Multiple organ carcinogenicity of inhaled chloroprene (2-chloro-1,3-butadiene) in F344/N rats and B6C3F1 mice and comparison of dose–response with 1,3-butadiene in mice

Ronald L. Melnick2, Robert C. Sills, Christopher J. Portier, Joseph H. Roycroft, Billy J. Chou1, Sondra L. Grumbein1 and Rodney A. Miller1

National Institute of Environmental Health Sciences, Research Triangle Park, PO Box 12233, NC 27709, USA and
1 Battelle Pacific Northwest Laboratories, Richland, WA 99352, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chloroprene (2-chloro-1,3-butadiene) is a high production chemical used almost exclusively in the production of polychloroprene (neoprene) elastomer. Because of its structural similarity to 1,3-butadiene, a trans-species carcinogen, inhalation studies were performed with chloroprene to evaluate its carcinogenic potential in rats and mice. Groups of 50 male and female F344/N rats and 50 male and female B6C3F1 mice were exposed to 0, 12.8, 32 or 80 p.p.m. chloroprene (6 h/day, 5 days/week) for 2 years. Under these conditions, chloroprene was carcinogenic to the oral cavity, thyroid gland, lung, kidney and mammary gland of rats, and to the lung, circulatory system (hemangiomas and hemangiosarcomas), Harderian gland, kidney, forestomach, liver, mammary gland, skin, mesentery and Zymbal's gland of mice. Survival adjusted tumor rates in mice were fit to a Weibull model for estimation of the shape of the dose–response curves, estimation of ED10 values (the estimated exposure concentration associated with an increased cancer risk of 10%) and comparison of these parameters with those for 1,3-butadiene. Butadiene has been identified as a potent carcinogen in mice and has been associated with increased risk of lymphatic and hematopoietic cancer in exposed workers. Shape parameter values for most of the neoplastic effects of chloroprene and 1,3-butadiene were consistent with linear or supralinear responses in the area near the lowest tested exposures. The most potent carcinogenic effect of 1,3-butadiene was the induction of lung neoplasms in female mice, which had an ED10 value of 0.3 p.p.m. Since the ED10 value for that same response in chloroprene exposed mice was also 0.3 p.p.m., we conclude that the carcinogenic potency of chloroprene in mice is similar to that of 1,3-butadiene. Cancer potency of chloroprene is greater in the mouse lung than in the rat lung, but greater in the rat kidney than in the mouse kidney and nearly equivalent in the mammary gland of each species.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chloroprene (2-chloro-1,3-butadiene; CAS no. 126-99-8) is a colorless volatile liquid (boiling point 59.4°C; vapor pressure 188 mm Hg at 20°C) produced either by dimerization of acetylene and addition of hydrogen chloride or by chlorination of 1,3-butadiene (1). Chloroprene is used almost exclusively as a chemical intermediate in the production of polychloroprene (neoprene) elastomer, which itself is used in the production of automotive rubber goods, wire and cable applications, and adhesives (1,2). There is no end product use of chloroprene monomer. The annual production of chloroprene in Western Europe, the United States and Japan is ~880 million pounds (3).

Chloroprene is highly reactive, forming peroxides and spontaneously polymerizing at room temperature and in the presence of oxygen. The recommended threshold limit value (TLV) for chloroprene in the work environment is 10 p.p.m. (4). The Occupational Safety and Health Administration standard for chloroprene, expressed as an 8 h time-weighted average, is 25 p.p.m.

Chloroprene was reported to be mutagenic in Salmonella typhimurium strains TA 100 (5) and TA 1530 (6) in the presence or absence of metabolic activation enzymes. In contrast, Zeiger et al. (7) found no evidence of mutagenic activity for chloroprene in four strains of S.typhimurium, including strain TA100. Westphal et al. (3) reported no mutagenic activity for freshly distilled chloroprene in strain TA100; however, a mutagenic response was observed with aged samples. Hence, previous positive mutagenicity findings for chloroprene were attributed to the formation and accumulation of mutagenic decomposition products, particularly cyclic chloroprene dimers that resulted during aerobic aging of the distilled compound (3). Chloroprene was not mutagenic in cultured V79 Chinese hamster cells (8).

