George M. Rusch

Biotransformation of 2,3,3,3-tetrafluoropropene (HFO-1234yf)

2,3,3,3-Tetrafluoropropene (HFO-1234yf) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential which has been developed as refrigerant. The biotransformation of HFO-1234yf was investigated after inhalation exposure. Male Sprague-Dawley rats were exposed to air containing 2000, 10,000, or 50,000 ppm HFO-1234yf for 6 h and male B6C3F1 mice were exposed to 50,000 ppm HFO-1234yf for 3.5 h in a dynamic exposure chamber (n=5/concentration). After the end of the exposure, animals were individually housed in metabolic cages and urines were collected at 6 or 12-hour intervals for 48 h. For metabolite identification, urine samples were analyzed by (1)H-coupled and 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 all urine samples, the predominant metabolites were two diastereomers of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine. In (19)F-NMR, the signal intensity of these metabolites represented more than 85% (50,000 ppm) of total (19)F related signals in the urine samples. Trifluoroacetic acid, 3,3,3-trifluorolactic acid, 3,3,3-trifluoro-1-hydroxyacetone, 3,3,3-trifluoroacetone and 3,3,3-trifluoro-1,2-dihydroxypropane were present as minor metabolites. Quantification of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine by LC/MS-MS showed that most of this metabolite (90%) was excreted within 18 h after the end of exposure (t(1/2) app. 6 h). In rats, the recovery of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine excreted within 48 h in urine was determined as 0.30+/-0.03, 0.63+/-0.16, and 2.43+/-0.86 micromol at 2000, 10,000 and 50,000 ppm, respectively suggesting only a low extent (<<1% of dose received) of biotransformation of HFO-1234yf. In mice, the recovery of this metabolite was 1.774+/-0.4 mumol. Metabolites identified after in vitro incubations of HFO-1234yf in liver microsomes from rat, rabbit, and human support the metabolic pathways of HFO-1234yf revealed in vivo. The obtained results suggest that HFO-1234yf is subjected to a typical biotransformation reaction for haloolefins, likely by a cytochrome P450 2E1-catalyzed formation of 2,3,3,3-tetrafluoroepoxypropane at low rates, followed by glutathione conjugation or hydrolytic ring opening.

Cardiac sensitization: methodology and interpretation in risk assessment.

An increased sensitivity of the heart to endogenous epinephrine (adrenaline), a phenomenon referred to as cardiac sensitization, has long been recognized as a risk during exposure to hydrocarbons, principally halogenated hydrocarbons. Cardiac sensitization, which can result in serious arrhythmia and death, requires a certain critical blood level of both the sensitizing agent and epinephrine. The original research and methods utilized an exogenous epinephrine challenge during inhalation exposure to a chemical to assess cardiac sensitization potential in Beagle dogs. These screening tests were developed about 30 years ago, although in the last 15 years some modifications of these methods have occurred in response to testing chlorofluorocarbon (CFC) replacements. Results from these experimental cardiac sensitization studies have been used for semi-quantitative risk evaluation for occupational exposures but now are being used more quantitatively for regulatory purposes. The risks associated with cardiac sensitization from CFC replacements are unknown but expected to be low based on cardiac sensitization studies in the 1970s where dogs were made to generate their own adrenaline. With the advent of physiologically based pharmacokinetic (PBPK) modeling, greater emphasis is being placed on quantitative risk assessment for cardiac sensitization. In this investigation, we have examined the various methodologies used for detection of cardiac sensitization and discussed their limitations and advantages. In addition, we examined the potential concerns involved in using experimental cardiac sensitization data and PBPK modeling to predict exposure scenarios.

Establishing a Point of Departure for Risk Assessment Using Acute Inhalation Toxicology Data

A simple method is presented for estimating a non-lethal level for inhalation toxicity studies. By reviewing 209 LC(50) studies representing 96 chemicals that also reported a non-lethal level, it has been shown that taking 1/3 of the LC(50) is a conservative estimate for a non-lethal exposure level. This approach was also compared to studies with LC(01) and BMCL(05) calculations. In the 38 studies that reported either of these values, again taking 1/3 of the LC(50) provided a more conservation estimate for the non-lethal threshold. The studies included time intervals from 5min out to 8h and utilized multiple species such as the rat, mouse, hamster, guinea pig and dog. In all but 13 cases, taking 1/3 of the LC(50) provided a more conservative estimate for a non-lethal exposure level compared to the experimentally observed value. In all but one of the 13 cases, the higher values were consequences of the selection of the exposure levels.

Biotransformation of trans-1,1,1,3-tetrafluoropropene (HFO-1234ze).

trans-1,1,1,3-Tetrafluoropropene (HFO-1234ze) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential and is developed as foam blowing agent. 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 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.

