2-chlorobuta-1,3-diene

Endpoint summary

Additional information

Toxicokinetics

The toxicokinetic behaviour of chloroprene or its metabolites by any route of exposure has not been addressed in human studies or experimental animals. This is largely because absorption, distribution, metabolism and elimination (ADME) experiments typically use radiolabeled test substance and for chloroprene, because of its volatility and uptake by the inhalation route and the inherent propensity for monomer reactivity, radiolabeled chloroprene could not be safely and reliably synthesized.   Consequently, alternative in vitro and in vivo approaches were used to understand the ADME properties for this chemical (e.g. in vitro studies of chloroprene in rodent and human tissue fractions). Human and animal physiologically-based pharmacokinetic (PBPK) models have been developed to predict the tissue dosimetry of chloroprene following inhalation exposures.

Absorption

No studies have reported quantitative data on the tissue distribution of chloroprene in vivo. Absorption following inhalation exposure can however be inferred from chronic inhalation studies in B6C3F1 mice and F344/N rats (NTP, 1998) which suggest chloroprene can induce non-neoplastic and neoplastic effects in multiple target organ and tissue sites (e.g. the nose, lungs, kidney, forestomach, Harderian gland and skin).  

Distribution

No studies have reported quantitative data on the tissue distribution of chloroprene in vivo. Systemic distribution following inhalation exposure can however be inferred from chronic inhalation studies in B6C3F1 mice and F344/N rats (NTP, 1998) which suggest chloroprene can induce non-neoplastic and neoplastic effects in multiple target organ and tissue sites (e.g. the nose, lungs, kidney, forestomach, Harderian gland and skin).  

Tissue partition coefficient values determined in mice, F334 and Wistar rats and hamsters using the vial equilibrium method described by Gargas et al, 1989 suggest that there are no significant species differences in the tissue distribution of chloroprene and that the substance will be preferentially distributed in adipose tissues, followed by lung, kidney, liver and muscle (Himmeslstein et al, 2004b).

Metabolism

The in vitro biotransformation of chloroprene has been studied to identify metabolites that might contribute to toxicity or mutagenicity. Early experiments indicated metabolism of chloroprene to reactive epoxide metabolites (Bartsch et al. 1979). In vivo reactivity with glutathione was reported based on altered non-protein sulfhydryl concentrations in lung and liver and urinary elimination of thiolether containing metabolites (Jeager et al. 1975; Plugge and Jeager; Sumner and Greim). More recently, in vitro metabolism methods have been applied that led to the isolation and confirmation of one epoxide metabolite, (1-chloroethenyl)oxirane, in lung and liver microsomal preparations which contain mixed function oxidase (CYP P450) activity (Himmelstein et al. 2001; Cottrell et al. 2001). The findings by Cottrell et al. (2001) and Munter et al (2003) provided the most thorough identification of chloroprene metabolite structures to date. In addition to confirming the identification of the 1-chloroethenyl)oxirane, these authors published evidence for a second transient epoxide, 2-chloro-2-ethenyloxirane, which subsequently undergoes rapid non-enzymatic hydrolysis to chlorinated ketone and aldehyde metabolites. Munter et al. (2003) further investigated species difference in the activation of chloroprene in liver microsomal preparations from mice, rats and humans using chloroprene incubation concentrations that ranged from 10 to 10,000 µM. The results showed significant differences between species in the amount of (1-chloroethenyl)oxirane present (mouse > rat > human). The oxidative formation of (1-chloroethenyl)oxirane and hydrolysis (detoxification by epoxide hydrolase to 3-chlorobut-3-ene-1,2-diol) was investigated. The hydrolysis pathway showed a distinct selectivity forS-(1-chloroethenyl)oxirane that resulted in an accumulation of the R-enantiomer; the ratios of the amounts of theR- between species were 20:4:1 for mouse:rat:human, respectively (Munter et al. 2003).

