Feasibility of using urinary TDGA as a biomarker for VCM exposures.

Thiodiglycolic acid (TDGA) is a major metabolite of vinyl chloride monomer (VCM), and it has been suggested as an exposure biomarker for VCM. The validity of this biomarker when the level of VCM is less than 5 ppm, however, is questionable. The objective of this article is to evaluate the feasibility of using urinary TDGA as a biomarker of VCM exposure in a community health risk assessment setting where the concentration of VCM in air is typically very low (likely below 1 ppm). To achieve this objective, we examine the fraction of urinary TDGA associated with different levels of VCM exposures of three studies from different countries, using estimations of the TDGA metabolite predicted by a PBPK model. It is demonstrated that differences in background TDGA have considerable effect on the adequacy of TDGA as a biomarker of VCM. We conclude that, in a community health assessment setting, TDGA should not be used as an exposure biomarker for VCM without having a proper control group, and a PBPK model can be used first to determine whether or not the amount of TDGA in urine is of concern.

Local cryoanalgesia is effective for tail-tip biopsy in mice.

Tail-tip biopsy for genotyping of genetically modified mice older than 21 d typically is performed by using isoflurane anesthesia. Isoflurane-induced changes in behavior and metabolism can result in unexpected complications and death. We investigated whether cryoanalgesia by using ethylene chloride spray would be an effective local anesthetic for tail-tip biopsies in mice. C57BL/6J mice were allocated randomly into 4 groups (n = 10 each) to receive isoflurane anesthesia with tail biopsy, ethylene chloride spray on the tip of the tail before biopsy, ethylene chloride spray without biopsy, or no treatment. Blood glucose was measured periodically in both groups undergoing tail biopsy, and the tail-pinch assay was performed in all mice that received ethylene chloride spray. Body weight, water, and food intake were measured daily for 2 wk. In both groups undergoing tail biopsy, blood glucose levels at 15 min were significantly higher than those after 2 min. This elevation was greater and more prolonged after 30 min in mice that received isoflurane compared with ethylene chloride spray. Tail-pinch latency at 20 min was greater than that after 2 min in all mice that received ethylene chloride spray. All mice gained weight, and there was no difference in food and water intake among groups. We conclude that ethylene chloride spray is an effective local anesthetic and a valuable alternative to isoflurane.

Estimation of benchmark dose for micronucleus occurrence in Chinese vinyl chloride-exposed workers.

In this study, we estimated the possibility of using benchmark dose (BMD) to assess the dose-response relationship between vinyl chloride monomer (VCM) exposure and chromosome damage. A group of 317 workers occupationally exposed to vinyl chloride monomer and 166 normal, unexposed control in Shandong Province northern China were examined for chromosomal damage in peripheral blood lymphocytes (PBL) using the cytokinesis-blocked micronucleus (CB-MN) assay of DNA damage. The exposed group (3.47 ± 2.65)‰ showed higher micronucleus frequency than the control (1.60 ± 1.30)‰ (P < 0.01). Occupational exposure level based on micronucleus occurrence in all individuals was analyzed with benchmark dose (BMD) methods. The benchmark dose lower limit of a one-sided 95% confidence interval (BMDL) for 10% excess risk was also determined. Results showed a dose-response relationship between cumulative exposure and MN frequency, and a BMDL of 0.54 mg/m3 and 0.23 mg/m3 for males and females, respectively. Female workers were more susceptible to MN damage than male workers.

Evaluation of the impact of the exposure route on the human kinetic adjustment factor.

The objective of this study was to assess the impact of the exposure route on the human kinetic adjustment factor (HKAF), for which a default value of 3.16 is used in non-cancer risk assessment. A multi-route PBPK model was modified from the literature and used for computing the internal dose metrics in adults, neonates, children, elderly and pregnant women following three route-specific scenarios to chloroform, bromoform, tri- or per-chloroethylene (TCE or PERC). These include 24-h inhalation exposure, body-weight adjusted oral exposure and 30 min dermal exposure to contaminated drinking water. Distributions for body weight (BW), height (BH) and hepatic cytochrome P450 2E1 (CYP2E1) content were obtained from the literature, whereas model parameters (flows, volumes) were calculated from BW and BH. Monte Carlo simulations were performed and the HKAF was calculated as the ratio of the 95th percentile value of internal dose metrics in subpopulation to the 50th percentile value in adults. On the basis of the area under the parent compound's arterial blood concentration vs time curve (AUC(pc)), highest HKAFs were obtained in neonates for every scenario considered, and were the highest for bromoform (range: 3.6-7.4). Exceedance of the default value based on AUC(PC) was also observed for an oral exposure to chloroform in neonates (4.9). In all other cases, HKAFs remained below the default value. Overall, this study has pointed out the dependency of the HKAF on the exposure route, dose metrics and subpopulation considered, as well as characteristics of the chemicals investigated.

