague Dawley rats and female New Zealand White rabbits were exposed by inhalation to 2,000, 5,000 and 10,000 ppm of trans-HCFO-1233zd for 6 hours and urine was collected for 48 hours after the end of the exposure. Study samples (urine, plasma, microsomal incubation supernatants) were analyzed for metabolites using 19F-NMR, LC-MS/MS and GC/MS. S-(3,3,3-trifluoro-trans-propenyl)-glutathione was identified as predominant metabolite of trans-HCFO-1233zd in rat, rabbit and human liver microsomes in the presence of glutathione. Products of the oxidative biotransformation of trans-HCFO-1233zd were only minor metabolites when glutathione was present. In rats, both 3,3,3-trifluorolactic acid and N-acetyl-(3,3,3 -trifluoro-trans-propenyl)-L-cysteine were observed as major urinary metabolites. 3,3,3-trifluorolactic acid was not detected in the urine of rabbits exposed to trans-HCFO-1233zd. Several minor metabolites formed by alternative reactions of S-(3,3,3-trifluoro-trans-propenyl)-glutathione were also excreted. Quantitation showed rapid excretion of both metabolites with urine t1/2 of less than 6 hours in both species. Based on metabolite recovery in urine and estimated doses of trans-HCFO-1233zd received by inhalation, the extent of biotransformation of trans-HCFO-1233zd was determined as approximately 0.01% of received dose in rabbits and approximately 0.002% in rats. The remaining material is likely exhaled due to its high volatility. The metabolite structures show that trans-HCFO-1233zd undergoes both oxidative biotransformation and glutathione conjugation at very low rates. The very low extent of biotransformation and the rapid excretion of metabolites formed are consistent with the very low potential for toxicity of trans-HCFO-1233zd in mammals.