 

Himmelstein et al (2004a) conducted an extensive quantitative comparison of the rates of total chloroprene oxidative metabolism in mouse, rat, hamster and human microsomes prepared from lung and liver tissues from each species. These tissues were selected because of the importance lung as a route of uptake (inhalation), the major role of the liver in xenobiotic metabolic clearance, and the presence of tumors in these tissues in rodents from the NTP bioassay (Melnick et al. 1999). Initial vial headspace concentrations ranged from 0.41 to 409 µM. The experimentally measured partition coefficient was 0.69:1 (liquid:air). Metabolism was measured based the declining concentration of chloroprene in the headspace using the partition coefficient to account for uptake into the liquid microsomal preparation. The objective was to develop Vmax and Km values that could be scaled for physiologically-based kinetic modeling. The intrinsic clearance rate (Vmax/Km) was similar among liver microsomes for each species with about a two-fold greater rate for the mouse vs rats or humans. For the lung, a much more sensitive tissue for tumor development in the mouse than the rat, the rate of chloroprene oxidative metabolism in the mouse was 50-fold greater than the rat, hamster, or human (Table 1). 

 

Himmelstein et al. (2004a) also investigated the metabolism of (1-chloroethenyl)oxirane by microsomal epoxide hydrolase. Because this reaction occurs in the microsomal protein fraction in proximity to oxidative P450 activity, it can be presumed to play a key role in the initial removal of the epoxide formed in vivo. Initial headspace vapor concentrations ranged from 0.37 to 330 µM. Metabolism was measured based the declining concentration of (1-chloroethenyl)oxirane in the headspace and an experimentally measured partition coefficient of 57.9:1 (liquid:air). A comparison of the intrinsic clearance (µmol/mL/mg microsomal protein) provides an important species comparison showing that the capacity for detoxification in human tissues is substantially greater relative to the mouse (Table 1).

 

Table 1. Species comparison of activation and detoxification potentials for chloroprene oxidation and (1-chloroethenyl)oxirane hydrolysis.
		Intrinsic clearance^a		Ratio^b	Ratio
		(Vmax/Km)		Activation/	Mouse/
Tissue	Speices	Oxidation	Hydrolysis	Detoxication	Other
Liver	Mouse	224	6.7	33.4	1
	Fischer rat	146	14.5	10.1	3.3
	Wistar rat	125	12.1	10.3	3.2
	Hamster	218	33.7	6.5	5.2
	Human	101	36.7	2.8	12.1
Lung	Mouse	66.7	2.1	31.8	1
	Fischer rat	1.3	1.3	1.0	32
	Wistar rat	1.3	1.7	0.76	42
	Hamster	1.3	7.1	0.18	173
	Human	1.3	8.0	0.16	195
a Values (mL/h/mg microsomal protein) from Himmelstein et al. (2004a) b Activation/Detoxication = Vmax/Km (Oxidation/Hydrolysis)

 

Species differences in the cytosolic glutathione-S-transferase reaction with (1-chloroethenyl)oxirane were also observed but they were less dramatic than the epoxide hydrolase rates. Initial headspace vapor concentrations ranged from 0.41 to 550 µM. The method relied on measuring the rate of decline of (1-chloroethenyl)oxirane in the headspace; individual metabolite structures can be presumed to match those reported by Cottrell et al. (2001). The fastest rate for liver cytosol was measured in the hamster > Fischer rat > Wistar rat > mouse > human; the difference between the hamster and human was about eight-fold. For lung cystosol, the rates were mouse > Fischer rat > human > Wistar rat > hamster with a four-fold difference between the mouse and hamster. The rates of metabolism were 3.2- and 1.8-fold faster in the mouse than human for liver and lung cytosol, respectively. An activation/detoxication ratio could not be calculated because the pseudo-second order rates (hour/mg protein) were different from those for oxidation and hydrolysis (mL/h/mg protein).

 

Himmelstein et al. (2004a), while measuring the kinetics of chloroprene oxidative metabolism, quantified the simultaneous formation and hydrolysis of (1-chloroethenyl)oxirane. A significant proportion of the chloroprene that was oxidized could not be accounted for by the portion of (1-chloroethenyl)oxirane formation by microsomal P450 and hydrolysis of (1-chloroethenyl)oxirane by microsomal epoxide hydrolase. This finding was consistent with the production of chlorinated ketone and aldehyde metabolites through spontaneous hydrolysis of the second chloroprene epoxide, 2-chloro-2-ethenyl)oxirane (Cottrell et al. 2001; Munter et al. 2003). Despite some uncertainty in the quantitative metabolic fate of each of the initial epoxides, total chloroprene oxidation rates effectively account for all possible reactive metabolites formed. For this reason, the total chloroprene oxidation rate was used to calculate an internal dose surrogate calculated by physiologically-based kinetic modeling. The internal dose (amount metabolized/gram tissue/day) was then used for dose response modeling (Himmelstein et al. 2004b). 