Assessing cancer risks from short-term exposures in children.

For the vast majority of chemicals that have cancer potency estimates on IRIS, the underlying database is deficient with respect to early-life exposures. This data gap has prevented derivation of cancer potency factors that are relevant to this time period, and so assessments may not fully address children's risks. This article provides a review of juvenile animal bioassay data in comparison to adult animal data for a broad array of carcinogens. This comparison indicates that short-term exposures in early life are likely to yield a greater tumor response than short-term exposures in adults, but similar tumor response when compared to long-term exposures in adults. This evidence is brought into a risk assessment context by proposing an approach that: (1) does not prorate children's exposures over the entire life span or mix them with exposures that occur at other ages; (2) applies the cancer slope factor from adult animal or human epidemiology studies to the children's exposure dose to calculate the cancer risk associated with the early-life period; and (3) adds the cancer risk for young children to that for older children/adults to yield a total lifetime cancer risk. The proposed approach allows for the unique exposure and pharmacokinetic factors associated with young children to be fully weighted in the cancer risk assessment. It is very similar to the approach currently used by U.S. EPA for vinyl chloride. The current analysis finds that the database of early life and adult cancer bioassays supports extension of this approach from vinyl chloride to other carcinogens of diverse mode of action. This approach should be enhanced by early-life data specific to the particular carcinogen under analysis whenever possible.

Embryo-fetal developmental and reproductive toxicology of vinyl chloride in rats.

Vinyl chloride (VC) exposure is primarily via inhalation in the workplace. The primary target organ of VC toxicity is the liver and occupational exposure to VC leads to hepatic angiosarcoma. However, based on epidemiological studies, researchers have been unable to ascertain the effect of occupational VC exposure on embryo-fetal development or reproductive function. A limited number of animal studies available in the literature have examined the effect of VC on embryo-fetal development, however, there are no published studies on the effect of VC exposure on reproductive capability. The current study was designed to assess the potential maternal and/or embryo-fetal developmental and 2-generation reproductive toxicity of inhaled VC in CD(R) Sprague-Dawley rats at exposure levels of 0, 10, 100, and 1100 ppm. In the embryo-fetal/developmental toxicity study, the female rats were exposed to VC daily from gestation day (GD) 6 through 19. In the reproductive toxicity study, the F(0) generation male and female rats were exposed to VC for a 10-week premating and 3-week mating periods. The F(0) generation male rats were exposed to VC until terminal euthanasia. The F(0) generation female rats were exposed from GD 0 through GD 20 and lactation day (LD) 4 through LD 25. Our results indicate that up to 1100 ppm VC exposure did not adversely affect embryo-fetal developmental or reproductive capability over 2 generations in rats. The primary target organ of VC, the liver, was affected as evidenced by an increase in liver weight and/or histologically identified cellular alterations, such as centrilobular hypertrophy at 100 and 1000 ppm. Based on the results of these studies, the no observed adverse effect level (NOAEL) for embryo-fetal/development is 1100 ppm, and the NOAEL for reproduction is 1100 ppm. The results from the current studies, which are a more comprehensive embryo-fetal/developmental and reproduction study, may be incorporated into future risk assessments of occupational exposure to VC where concerns regarding the effects of VC exposure remain.

Increased numbers of microchimeric cells of fetal origin are associated with dermal fibrosis in mice following injection of vinyl chloride.

