Industrial Health 1997, 35, 301-324

REVIEW ARTICLE

Assessment of the Health Effects of Trichloroethylene

Takashi KANEKO, Pei-Yu WANG and Akio SATO*

Department of Environmental Health, Medical University of Yamanashi, Tamaho, Yamanashi 409-38, Japan

Received March 22, 1997 and accepted May 7, 1997

Abstract: The epidemiological studies performed thus far have presented only limited evidence for the carcinogenicity of trichloroethylene (TRI) to humans. However, these studies had drawbacks such as insufficient size of cohort, short observation period, and inadequate TRI exposure assessment; therefore, no concrete conclusion has been reached concerning TRI carcinogenicity to humans. Despite the limited epidemiological evidence as to the carcinogenicity of TRI, the International Agency for Research on Cancer (IARC) has changed the carcinogenicity classification of TRI from Group 3 (not classifiable as to carcinogenicity to humans) to Group 2A (probably carcinogenic to humans). In regard to the new classification by the IARC, the committee for occupational exposure limits of the Japan Society for Occupational Health has made a proposal that it is too early to classify the carcinogenicity of TRI as Group 2A and that it is proper to promote exposure control with the carcinogenicity being classified as 2B for the moment. There are species differences in TRI carcinogenicity, particularly between rats and mice. Although experimental studies have found no evidence that TRI induces liver cancer in rats, there is ample evidence that TRI promotes the development of liver cancer in mice, particularly in B6C3F~ mice. The carcinogenicity of TRI in this strain of mice may be based on an epigenetic mechanism rather on a genotoxic mechanism and the liver cancer may be induced only after TRI has been inhaled for a long period of time at concentrations high enough to cause cytotoxicity. Conversely, with no reports showing TRI-induced renal tumors in mice, the possibility has been suggested that this chemical induces such tumors in male rats. The species differences are mainly accounted for by differences in the metabolism of TRI between rats and mice. From a general survey of the literature, it can be concluded that TRI itself is not mutagenic. However, the conjugation of TRI with glutathione (GSH), a minor pathway of TRI metabolism, results in mutagenic metabolites in the kidney of rats. The acute toxicity of TRI is neurotoxicity based on its anesthetic action. An exposure to extremely high levels of TRI may cause the liver and kidney disorders. Repeated exposures to high levels of TRI may result in neuro-, hepato-, and/or nephrotoxicity. The main symptoms appearing after chronic exposure at low levels are neurological changes represented by subjective symptoms relating to central and autonomic nervous systems, or by a lowered conduction velocity of the nerves or a prolonged latency of the nerve responses. For the present, it is reasonable to use the neurological findings for establishing the reference values of TRI for both work and general environments. A value of 25 ppm (135 mg/m3) is proposed as a reference value for work environments, and 25-50 ppb (135-270 pug/m3)for the general environment (111,000 of the value for work environment).

Key words: Trichloroethylene, Carcinogenicity, Mutagenicity, Risk assessment

*To whom correspondence should be addressed . 302 T KANEKO et al.