Chloroprene induced sex-linked recessive lethal mutations in Drosophila (9) and caused transformation of hamster lung cells (10). However, Foureman et al. (11) reported no significant increase in sex-linked recessive lethal mutations in germ cells of male flies exposed to chloroprene. Exposure of B6C3F1 mice to 12, 32 or 80 p.p.m. chloroprene vapors, 6 h/day for 12 days, did not induce increases in chromosomal aberrations or sister chromatid exchanges in bone marrow cells or increase the frequency of micronucleated erythrocytes in peripheral blood (12). With a similar exposure protocol, 1,3-butadiene produced increases in sister chromatid exchanges at 6.25 p.p.m., micronucleated erythrocytes at 62.5 p.p.m. and chromosomal aberrations at 625 p.p.m. (13).

No definitive studies on the biotransformation of chloroprene have been reported; however, a volatile alkylating metabolite produced by mouse liver microsomes was trapped with 4-(4-nitrobenzyl)pyridine (14). By analogy to vinyl chloride, it was suggested that 2-chloro-1,2-epoxybutene-3 and/or 2-chloro-3,4-epoxybutene-1 may be intermediates in chloroprene biotransformation. These epoxide intermediates may be detoxified by conjugation with glutathione (15). In support of this hypothesis, Summer and Greim (16) found that oral treatment of rats with chloroprene led to rapid depletion of hepatic glutathione and increased excretion of urinary thioethers. Hydrolysis by epoxide hydrolase may provide an alternative detoxification pathway (17).

In inhalation toxicity studies lasting 13 weeks, exposure of rats to 80 or 200 p.p.m. chloroprene caused degeneration and metaplasia of the olfactory epithelium, while anemia, hepatocellular necrosis and reduced sperm motility were seen only at 200 p.p.m. (18). In mice, the only significant histopathological effect was an increased incidence of epithelial hyperplasia of the forestomach with exposure to 80 p.p.m. for 13 weeks (18). Results of these studies in rats and mice were used to select the chloroprene exposure concentrations for the 2 year inhalation studies reported here.

In a 4 week inhalation study in which Wistar rats and Syrian golden hamsters were exposed to 0, 40, 160 or 625 p.p.m. chloroprene, chemically induced mortality was observed at the two highest exposures in both species (19). Histopathological changes included centrilobular degeneration and necrosis of the liver in both species exposed to 160 or 625 p.p.m., renal tubular epithelial degeneration in rats exposed to 625 p.p.m., small hemorrhages and perivascular edema in the lungs of rats that died during the study and irritation of the mucous membranes of the nasal cavity in hamsters exposed to 40 or 160 p.p.m. No significant hematological changes were observed in either rats or hamsters.

In a 7 month inhalation study, a dose-dependent increase in the incidence and multiplicity of lung tumors was observed in Kunming albino mice exposed to 0.8–52 p.p.m. chloroprene (20). A chronic inhalation study of chloroprene in Wistar rats (24 month exposure) and Syrian golden hamsters (18 month exposure), performed 20 years ago, reported no evidence of carcinogenicity in either species at exposure concentrations up to 50 p.p.m. (21). Limited epidemiological studies suggest that occupational exposure to chloroprene may increase cancer risk for digestive and lymphatic/hematopoietic tumors (22) and for liver, lung and lymphatic tumors (23).

The 2 year inhalation studies on chloroprene reported here were initiated because of the structural similarity between this chemical and 1,3-butadiene, a trans-species carcinogen in laboratory animals (2426). In addition, separate cohort studies of 1,3-butadiene production workers found significant excess mortality for lymphosarcoma (27,28), while separate evaluations of workers exposed to 1,3-butadiene in the styrene–butadiene rubber industry found significant increased risk of leukemia (29,30). The present studies were designed to evaluate and compare the potential carcinogenicity of chloroprene in rats and mice and to characterize exposure–response relationships.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Test chemical and exposure system
Chloroprene, containing 0.1% t-butylcatechol and 0.1% phenothiazine to inhibit peroxide and polymer formation, was obtained from Denka Chemical Co. (Houston, TX) and stored under nitrogen at –20°C. Gas chromatographic analyses indicated that the purity of the test material was >96% during these studies. The major impurities were chlorobutene, 1-chloro-1,3-butadiene, and chloroprene dimers. Peroxide content was <1 milliequivalent per kg of sample during the study.

Chloroprene vapors were generated in a rotating evaporation flask (immersed in a hot water bath maintained at 66°C), carried in nitrogen through a chilled water condensing column (~1°C) to a distribution manifold, and then separately metered to each stainless steel Hazelton 2000 exposure chamber (Lab Products, Aberdeen, MD) used in this study. Chamber concentrations of chloroprene were regulated by adjusting the metering valves which controlled individual delivery lines from the distribution manifold and by adjusting the pressure of the compressed air to the vacuum pumps in which the chloroprene vapors were diluted prior to entry into the exposure chambers. Concentrations of chloroprene in each chamber were measured at least once every hour during the exposures, with a Hewlett-Packard 5890 gas chromatograph equipped with a flame ionization detector. The mean concentrations of chloroprene in each chamber for the entire study were between 99 and 100% of the target, with a % relative standard deviation of <=4%.