OBJECTIVE: To develop a murine model for use in examining the role of microchimeric cells and certain chemical exposures in the pathogenesis of systemic sclerosis (SSc). METHODS: Female BALB/cJ retired breeder mice were bled before and after vinyl chloride injection. The DNA from their white blood cells was obtained, and the number of microchimeric cell equivalents was determined by quantitative polymerase chain reaction using DNA primers specific for the H-2Kb gene, a sequence not found in BALB/cJ mice. Skin was obtained at autopsy, embedded in paraffin, sectioned, and stained with Masson's trichrome. Hydroxyproline analyses were performed on 4-mm skin biopsy samples. RESULTS: Microchimeric cells were identified and quantitated before and after 20 daily intraperitoneal injections of vinyl chloride. The number of microchimeric cells in the peripheral blood increased an average of 48-fold after treatment with vinyl chloride. Histologic examination of the skin of these same mice (which had an increased number of microchimeric cells) showed inflammation, with abundant fibroblasts and a heavy mononuclear infiltration in the dermis. The collagen fibers appeared densely packed and disorganized. Histologic examination of the skin of untreated retired breeder mice and treated virgin mice appeared normal. Quantitative assays to determine the collagen content of skin biopsy samples obtained from treated microchimeric mice compared with nontreated microchimeric or with treated nonmicrochimeric mice showed a 2-3-fold increase in collagen content in the treated microchimeric mice. Extraordinary splenomegaly was present in the vinyl chloride-treated microchimeric mice, accompanied by cellular infiltration and fibrosis. CONCLUSION: The results suggest that vinyl chloride injections into BALB/cJ retired breeder mice lead to activation of microchimeric cells, which causes the cells to divide and multiply. The correlation between the 48-fold increase in microchimeric cells and the appearance of dermal inflammation and fibrosis similar to that of graft-versus-host disease suggests that activated microchimeric cells may be a necessary factor in the pathogenesis of autoimmune diseases such as SSc.

Metabolic activation of vinyl chloride by rat liver microsomes: low-dose kinetics and involvement of cytochrome P450 2E1.

The metabolism and pharmacokinetics of vinyl chloride (VC) have been extensively studied in rodents and humans, but the maximum velocity (Vmax) and Michaelis constant (K(m)) for the activation of VC by microsomal monooxygenases in vitro have not yet been determined. Using a new sensitive assay, the epoxidation of VC by rat liver microsomes (adult Sprague-Dawley) at concentrations from 1 ppm to 10(6) ppm in the gas phase was measured. In the assay, the reactive VC metabolites chloroethylene oxide and 2-chloroacetaldehyde were trapped with excess cAMP, yielding, 1,N6-etheno-cAMP (epsilon cAMP) which was quantitated by HPLC fluorimetry. The trapping efficiency of electrophilic VC metabolites by cAMP was close to 10%. The specificity of the method was confirmed by purification of epsilon cAMP on an immunogel. The VC concentration in the gas phase was measured by GC/flame ionization detection, while in the aqueous phase it was calculated from the partition coefficient between air and the microsomal suspension. Activation of VC by rat liver microsomes followed Michaelis-Menten kinetics with K(m) = 7.42 +/- 0.37 (+/- SD) microM and Vmax = 4674 +/- 46 pmol.mg protein-1.min-1. Inhibitor studies and immunoinhibition assays showed that VC was activated by cytochrome P450 (CYP) 2E1 down to 1 ppm in the air phase. Based on the metabolic parameters determined, the uptake of VC by rats in vivo can be accurately predicted.

[Quantitative evaluation of health risk associated with occupational inhalation exposure to vinyl chloride at production plants in Poland]. / Ilosciowa ocena ryzyka zdrowotnego zwiazanego z zawodowym, inhalacyjnym narazeniem na chlorek winylu w zakladach produkcyjnych w Polsce.

Vinyl chloride is classified by the IARC in group 1-human carcinogens. In Poland occupational exposure to vinyl chloride is found among workers employed in many branches of industry, among others in the industry of vinyl chloride synthesis and polymerization as well as in the plastics, footwear, rubber, pharmaceutical and metallurgical industries. Concentrations observed range from the noon-determinable level to 90 mg/m3, at the MAC value equal to 5 mg/m3. Neoplasm of liver is a major carcinogenic effect of vinyl chloride. Hence, the health assessment focused on this critical risk. Four different linear dose-response models, developed by several authors and based on results of different epidemiological studies, were used to characterise the extent of cancer risk depending on the level of vinyl chloride concentrations. The estimated risk related to a forty-year employment under exposure equal to MAC values (5 mg/m3) fell within the range from 2.9.10(-4) to 2.6.10(-3). As the figures depict it did not exceed the acceptable level (10(-3)).