Introduction the kidney [1.26]), and benign brain tumors or brain tumors not evaluated for the degree of malignancy (1.10, brain tumor Trichloroethylene (TRI) is an industrial solvent used not evaluated for the degree of malignancy [1.21]). The mainly for degreasing of metal parts. Its use in dry cleaning lower limit of the 95% confidence interval (95% CI) of SMR is now uncommon. TRI may be found in printing inks, did not exceed 1.0 for any of these lesion sites. Garabrant varnishes, adhesives, paints, and lacquers. TRI has attracted et al. concluded that no significant excesses in cause-specific great attention among the general public because of its mortality was found. However, they also stated that because leakage from industrial settings into the general environment, of the study design, excess risks could not be detected for particularly into underground water. The greatest concern those diseases that had latency periods in excess of 20 to 30 raised by both occupational and environmental health years. In this study, 37% of the subjects were exposed professionals has been whether or not TRI is a human exclusively to TRI, but workers exposed to other various carcinogen, i.e., whether long-term exposure to low doses compounds were also included in the cohort. of TRI causes cancer in humans. In the present paper, the Spirtas et al.6~carried out a retrospective cohort study at health effects of TRI are discussed with a special emphasis an aircraft maintenance facility of an air force base in Utah, on its carcinogenicity. the U.S., that employed 14,457 workers. Of these workers, 6,929 were exposed to TRI in the 1950s to 1960s. The Carcinogenicity of TRI observed deaths among white workers were compared with the expected number of deaths, based on the Utah white (1) Epidemiological studies population, and adjusted for age, sex, and calendar period. The results of the epidemiological studies of TRI Significant decreases were found in mortality from all causes carcinogenicity are summarized in Table 1'~. The first cohort (SMR 0.92; 95% CI 0.90-0.95), all malignant neoplasms study on the carcinogenicity of TRI was conducted in 1978 (0.90; 0.83-0.97), ischemic heart disease (0.93; 0.88-0.98), by Axelson et a1.2~in Sweden. This study was carried out non-malignant respiratory disease (0.87; 0.76-0.98), and with a cohort of 518 persons exposed to TRI; no excess accidents (0.61; 0.52-0.70). Mortality was increased for death from cancer was demonstrated. A cohort study multiple myeloma (MM) among the women (2.36; 0.87- conducted in Finland thereafter with 2,084 workers 5.14), non-Hodgkin's lymphoma (NHL) among the women occupationally exposed to TRI also found no evidence of (2.12; 1.02-3.90), and cancer of the biliary passages and TRI carcinogenicity3~. Excess death from cancer was not liver among the men who died after 1980 (3.58;1.16-8.36). found in a cohort consisting of 2,646 employees of a The detailed analysis of the 6,929 employees occupationally manufacturing plant using TRI4~. In this study, the cohort exposed to TRI, the most widely used solvent at the base had an observed cancer mortality of 21 as opposed to an during the 1950s and 1960s, did not show any significant expected mortality of 36.7, with a standardized mortality or persuasive association between several measures of ratio (SMR) of 0.57. exposure to TRI and any excess of cancer. Among the women Garabrant et al.5~conducted a retrospective cohort mortality employed in the departments in which fabric cleaning and study with 14,067 workers of both sexes employed for four parachute repair operations were performed, more deaths or more years between 1958 and 1982 at an aircraft than expected occurred from MM and NHL. The inconsistent manufacturing company in San Diego County, California. mortality patterns by sex, multiple and overlapping The follow-up of the cohort members for a mean duration exposures, and small numbers made it difficult to ascribe of 15.8 years from the date of first employment resulted in these excesses to any particular substance. the successful tracing of 95% of the cohort and found 1,804 Axelson et al.'s expanded their initial epidemiological deaths through 1982. The mortality rates thus obtained were study2>.The cohort of the updated study was 1,727 workers compared with the U.S. national data and San Diego County who had used TRI and had undergone a urinalysis for data in terms of race (white and nonwhite), sex, age, calendar trichloroacetic acid (TCA) at least once during the period year, and cause of death. Mortality due to all cancers was 1955 through 1975. Of these workers, 57 were excluded low (SMR 0.84). When the data were analyzed according from the data analysis because of failure in the follow-up to the site of lesion, SMR with a value more than 1.0 was (e.g., incomplete documents); the remaining 1,670 workers calculated for the esophagus (1.14), pancreas (1.19), urinary (1,421 males and 249 females) were enrolled in the study. tract (1.10, malignant tumors in the urinary tract other than The mean TCA value was used as the level of exposure to