Fresh samples of chloroprene were used each day of exposure. The stabilizers in the bulk chemical, most of the impurities and most of the degradation products were expected to be removed by the evaporation flask/chilled condenser method of vapor generation because they are less volatile than chloroprene. Vapor samples were collected from the distribution line and found to contain chlorobutene and 1-chlorobutadiene (GC peak areas were ~0.5 and 0.2% that of chloroprene, respectively); no peroxides or chloroprene dimers were detected.

Animal maintenance
Four-week-old male and female F344/N rats and B6C3F1 mice were obtained from Simonsen Laboratories (Gilroy, CA) and quarantined for 14 days prior to the start of the study. Animals were housed in individual stainless steel wire mesh cage units within the exposure chambers. Chambers for control and exposed groups were maintained at 75 ± 3°C and 55 ± 15% relative humidity with ~15 air changes per hour. City water (Richland, WA) and NIH-07 diet were available ad libitum, except during the exposure periods when the feed was removed. Other than exposure to chloroprene, control and exposed animals were treated similarly.

Exposure regimen
Groups of 50 male and female rats and 50 male and female mice were exposed to chloroprene vapors by whole-body exposures at target concentrations of 0 (control chamber), 12.8, 32 or 80 p.p.m. for 6 h + T90 (12 min, time to reach 90% of the target concentration) per day, 5 days per week for 104 weeks (excluding holidays).

Histopathology
All animals that died during the study or that were killed at the end of the exposure period received a complete necropsy and histopathological examination. All major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 5–6 µm, and stained with hematoxylin and eosin for microscopic examination. The following tissues were examined microscopically: gross lesions and tissue masses, adrenal gland, bone and marrow, brain, clitoral gland, esophagus, gallbladder (mice), heart, large intestine (cecum, colon and rectum), small intestine (duodenum, jejunum and ileum), kidney, larynx, liver, lung, lymph nodes (bronchial, mandibular, mediastinal and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and glandular stomach), testes with epididymides and seminal vesicles, thymus, thyroid gland, trachea, urinary bladder and uterus.

Statistical analyses
Differences in survival were analyzed by life table methods (31,32), and incidences of neoplasms and non-neoplastic lesions (the ratio of the number of animals bearing such lesions at a specific anatomical site to the number of animals in which that site was examined) including information on time of death were analyzed by logistic regression analysis for lesions incidental to the cause of death or not rapidly lethal (33) and by life table tests for rapidly lethal neoplasms (31,32). Tumor rates in both species were also analyzed after adjustment for intercurrent mortality using the survival-adjusted `poly-3' quantal response test described by Portier and Bailer (34), with a power value of k = 3 (35).

Dose–response shape and ED10
For those neoplasms showing chemical-related effects in mice, the shape of the dose–response curve was estimated by fitting the following modified Weibull model (35) to the survival-adjusted tumor data:


where P(dose) is the probability of a neoplasm prior to study termination for animals administered a defined dose of chloroprene. The parameters intercept, scale and shape are estimated via maximum likelihood estimation using a binomial likelihood based upon the `poly-3' adjusted tumor data. The ED10 values obtained from these dose–response curves represent the exposure concentrations associated with an excess cancer risk of 10% at each site. A likelihood ratio test was used to test the hypothesis that the shape parameter equals 1 and likelihood-based confidence intervals were used to estimate lower (highest scale, lowest shape) and upper (lowest scale, highest shape) confidence bounds on the ED10. If the estimated shape parameter is >1, the resulting dose–response has more curvature than a linear model (shape parameter equal to one) and exhibits sublinear behavior. If the estimated shape parameter is <1, then the dose–response curve is very steep (supralinear) in the low-dose region.

In some cases, the dose–response appeared to have a maximum response of <100% which could artificially lower the estimated shape. In these cases a Hill model was fit to the survival-adjusted tumor data:


This model is fully specified for these data and has as many parameters as there are data points. The estimated values of shape and ED10 from this model are intended as a guidance value to indicate what changes may be possible in these values due to model misspecification. This model achieves a maximal response of <100%.