Transport of reactive metabolites of procarcinogens between different liver cell types, as demonstrated by the single cell microgel electrophoresis assay.

Procarcinogens have to be activated by specific cytochromes before showing adverse effects. Freshly isolated hepatocytes (parenchymal liver cells, PC) are characterized by a high content of such xenobiotic enzymes and are widely used to investigate chemically induced DNA damage. But in many cases liver tumors caused by indirect acting carcinogens can also originate from non-parenchymal liver cells (NPC). We used freshly isolated rat PC and NPC to demonstrate that only PC have activation capacity when treated in vitro with different genotoxic procarcinogens (N-nitrosodimethylamine, NDMA; vinyl chloride, VC). The alkaline single cell microgel electrophoresis assay was applied to measure the genotoxic activity of the activated compounds. In order to test the hypothesis that reactive metabolites can be transported from PC to NPC, we performed additional in vivo studies as well as studies in which PC were incubated together with NPC, only separated by a dialysis tube (in vitro coincubation). The results indicate that reactive metabolites of both NDMA and VC are stable enough to be transported intercellularly from PC to NPC.

Lifetime (149-week) oral carcinogenicity study of vinyl chloride in rats.

A lifetime (149-wk) oral carcinogenicity study of vinyl chloride monomer (VCM) was carried out. Four groups of Wistar rats were used, each consisting of 100 males and 100 females, except for the high-dose group, which comprised 50 males and 50 females. VCM was administered by incorporating polyvinyl chloride powder with a high content of VCM into the diet. The actual exposure levels of VCM were 0 (control), 0.014, 0.13 and 1.3 mg VCM/kg body weight/day. Detailed histopathological examination was restricted to the liver. In the final stage of the study, the mortality in the high-dose group was slightly higher than in controls. A variety of VCM-related liver lesions was found in the high-dose group. The lesions included increased incidences of liver-cell polymorphism, hepatic cysts, foci of cellular alteration, neoplastic nodules, hepatocellular carcinomas and angiosarcomas. Compared with controls, there were increased incidences of hepatic foci of cellular alteration in females of the mid-dose group and of basophilic foci of hepatocellular alteration in females of both the low- and mid-dose groups. There was no evidence that feeding of VCM affected the incidence of tumours in organs other than the liver. Thus, the present study showed that the feeding of VCM at a level of 1.3 mg/kg body weight/day can induce neoplastic and non-neoplastic changes in the livers of male as well as female rats. The feeding of 0.014 or 0.13 mg VCM/kg body weight/day may result in an increased incidence of (basophilic) foci of cellular alteration in the liver of female rats. It was concluded that 0.13 mg VCM/kg body weight/day is the no-observed-adverse-effect level with respect to the induction of tumours in rats.

Incorporation of biological information in cancer risk assessment: example--vinyl chloride.

Vinyl chloride (VC) is used as an example to demonstrate how biological information can be incorporated into quantitative risk assessment. The information included is the pharmacokinetics of VC in animals and humans and the data-generated hypothesis that VC primarily affects the initiation stage of the multistage carcinogenesis. The emphasis in this paper is on the improvement of risk assessment methodology rather than the risk assessment of VC per se. Sufficient data are available to construct physiologically-based pharmacokinetic models for both animals and humans. These models are used to calculate the metabolized dose corresponding to exposure scenarios in animals and in humans. On the basis of the data on liver angiosarcomas and carcinomas in rats, the cancer risk per unit of metabolized dose is comparable, irrespective of routes (oral or inhalation) of exposure. The tumor response from an intermittent/partial lifetime exposure is shown to be consistent with that from a lifetime exposure when VC is assumed to affect the first (initiation) stage of the multistage carcinogenic process. Furthermore, the risk estimates calculated on the basis of animal data are shown to be consistent with the human experience.

Carcinogenicity of vinyl chloride in Sprague-Dawley rats after prenatal and postnatal exposure.

Vinyl chloride was administered by inhalation, 7 hours daily, 5 days weekly, at concentrations of 2500 and 0 ppm, to Sprague-Dawley rats. The treatment was started on 13-week-old breeders and male and female offspring (12-day embryos). The breeders and part of the offspring were exposed for 104 weeks; the other part of the offspring was exposed for 15 weeks only. Under the experimental conditions, vinyl chloride caused an exceptionally high incidence of brain neuroblastomas, liver angiosarcomas, and hepatocarcinomas. The age at start and/or length of treatment may affect the onset of these tumors in different ways.