Hepatotoxicity Associated with Overexposure to 1,1-Dichloro-2,2,2-Trifluoroethane (HCFC-123)

1,1-Dichloro-2,2,2-trifluoroethane (HCFC-123) was evaluated as a substitute for trichlorofluoromethane (CFC-11), and it appeared that a permissible exposure limit of 50 ppm was justified. When HCFC-123 was introduced as a precision cleaning agent in a controlled operation, marked elevations in serum alanine transaminase and serum aspartase transaminase were noted in exposed workers. Sampling taken during start-up documented personal samples from 24-480 ppm (375 and 21 min, respectively) and area samples of 18-180 ppm (375 and 21 min, respectively). Personal and area samples collected after the liver abnormalities were identified ranged from 5-12 ppm. Exposure data were not available for the period when the abnormalities are suspected to have developed. Two models were developed to estimate exposure during the unmonitored period: (1) the entire plant as a homogenous box and (2) evaporation into smaller work zones. Modeling using the entire building estimated 8-hour time-weighted average (TWA) exposures of 10-35 ppm. Modeled estimates of work area and air exchange rates indicated that degreaser exposed workers could have experienced peak levels of 280-2,100 ppm (8-hour TWAs 252-1,630 ppm). Modeling of the work environment, estimated to be one-third of the volume of the entire open building, indicated peak exposures of 28-210 ppm (8-hour TWAs 25-163 ppm). These ranges estimate the minimum and maximum exposure levels. The best estimates, using 12 air changes per day, suggest peak levels around the degreaser of 635-2,100 ppm (8-hour TWA 499-1,630 ppm) and 63-207 ppm (8-hour TWAs 50-163 ppm) in the work area. These are the first estimates of exposure level associated with these hepatotoxic effects; all are significantly higher than personal and area samples collected for HCFC-123 after the liver abnormalities were identified.

Biotransformation of 2,3,3,3-tetrafluoropropene (HFO-1234yf) in male, pregnant and non-pregnant female rabbits after single high dose inhalation exposure.

2,3,3,3-Tetrafluoropropene (HFO-1234yf) is a novel refrigerant intended for use in mobile air conditioning. It showed a low potential for toxicity in rodents studies with most NOAELs well above 10,000 ppm in guideline compliant toxicity studies. However, a developmental toxicity study in rabbits showed mortality at exposure levels of 5,500 ppm and above. No lethality was observed at exposure levels of 2,500 and 4,000 ppm. Nevertheless, increased subacute inflammatory heart lesions were observed in rabbits at all exposure levels. Since the lethality in pregnant animals may be due to altered biotransformation of HFO-1234yf and to evaluate the potential risk to pregnant women facing a car crash, this study compared the acute toxicity and biotransformation of HFO-1234yf in male, female and pregnant female rabbits. Animals were exposed to 50,000 ppm and 100,000 ppm for 1h. For metabolite identification by (19)F NMR and LC/MS-MS, urine was collected for 48 h after inhalation exposure. In all samples, the predominant metabolites were S-(3,3,3-trifluoro-2-hydroxypropanyl)-mercaptolactic acid and N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-L-cysteine. Since no major differences in urinary metabolite pattern were observed between the groups, only N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-L-cysteine excretion was quantified. No significant differences in recovery between non-pregnant (43.10 ± 22.35 μmol) and pregnant female (50.47 ± 19.72 μmol) rabbits were observed, male rabbits exposed to 100,000 ppm for one hour excreted 86.40 ± 38.87 μmol. Lethality and clinical signs of toxicity were not observed in any group. The results suggest that the lethality of HFO-1234yf in pregnant rabbits unlikely is due to changes in biotransformation patterns or capacity in pregnant rabbits.

The acute, genetic, developmental and inhalation toxicology of trans-1-chloro,3,3,3-trifluoropropene (HCFO-1233zd(E))

Abstract Trans-1-chloro,3,3,3-trifluoropropene (HCFO-1233zd(E)) is being developed as a foam blowing agent, refrigerant and solvent because it has a very low global warming potential (<10), as contrasted to the hydrofluorocarbons (>500). The toxicology profile is described. The acute 4-hour 50% lethal concentration value in rats receiving HCFO-1233zd(E) was 120 000 ppm. The no observed effect level (NOEL) in cardiac sensitization studies in dogs was 25 000 ppm. In a 2-week range-finding study, rats were exposed to HCFO-1233zd(E) at levels of 0, 2000, 7500 and 20 000 ppm 6 hours/day for 5 days/week. Histopathological changes in the heart described as multifocal mononuclear infiltrates were observed in males (mid- and high-exposure group) and females (high-exposure group), suggesting this organ was the target for HCFO-1233zd(E) toxicity. In a 4-week study, rats were exposed to 0, 2000, 4500, 7500 and 10 000 ppm. The only finding was an increase in potassium (mid- and high-exposure males). No increase was observed after a 2-week recovery period, nor in a subsequent 13-week toxicity study. In a 13-week study, rats were exposed to 4000, 10 000 and 15 000 ppm 6 hours/day for 5 days/week. Findings consisted of multifocal mononuclear cell infiltrates in the heart with a NOEL/lowest observed adverse effect level of 4000 ppm. No genetic toxicity was observed in a battery of genetic toxicity studies. In a rat prenatal developmental toxicity study, dilated bladders were observed in the high-exposure group fetuses (15 000 ppm), a finding of unclear significance. HCFO-1233zd(E) was not a developmental toxin in rabbits, even at exposure levels up to 15 000 ppm.