    Results
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 Materials and methods
 Results
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 References
 
Chronic effects of chloroprene in rats
Survival of male rats exposed to 32 or 80 p.p.m. chloroprene was significantly lower than that of the chamber control group; there was no significant effect of exposure on survival of female rats (Figure 1Go). The only body weight effect observed in rats was a slight decrease in males in the high exposure group compared with chamber controls after 93 weeks of exposure.



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Fig. 1. Kaplan–Meier survival curves for male and female rats exposed to chloroprene by inhalation for 2 years.

 
Increased incidences and exposure-related positive trends were observed for neoplastic lesions in the oral cavity, thyroid gland, lung, kidney and mammary gland (Table IGo). Non-neoplastic lesions observed in rats following exposure to chloroprene are shown in Table IIGo. The incidences of squamous cell papilloma and the combined incidences of squamous cell papilloma or squamous cell carcinoma of the oral cavity were significantly increased in male rats exposed to 32 p.p.m. and in male and female rats exposed to 80 p.p.m. The increased incidences in the 32 and 80 p.p.m. groups exceeded the NTP historical control range for these lesions. The historical incidence of squamous cell papilloma or carcinoma of the oral cavity in chamber control F344/N rats from previous 2 year inhalation studies is 1.4% (0–6% range) in males and 1.2% (0–6%) in females. Oral cavity neoplasms were detected in the palate, pharynx, gingiva, cheek and tongue. No squamous cell carcinomas were observed in control rats, whereas three were observed in exposed male rats and eight were observed in exposed female rats.


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Table I. Incidence of primary neoplasms in male and female F344/N rats exposed to chloroprene for up to 2 yearsa
 

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Table II. Incidence of non-neoplastic lesions in male and female F344/N rats exposed to chloroprene for up to 2 yearsa
 
The incidences of follicular cell adenoma or carcinoma of the thyroid gland were increased in males exposed to 32 or 80 p.p.m. and exceeded the historical control range. Although the incidence in female rats exposed to 80 p.p.m. was not significantly greater than that of the control group, it did exceed the historical control range.

An exposure-related increased trend for alveolar/bronchiolar adenoma or carcinoma (combined) was observed in male rats. Although none of the pairwise comparisons between exposed groups and controls was significant, the incidence in the 80 p.p.m. group exceeded the historical control range. The incidence of alveolar/bronchiolar carcinomas in the 80 p.p.m. exposure group of males (4/50; 8%) exceeded the historical incidence of 0.9% (0–2% range) in chamber control male F344/N rats. In addition, incidences of alveolar epithelial hyperplasia were significantly greater in all exposed groups of males and females compared with chamber controls (Table IIGo).

Slight increases in the incidences of renal tubule hyperplasia (data not shown) and of renal tubule adenoma were observed in the kidneys of male and female rats after single section evaluations (two adenomas in the high dose group of each sex and one in the mid-dose group of males versus zero in the chamber control groups). Because of the slight increased trends in renal proliferative lesions and because renal tubule neoplasms are uncommon in control F344/N rats, eight additional sections of kidney were examined from each control and exposed rat to provide a clearer indication of the potential effects of chloroprene in this organ. The historical incidence of renal tubule adenoma in chamber control rats is 0.9% (0–4% range) in males and 0.3% (0–2%) in females. After step-sectioning, the incidence of renal tubule hyperplasia was increased significantly in the 32 and 80 p.p.m. exposure groups of male rats and in the 80 p.p.m. exposure group of female rats (Table IIGo) and the incidence of renal tubule carcinoma or adenoma was increased in all exposure groups of males (Table IGo). Also unusual was the finding of four renal tubule adenomas in the 80 p.p.m. exposure group of female rats.

The incidence of mammary gland fibroadenomas was increased in female rats exposed to 32 or 80 p.p.m. chloroprene; however, mammary gland carcinomas were not increased.

Several exposure-related non-neoplastic nasal lesions were observed in male and female rats including atrophy, fibrosis, adenomatous hyperplasia, basal cell hyperplasia, chronic active inflammation, metaplasia and necrosis of the olfactory epithelium (Table IIGo). For the most part, these lesions were mild to moderate in severity. Although the incidence of many of these lesions approached or reached 100% at the 80 p.p.m. exposure concentration, there was no evidence of progression to neoplasia.