Neoplastic effect of vinyl chloride in mouse lung--lower doses and short-term exposure.

Neoplastic pulmonary effects of lower doses of vinyl chloride (0 = control, 1, 10, 100, 300, and 600 ppm) and short-term exposure (4 weeks) by inhalation have been studied by light and electron microscopy in 220 mice. Except for dead or seriously sick animals, a large majority of the animals were sacrificed at three different stages: immediately after exposure, 12 weeks later, and 40 or 41 weeks after exposure. Six mice (4: 600 ppm, 2: 0 ppm) were kept longer than 41 weeks to examine the effects of the chemical after a long-term recovery period. Alveologenic tumors were first observed 10 weeks after exposure to 600 ppm. In the subgroups exposed to higher concentrations (600 and 300 ppm) the incidence of tumors was higher and their appearance was earlier than in the subgroups exposed to lower concentrations (100, 10, and 1 ppm). These findings indicated a dose-response relationship for incidence of alveologenic tumors, and the latency period was inversely related to dose. By light and electron microscopy, there was no obvious evidence that tumor cells were derived from Clara cells of the terminal bronchioles. Rather, neoplastic cells in both the tubulopapillary and adenomatous forms of the pulmonary tumors possessed all or some of the ultrastructural characteristics of type II alveolar cells, based on observations of mitochondria, microvilli, osmiophilic lamellar bodies, and other criteria. Type II alveolar cells are therefore considered to be the most sensitive in mice to the neoplastic effect of vinyl chloride.

Non-reversibility of vinyl chloride carcinogenesis in rodents.

A review of the data obtained from various studies on carcinogenicity of vinyl chloride (VC) in rodents, particularly on the effect of dose, age, duration of exposure and potential reversibility of lesions, revealed that vinyl chloride-induced carcinogenicity in rodents was dose and time related; no recovery occurred in mice even after only 1 month of VC exposures or in rats after 6-month exposures. In addition, younger animals (2 months old) were more susceptible to VC-induced carcinogenicity than animals held for 6 or 12 months prior to exposure. Initial 6 or 12 month exposures were adequate to detect the carcinogenic potential of VC. The above information was used as a basis for discussion on design of carcinogenicity studies. Possibility of determining the carcinogenic potential of a compound in a shorter period than the traditional 2 year studies in rodents was discussed in consideration with appropriate doses, species, age and exposure duration. Although this approach may be applicable to a strong carcinogen, it was not considered practicable in case of weak or unknown carcinogens.

Vinyl chloride: inhalation teratology study in mice, rats and rabbits.

These studies evaluated the effects of inhaled vinyl chloride monomer (VCM) on mouse, rat and rabbit embryonal and fetal development. Groups of pregnant CF-1 mice, Sprague-Dawley rats and New Zealand white rabbits were exposed to 500 ppm VCM for 7 hr daily during the period of major organogenesis. Subsequently, other groups of mice were similarly exposed to 50 ppm VCM, and rats and rabbits were exposed to 2500 ppm. While maternal toxicity was observed, exposure to VCM did not cause significant embryonal or fetal toxicity and was not teratogenic in any of the three species at the concentrations tested. Simultaneous exposure of some of the pregnant animals to VCM by inhalation plus 15% ethanol in the drinking water resulted in toxic effects greater than those associated with exposure to VCM alone in the three species. The fetal effects observed were similar to those reported for these three species following administration of ethanol without VCM exposure.

Cancer induction following single and multiple exposures to a constant amount of vinyl chloride monomer.