Biotransformation of trans-1-chloro-3,3,3-trifluoropropene (trans-HCFO-1233zd)

trans-1-Chloro-3,3,3-trifluoropropene (trans-HCFO-1233zd) is a novel foam blowing and precision cleaning agent with a very low impact for global warming and ozone depletion. trans-HCFO-1233zd also has a low potential for toxicity in rodents and is negative in genotoxicity testing. The biotransformation of trans-HCFO-1233zd and kinetics of metabolite excretion with urine were assessed in vitro and in animals after inhalation exposures. For in vitro characterization, 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 to 2,000, 5,000 and 10,000ppm for 6h and urine was collected for 48h after the end of the exposure. Study specimens were analyzed for metabolites using (19)F 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 all microsomal incubation experiments 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. Quantitation showed rapid excretion of both metabolites in both species (t1/2<6h) and 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. trans-HCFO-1233zd undergoes both oxidative biotransformation and glutathione conjugation at very low rates. The 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.

The acute, genetic, developmental and inhalation toxicology of trans-1,3,3,3-tetrafluoropropene (HFO-1234ze)

HFO-1234ze is being developed as a refrigerant, propellant, and foam-blowing agent because it has a very low global warming potential (less than 10), as contrasted to the hydrofluorocarbons with values of over 500. Several toxicology studies were conducted to develop a toxicology profile for this material. There was no lethality in mice and rats receiving single 4-hour exposures up to 103,300 or 207,000 ppm, respectively. Exposures up to 120,000 ppm did not induce cardiac sensitization to adrenalin. Rats were exposed to HFO-1234ze at levels of 5,000, 20,000, and 50,000 ppm 6 hours/day 5 days/week for 2 weeks. Predominate findings of increased liver and kidney weights and histopathological changes in the liver and heart suggested that these organs were the targets for HFO-1234ze toxicity. In a 4-week study at 1000, 5000, 10,000, and 15,000 ppm, the only organ showing treatment-related effects was the heart. In a 90-day study with exposures of 1500, 5000, and 15,000 ppm 6 hours/day 5 days/week, again, the heart was the only target organ. The findings consisted of focal and multifocal mononuclear cell infiltrates in the heart. There was no evidence of fibrosis, and, when compared to the 2- and 4-week studies, there did not appear to be an increase in severity with length of exposure. HFO-1234ze was inactive in a mouse and rat micronucleus assay, an Ames assay, and an unscheduled DNA synthesis assay and was not clastogenic in human lymphocytes. It was also not a developmental toxin in either the rat or rabbit, even at exposure levels up to15,000 ppm.

The acute, developmental, genetic and inhalation toxicology of 2,3,3,3-tetrafluoropropene (HFO-1234yf).

Abstract 2,3,3,3-Tetrafluoropropene (HFO-1234yf) is being developed as a refrigerant because it has a very low global warming potential (less than 10), as contrasted to the hydrofluorocarbons, which is intended to replace with values of over 500. Several toxicology studies were conducted to develop a toxicology profile for this material. There was no lethality in mice and rats receiving single 4-hour exposures up to 101 850 or 405 800 ppm, respectively. Additionally, there was no mortality or clinical signs of toxicity when rabbits were exposed to 100 000 ppm for 1 hour. Exposures up to 120 000 ppm did not induce cardiac sensitization to adrenalin in dogs. Rats were exposed to HFO-1234yf at levels of 5000, 20 000 and 50 000 ppm 6 hours/day 5 days/week for 2 weeks and at levels of 5000, 15 000 and 50 000 ppm for 4 weeks and for 90 days. No treatment-related adverse effects were noted in these studies. HFO-1234yf was not genotoxic in a mouse and a rat micronucleus assay, and unscheduled DNA synthesis assay and was not clastogenic in human lymphocytes. HFO-1234yf was mutagenic to Salmonella typhimurium TA 100 and Escherichia coli (WP2 uvrA) at concentrations of 20% and higher in the presence of metabolic activation only. There were no biologically significant effects in a rat developmental toxicity study with exposures up to 50 000 ppm.