Chronic effects of chloroprene in mice
Survival was reduced in male mice exposed to 32 or 80 p.p.m. chloroprene and in all exposed groups of females compared with chamber controls (Figure 2Go). Most early deaths and moribund sacrifices were attributed to chloroprene-induced neoplasms, including hemangiosarcomas, mammary gland carcinomas and sarcomas of the skin. Mean body weight of female mice exposed to 80 p.p.m. chloroprene was lower than that of controls after week 75.



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Fig. 2. Kaplan–Meier survival curves for male and female mice exposed to chloroprene by inhalation for 2 years. P(dose) = 1-e -(interceptscale{bullet}doseshape kshape + doseshape)

 
Increased incidences and exposure-related positive trends were observed for neoplastic lesions in the lung, circulatory system, Harderian gland and forestomach in male mice (Table IIIGo) and in female mice (Table IVGo). In addition, incidences of kidney tumors were increased in exposed male mice (Table IIIGo) and neoplasms of the liver, mammary gland, skin and mesentery were increased in exposed female mice (Table IVGo). Because of the large number of early deaths in mice exposed to chloroprene, the poly-3 survival-adjusted neoplasm rates provide a clearer indication of exposure–response relationships. Non-neoplastic lesions induced by chloroprene in mice are shown in Table VGo.


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Table III. Incidence of primary neoplasms in male B6C3F1 mice exposed to chloroprene for up to 2 yearsa
 

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Table IV. Incidence of primary neoplasms in female B6C3F1 mice exposed to chloroprene for up to 2 yearsa
 

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Table V. Incidence of non-neoplastic lesions in male and female B6C3F1 mice exposed to chloroprene for up to 2 yearsa
 
The incidences of alveolar/bronchiolar adenoma or carcinoma were increased in all exposed groups of male and female mice and greatly exceeded the historical ranges of these neoplasms in chamber control mice (Table IIIGo and Table IVGo). In addition, the incidences of multiple alveolar/ bronchiolar adenoma and carcinoma, as well as the incidences of bronchiole hyperplasia, were increased in all exposed groups of males and females (Tables III–VGoGoGo).

Using a PCR-based assay, male and female mice from this study were concluded to be infected with Helicobacter hepaticus (36); however, only male mice contained the spectrum of lesions associated with Helicobacter infection, including bile duct hyperplasia, karyomegaly and regeneration of hepatocytes. In addition, silver staining of male mouse liver sections revealed the presence of helical microorganisms. Because Helicobacter-associated hepatitis has been shown to be associated with increased incidences of liver neoplasms in male mice (36,37), hemangiosarcomas of the liver were excluded from the analyses of circulatory (endothelial) neoplasms in male mice in the present study. Even with this exclusion, the combined incidences of hemangiosarcoma and hemangioma at other sites were increased in all exposed groups of male mice (Table IIIGo). The incidence of these lesions was also increased in the 32 p.p.m. exposure group of female mice. The incidences of neoplasms in female mice or at other sites in male mice were not considered to have been impacted by infection with H.hepaticus or its associated hepatitis (36). The incidence of hepatocellular carcinoma was increased in all exposed groups of female mice compared with chamber controls (Table IVGo). The incidence of hepatocellular adenoma was not increased in any of the exposed groups of female mice.

Marginal increases in the incidences of renal tubule hyperplasia and of renal tubule adenoma were observed in the kidney of male mice after single section evaluations (data not shown). Because of the increased trends in renal proliferative lesions and because the occurrence of renal tubule adenomas in control male B6C3F1 mice is rare (0.2%), eight additional kidney sections were examined from each control and exposed male mouse to provide a clearer indication of the potential effects of chloroprene in this organ. After step sectioning, the incidence of renal tubule hyperplasia was increased significantly in all of the exposed groups of male mice (Table VGo) and the incidence of renal tubule adenoma was increased in the 32 and 80 p.p.m. exposure groups (Table IIIGo). No renal tubule neoplasms were seen in chamber control male mice even after step sectioning.

As with the rat study, the mammary gland was a target of chloroprene-induced neoplasia in female mice. The incidence of mammary gland carcinoma or adenoacanthoma was increased in the 32 and 80 p.p.m. exposure groups, and the incidence in all of the exposed groups exceeded the historical control range (Table IVGo).

Similar to the study in rats, high incidences of non-neoplastic nasal lesions were observed in male and female mice exposed to 80 p.p.m. chloroprene (including atrophy, adenomatous hyperplasia and metaplasia of the olfactory epithelium), without evidence of neoplasia.