 

An additional component of the alternative ADME approach was the collection of in vivo metabolism data from rodents for testing of the physiologically-based kinetic model (Himmelstein et al. 2004b). An extensive set of experiments involved closed chamber gas uptake experiments in mice, rats, and hamsters. Introduction of chloroprene into the re-circulating system clearly demonstrated absorption via the respiratory system. Tissue distribution was modeled using experimentally measured tissue-to-blood partition coefficients with key tissues from each of the rodent species and representative human blood samples. Although these experiments did not track elimination of metabolites in exhaled breath, urine or feces, the blood-to-air partition coefficients (ranging from 4.5:1 for humans and 7.3 to 9.0:1 for rodents) indicate that exhalation of un-metabolized chloroprene could be an expected route of elimination after cessation of inhalation exposure. Oxidative metabolism of course would also be a significant route for chloroprene elimination in vivo. The physiologically based kinetic model by Himmelstein et al. (2004b) successfully described the closed chamber gas uptake concentrations of chloroprene with and without pretreatment of animals with cytochrome P450 inhibitor.

 

Taken as a whole the in vitro metabolism results show why the mouse is at considerably more risk for toxicity. Although the full mechanism leading to tumor development in the lung or other tissues is not completely understood, chloroprene metabolism, particularly its greater oxidation leading to epoxides or other reactive metabolites, slower epoxide hydrolysis, and the potential for glutathione depletion in the mouse relative to the other rodent species indicates that the mouse is not an appropriate model for human health risk assessment. Species differences in tumor outcome are also supported by the lack of lung and liver tumors in other rodent species where observed activation:detoxication ratios are lower than for mice (Table 1); e.g. Wistar rat and Syrian Golden hamster (Trochimowicz et al., 1998). This lack of species similarity in response is also seen in the outcome of the latest human epidemiology studies were no significant increase in lung or liver cancer deaths were reported (Marsh et al. 2007). Collectively, these data show that the mouse is not a good predictor for human outcome. While quantitative response factors cannot be derived with certainty, intrinsic clearance values for lung and liver address species and tissue specific concerns for sensitivity. The activation:detoxication ratios suggest that a modification factor of at least 10 for liver effects and at least 100 for lung effects appears appropriate if the mouse is used for derivation of the effect levels.

Excretion

Excretion of urinary thioethers (presumably glutathione conjugates) by Wistar rats administered chloroprene at 100 or 200 mg/kg bw by gavage is rapid, with clearance reaching a threshold at 24 hours after dosing.

PBPK modelling

Himmelstein et al (2004b) have developed physiologically-based pharmacokinetic (PBPK) models for chloroprene in rodents and humans. Mathematical models were constructed using physicochemical, physiological and metabolic parameters from mouse, rat, hamster and humans and consisted of five distinct compartments for the lungs, liver, fat, richly-perfused tissues and slowly-perfused tissues. Metabolism of chloroprene in the lungs and the liver was described by Michaelis-Menten type saturable kinetics.

The models were used by Himmelstein et al to investigate the cancer dose-response relationship for combined adenoma and carcinoma lung tumours in rodents as the basis for predicting a human equivalent dose, reflecting a 10% excess lifetime risk. The PBPK model was used to predict the internal dose in rodents corresponding to the exposure concentrations used in the bioassay studies, where the internal dose-metric was determined as the total daily amount of chloroprene metabolised per gram of lung. In dose response-analysis, a multistage model was used to determine the benchmark internal dose corresponding to a tumour response of 10% in animals (e.g. 95% lower bound, BMDL10%). PBPK modelling was used to convert the BMDL10% determined in rodents to a human equivalent inhalation exposure concentration. Himmelstein et al predicted that continuous lifetime inhalation exposures to chloroprene at 23 ppm would be associated with a 10% extra risk of developing tumours. 

The study did not report in vivo blood or tissue time-course concentration data for model validation.Thus this limitation does not justify its sole use in predictive human cancer risk assessment; human cancer mortality data are available and more appropriate for risk assessment (see D(N)MEL derivation)

References

 

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