Vinyl chloride monomer (VCM), already identified as a human animal carcinogen, was selected as a model agent to explore an area of concern for single and intermittent low level exposure. In traditional cancer bioassay, animals are repeatedly exposed over their lifespan to a dose of suspected chemical. In the current studies rats and mice were exposed in an inhalation chamber to single one-hour doses of VCM ranging from 50 to 50,000 ppm. A second group was given 10 one-hour exposures to 500 ppm or 100 one-hour exposures to 50 ppm of the same chemical. All animals were then observed for the remainder of their lives, generally 18-24 months. Moribund animals were euthanized, and survivors were sacrificed on schedule and their tissues examined for pathological changes. Specifically, the oncogenic study demonstrated dose related effects for single one-hour exposure of VCM at high levels, i.e., 5,000 and 50,000 ppm. These concentrations increased the incidence of pulmonary adenomas and carcinomas in mice. Repeated exposure of A/J mice to the same chemical at 500 ppm X 10 one-hour exposures also increased the incidence of pulmonary adenomas and carcinomas which are considered highly one-hour exposure, no significant increase in tumors was observed. Rats exposed to identical concentrations of VCM failed to elicit a tumorigenic response.

Carcinogenicity bioassays of vinyl chloride monomer: a model of risk assessment on an experimental basis.

Data are presented regarding the final results of the Bentivoglio (Bologna) project on long-term carcinogenicity bioassays of vinyl chloride (VC). The experimental project studied the effects of the monomer, administered by different routes, concentrations and schedules of treatment, to animals (near 7000) of different species, strains, sex and age. To our knowledge this is the largest experimental carcinogenicity study performed on a single compound by a single institution. The results indicate that VC is a multipotential carcinogen, affecting a variety of organs and tissues. In the experimental conditions studied, the neoplastic effects of the monomer were also detected at low doses. The experimental and biological factors greatly affect the neoplastic response to VC. Long-term carcinogenicity bioassays are, at present, a unique tool for the identification and quantification of environmental and occupational risks. Precise and highly standardized experimental procedures are needed to obtain data for risk assessment.

[Biochemical changes in vinyl chloride poisoning. I. Effect of different conditions of vinyl chloride exposure on lipid metabolism in rats]. / Zmiany biochemiczne w zatruciach chlorku winylu. I. Wplyw róznych warunków ekspozycji na chlorek winylu na przemiane lipidowa u szczurów.

Lipids metabolism was evaluated in rats chronically exposed to vinyl chloride concentrations of 50, 500 and 20 000 ppm. The studies involved: 1) estimation of lipids content in serum; 2) estimation of cholesterol and triglycerides content in some tissues (liver, muscles, connective tissue, aortic wall), 3) evaluation of the rate of incorporating 14C-acetate into 14C-cholesterol in liver and the rate of its transition into blood. The studies were carried out after 1, 3, 6 and 10 months of vinyl chloride exposure. The results prove that under effects of vinyl chloride exposure some tendencies of an increase in the total and esterified cholesterol in serum vary with the magnitude of exposure (vinyl chloride airborne concentration, intoxication time). As to triglycerides and phospholipids at the final phase of intoxication (after 10 months) a significant increase in their serum content is found at the 20 000 ppm. concentration, whereas there are no changes in the free fatty acids level. As to the examined tissue only in liver some tendencies are found of a decrease in cholesterol content and much greater decrease in triglycerides level, which is progressing with intoxication time and exposure magnitude. No effect of vinyl chloride on the level of lipids in muscles, aortic wall and connective tissue were found. Studies of cholesterol synthesis in liver showed some increase in the rate of building 14C-acetate into cholesterol only in two highest vinyl chloride concentrations (500 and 20 000 ppm) after 10 months of exposure. The results demonstrate that chronic vinyl chloride exposure only slightly affects lipid metabolism. The most significant changes occur only after 10 months of exposure at the concentration of 20 000 ppm. It is supposed that the disturbances in lipid metabolism have no important contribution to vinyl chloride poisonings pathology.

Vinyl chloride: an example for evaluating mutagenic effects in mammals in vivo after exposure to inhalation.

As part of a programme of investigations on the mutagenic effects in mammals in vivo after inhalation of environmental chemicals, the effects of the industrial compound vinyl chloride (VC) was analysed. Chinese hamsters were exposed to 1.25%, 2.5% or 5% (v/v) VC in air for 6, 12 or 24 hr. Bone marrow chromosomes were analysed for induced chromosome aberrations and sister-chromatid exchanges (SCEs) 26 h after beginning of exposure. The frequency of VC-induced chromosome aberrations and SCEs both depend on dose and length of exposure. The highest measured effects were 33.25 SCEs/ cell after an exposure to 2.5% VC for 24 h and 25.7% metaphases with aberrations, when exposed to 5% VC for 24 h.