Comparison of dose–response for neoplasms induced by chloroprene in rats and mice and by 1,3-butadiene in mice
The shapes of the dose–response curves and ED10 values for neoplasms induced by chloroprene in mice were estimated by fitting a modified Weibull model (35) to the poly-3 survival-adjusted tumor rates. The ED10 was chosen because in most cases in mice it represents a very slight extrapolation to lower doses, consistent with the recommendations of Murrell et al. (38). The shape parameter and ED10 values for chloroprene in rats are shown in Table VIGo, and the estimates for chloroprene in mice along with the corresponding estimates for 1,3-butadiene (based on data from ref. 39), are shown in Table VIIGo. If the estimated shape parameter is significantly >1, then the resulting dose–response has more curvature than a linear model (shape parameter equal to 1) and exhibits sublinear behavior. If the estimated shape parameter is <1, then the dose–response curve is very steep (supralinear) in the low-dose region. The ED10 values represent the estimated exposure concentration associated with an increased cancer risk of 10% at each site. In cases where there may have been <100% possible maximal response, estimates of these values using the Hill model are also presented.


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Table VI. Dose–response shape parameter and ED10 values for chloroprene in F344/N ratsa
 

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Table VII. Comparison of dose–response shape parameter and ED10 values for chloroprene (Chl) and 1,3-butadiene (BD) in B6C3F1 micea
 
For many of the chloroprene-induced neoplastic effects that were evaluated, the dose–responses were consistent with a linear model. In most instances in which a departure from linearity was evident, the shape parameter indicated a dose–response curve that was supralinear (concave downward) in the low-dose region (kidney in male rats, hemangioma and hemangiosarcomas in male and female mice, and alveolar/bronchiolar adenomas or carcinomas, skin sarcomas and mesentery sarcomas in female mice). Only squamous cell papillomas or carcinomas of the forestomach in female mice had evidence of a sublinear (concave upward) dose–response. For 1,3-butadiene, the shape parameter indicated a supralinear dose–response for lung, Harderian gland and liver neoplasms in male and female mice and for mammary gland neoplasms in female mice.


    Discussion
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 Abstract
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 Materials and methods
 Results
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 References
 
Chloroprene was selected for study because it is an important high production chemical with a potential for human exposure, there is limited information available on its carcinogenic potential in experimental animals and humans and because it is the 2-chloro analogue of 1,3-butadiene. Chloroprene is also structurally similar to vinyl chloride, a human carcinogen known for its induction of angiosarcomas of the liver in laboratory animals and exposed workers.

In long-term inhalation studies of 1,3-butadiene in B6C3F1 mice (24,25,39,40), particularly noteworthy findings included the induction of early malignant lymphomas, uncommon hemangiosarcomas of the heart and malignant lung neoplasms in female mice at exposure concentrations as low as 6.25 p.p.m. Other sites of tumor induction in mice included the liver, forestomach, Harderian gland, ovary, mammary gland and preputial gland. Exposure of Sprague–Dawley rats to 1000 or 8000 p.p.m. 1,3-butadiene produced carcinogenic effects in the mammary gland, brain, Zymbal's gland, uterus, pancreas, testis and thyroid gland (26). These studies established 1,3-butadiene as a multi-species, multi-organ carcinogen, with the mouse eliciting the more striking response. Unlike the genotoxic effects induced by 1,3-butadiene in mice (13), chloroprene did not produce cytogenetic damage in bone marrow cells of mice exposed to concentrations up to 80 p.p.m. (12).

Epidemiological studies have consistently found excess mortality from lymphatic and hematopoietic cancers associated with occupational exposure to 1,3-butadiene. Significant excess mortality from lymphosarcoma has been observed in separate studies of workers in the 1,3-butadiene production industry (27,28), while increases in leukemia in the styrene–butadiene rubber manufacturing industry have been associated with exposure to 1,3-butadiene (29,30).

In the present study, 2 year exposures to chloroprene produced multiple organ carcinogenic responses in rats and mice with several sites being the same as those targeted by 1,3-butadiene (Table VIIIGo). Exposure-related carcinogenic effects of chloroprene in rats were seen in the lung, oral cavity, thyroid gland, mammary gland and kidney. The mammary gland and thyroid gland were also sites of tumor induction by 1,3-butadiene in rats (26). Exposure to chloroprene or 1,3-butadiene increased the incidence as well as the multiplicity of mammary gland fibroadenomas in female rats. The finding of oral cavity, lung and kidney neoplasms in chloroprene-exposed rats and the lack of similar effects with 1,3-butadiene at much higher exposure concentrations (8000 p.p.m.) suggests that chloroprene carcinogenesis in rats involves processes beyond those associated with 1,3-butadiene carcinogenesis.


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Table VIII. Sites of increased incidences of neoplasms in the 2 year inhalation studies of chloroprene and 1,3-butadiene in rats and mice
 
Similar to 1,3-butadiene, species differences in sites of tumor induction were observed in rats and mice exposed to chloroprene. Sites of tumor induction by chloroprene that were observed in mice but not in rats included the female mouse lung, circulatory system, Harderian gland, forestomach, skin, mesentery, Zymbal's gland and liver. Neoplasms of the oral cavity and thyroid gland were induced in rats but not in mice.

Because these studies involved whole-body exposure, some of the absorbed chloroprene dose might have resulted from oral exposure (from grooming) or skin penetration. There are insufficient data to estimate relative doses from the latter routes. In any case, it is possible that oral and skin exposure contributed in part to the induction of oral cavity tumors in rats and skin tumors in mice that were observed in this study.

In contrast with our findings of multisite carcinogenicity of chloroprene in F344/N rats, a 2 year study in Wistar rats and an 18 month study in Syrian golden hamsters were reported to show no evidence of carcinogenicity at chloroprene concentrations up to 50 p.p.m. (21). However, one finding that was similar to the present study was the increased incidence and multiplicity of mammary gland fibroadenomas in female rats exposed to 50 p.p.m. chloroprene. The apparent lack of concordance between these studies at other sites in rats may in part be due to differences in exposure concentration (up to 80 versus 50 p.p.m.), strain differences in response (F344/N versus Wistar), the additional sectioning of male and female rat kidneys in our study to provide a clearer indication of potential carcinogenic effects in this organ, and the multiple levels of pathology review in the present studies. Differences in the methods of vapor generation were probably not a critical factor since neither study detected peroxides or chloroprene dimers in the distribution line (present study) or in the vapor generator reservoir (21).

Exposure to chloroprene caused chronic degeneration and hyperplasia of the olfactory epithelium in rats and mice without evidence of neoplasia. The lack of an association between proliferative nasal lesions and nasal carcinogenesis has been observed with several chemicals studied by the National Toxicology Program (41).

Several organs in which 1,3-butadiene was carcinogenic in mice were similarly affected by chloroprene (Table VIIIGo), including the lung, Harderian gland, liver, forestomach and mammary gland. Isoprene, the 2-methyl analogue of 1,3-butadiene, has also been shown to induce neoplasms of the lung, liver, Harderian gland, and forestomach in mice (42). The carcinogenicity of 1,3-butadiene and its congeners (chloroprene and isoprene) at these four sites may involve a common mechanism. Comparisons between chloroprene and vinyl chloride are also worth noting because the latter compound is metabolized to a chloroepoxide intermediate. Common sites of tumor induction by vinyl chloride (43) and chloroprene include hemangiosarcomas, as well as neoplasms of the liver, lung, mammary gland and forestomach in mice, and mammary gland neoplasms in rats.

Some carcinogenic effects of 1,3-butadiene in mice were not seen in the present study. Most notable was the lack of lymphomas in mice exposed to chloroprene compared with the early occurrence and extensive development of lymphocytic lymphomas in mice exposed to 625 p.p.m. 1,3-butadiene (24,25). This difference may be related to differences in exposure concentrations and to differences in target organ dosimetry and/or reactivity of metabolic intermediates. Exposure of mice to 1,3-butadiene also induced hemangiosarcomas of the heart. Although exposure to chloroprene did not induce hemagiosarcomas of the heart, there were exposure-related increases in hemangiomas and hemangiosarcomas at multiple organ sites. In addition, 1,3-butadiene, but not chloroprene, induced granulosa cell tumors of the ovary, and chloroprene, but not 1,3-butadiene, induced skin and mesentery sarcomas in female mice. Small numbers of renal tubule adenomas were observed in male mice exposed to either 1,3-butadiene or chloroprene.

Kidneys from rats and mice were step sectioned because we observed marginal increased trends and incidences of renal tubule hyperplasia and adenoma with the standard histopathological evaluations. The results from the additional kidney sections indicate that chloroprene induces proliferative renal tubule lesions, including neoplasms, in rats and mice. Hence, this study identified chloroprene as a trans-species renal carcinogen.

Several sites of tumor induction identified in this study have been reported as sites suggestive of increased risk of human cancer associated with occupational exposure to chloroprene, including the lung, skin and liver (1,23). The lack of evidence for increased risk of mammary carcinogenesis by chloroprene or 1,3-butadiene in workers may be related to the fact that most epidemiological studies of these chemicals excluded analyses of females because of the small numbers who were potentially exposed.

The analyses of dose–response for tumors induced by chloroprene in rats or mice or by 1,3-butadiene in mice (Tables VI and VIIGoGo) were for the most part consistent with a linear model or indicative of a supralinear response in the low-dose region. ED10 values, which are central tendency estimates generally just below the region of experimental observation, represent the estimated exposure concentration associated with an increased cancer risk of 10% at each site and have been used to compare cancer potency between carcinogens. The data in Table VIIGo indicate that chloroprene is more potent than 1,3-butadiene at inducing hemangiomas or hemagiosarcomas in male and female mice (although under the Hill model they are equivalent); however, the two chemicals were nearly equivalent in their potency to induce lung and Harderian gland neoplasms in male and female mice, and mammary gland, liver and forestomach neoplasms in female mice. For both chemicals, one of the most potent responses was for alveolar/bronchiolar adenoma or carcinoma in female mice, with ED10 values of 0.3 p.p.m. ED10 values for neoplasms that were induced by 1,3-butadiene but not by chloroprene (e.g. malignant lymphoma and granulosa cell tumors of the ovary) were higher than those estimated for 1,3-butadiene-induced lung neoplasms, Harderian gland neoplasms or hemangiomas and hemangiosarcomas. Hence, the overall carcinogenic potency of chloroprene in mice appears to be similar to that of 1,3-butadiene.

Three common sites of neoplasm induction by chloroprene in rats and mice include the lung and kidney in males and the mammary gland in females. A comparison of ED10 values for these neoplasms (Tables VI and VIIGoGo) indicates that the cancer potency of chloroprene is greater in the male mouse lung than in the male rat lung, greater in the male rat kidney than in the male mouse kidney, and nearly equivalent in the female mammary gland of each species.

The mechanism of chloroprene-induced carcinogenicity is not known. Although chloroprene was reported to be mutagenic to Salmonella (5,6), follow-up studies found no evidence of its mutagenicity in Salmonella (3,7) especially when freshly distilled samples were used. These results indicate that either chloroprene is not mutagenic to Salmonella or that in the systems used to determine its mutagenicity the reactive alkylating intermediate did not reach the target DNA. In addition, chloroprene did not induce chromosomal aberrations, sister chromatid exchanges or micronucleated erythrocytes in bone marrow or peripheral blood of mice exposed to concentrations as high as 80 p.p.m. (12). Clearly, the genotoxicity assays cited above were not predictive of the potent multisite carcinogenic effects of chloroprene. The finding of a high frequency of K-ras mutations in chloroprene-induced lung and Harderian gland neoplasms from mice in this study, which were predominantly A->T transversions at codon 61 (44), suggests the possible involvement of a mutagenic event in chloroprene-induced neoplasia. Interestingly, Harderian gland neoplasms induced by isoprene also had a high frequency of K-ras codon 61 A->T transversions (45).

The carcinogenic effects of 1,3-butadiene have been attributed to its mutagenic epoxide intermediates (46). Similarly, the mutagenic and carcinogenic effects of vinyl chloride have been attributed to its epoxide metabolite, chloroethylene oxide, and the rearrangement product, chloroacetaldehyde, both of which can react with DNA to form a variety of adducts (47,48). Neither the metabolic fate of chloroprene nor the biological properties of its metabolic intermediates have been well studied. Oxidation of chloroprene to epoxide intermediates (2-chloro-1,2-epoxybutene-3 and 2-chloro-3,4-epoxybutene-1) was suggested to occur based on the detection of alkylated 4-(4-nitrobenzyl)pyridine in incubations of chloroprene and mouse liver microsomes (14,15). Analogous to the formation of chloroacetaldehyde subsequent to the oxidation of vinyl chloride to chloroethylene oxide, it is possible that 2-chloro-1,2-epoxybutene-3 could undergo rearrangement to form an unsaturated chloroketone. These postulated oxidative intermediates of chloroprene metabolism may be protein and/or DNA reactive and account for the cytotoxicity and carcinogenic effects of this compound. Differences in stability, disposition and reactivity of these various intermediates may account for differences in dose–response carcinogenic effects of chloroprene and 1,3-butadiene. Clearly, further studies are needed to understand the processes involved in chloroprene carcinogenesis.


    Notes
 
2 To whom correspondence should be addressed Email: melnickr{at}niehs.nih.gov Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 16, 1998; revised January 5, 1999; accepted January 25, 1999.