Industrial Health 1997, 35, 301-324 HEALTH EFFECTS OF TRICHLOROETHYLENE 303

Table 1. Epidemiological studies on the carcinogenicity of trichloroethylene

TRI when more than one TCA values were available. The was 1.17 (1.00-1.37), showing that the mortality rate for interval from the date when the first urinalysis for TCA was circulatorydiseases was marginally higher in persons exposed made until retirement or 1979 was used as the duration of to TRI. However, no significant relationship between the exposure. The cohorts were monitored for deaths until 1986 level of exposure, as determined from urinary TCA levels, (or 1987 for cancer death or cancer incidence). Risk was and the mortality from circulatory disorders was observed. expressed in terms of SMR or standardized incidence ratio When the duration of exposure was divided into less than 2 (SIR) by comparing the cancer mortality or incidence in years and not less than 2 years, the subjects exposed to TRI the cohorts with those in the nationwide population. Among for less than 2 years showed a higher SMR value for all the men (n=1,421), the SMR for all mortalities was 0.97 mortalities (1.36; 1.241.72) as well as for the mortality (95% CI 0.86-1.10) and the SMR for mortalities from cancer from circulatory disorders (1.68; 1.242.27). In addition, was 0.65 (0.47-1.10). The SMR for mortalities from in the low exposure level group (TCA<49 mg/1), the SMR circulatory disorders (cardiovascular and cerebrovascular) was significantly higher both for all mortalities (1.49;1.13- 304 T KANEKO et al.

1.97) and for mortalities from circulatory disorders (1.79; was significantly higher than 1.0 in the following: total 1.23-2.57). The incidence of all cancers in the exposed cancers (number of patients observed 60, SIR 1.97, 95% CI cohort was similar to that in the general population (SIR 1.20-2.20), stomach cancer (7, 2.98,1.20-2.20), liver cancer 0.96; 0.80-1.16). When the incidence of all cancers was (males: 3, 6.07,1.25-17.7; females: 3, 13.0, 2.68-37.9), analyzed according to the site of the lesion, the incidence prostatic cancer (8, 3.57, 1.54-7.02), and of nonmelanocytic skin cancer was higher in the exposed lymphohematopoietic tissue tumors (7, 2.98,1.20-6.14). cohort than in the nationwide population (SIR 2.36; 1.02- When the data were analyzed by taking into account the 4.65) with the higher incidence being particularly noted in level of TRI exposure, an increase in the incidence was noted the low-exposure-level group (TCA<49 mg/l). The for rectal cancer (9, 2.34,1.07-4.44) in the group having incidences of liver cancer (SIR 1.14), larynx cancer (1.39), urinary TCA<100 mgll and for cancer of the cervix uteri prostate cancer (1.39), testis tumor (1.25), and non-Hodgkin (5, 4.35, 1.41-10.1) in the group with urinary TCA> 100 lymphoma (1.56) were higher than the expected values, but mgll. However, when all the members of the exposed cohort no significant increase was found in the exposed cohort. were used for comparison, an increased incidence was found The number of subjects in the female subcohort was small only in the cancer of cervix uteri (8, 2.4,1.1-4.8). Since (249) and the information available was limited. The total the study of Anttila et al. was carried out in Finland, where mortality rate in the exposed female cohort was higher than the cancer registry and the dynamic statistics of the population that in the nationwide population (SMR 1.55; 1.02-2.31). are accurately conducted with individual identification codes, The observed number of mortalities was larger for malignant the obtained results are most likely reliable. However, no tumors (SMR 1.53) and circulatory disorders (2.02) than account has been given in the report of Anttila et al. the expected number of mortalities, with no significant concerning the increased incidence of stomach or uterine increase in the exposed cohort. In all, for both genders, the (cervix) cancer in the TRI-exposed cohort, which has not excess risks were largely confined to groups of workers with been found in animal experiments or in other epidemiological lower exposure levels or short duration of exposure or both. studies. Whether or not the members in the exposed cohort Axelsonet al. concludedthat their study provided no evidence were representative of the Finnish people concerning the that TRI is a human carcinogen. living environment is unknown because the details of the Anttila et al.8~compared workers occupationally exposed cohort are not given in the report. to TRI, tetrachloroethylene (TETRA), and 1,1,1- Henschler et al.9~conducted a retrospective cohort study trichloroethane (1,1,1-TRI) whose exposure was identified on the relationship between exposure to TRI and renal cell by analyzing biological samples (TRI, urinary TCA; TETRA, cancer in a cardboard factory in Germany. The study group blood TETRA; 1,1,1-TRI, blood 1,1,1-TRI) with the consisted of 169 men who had been exposed to TRI for at population in Finland with respect to the incidence of cancers least 1 year between 1956 and 1975. The average observation (SIR). Only the epidemiological study of TRI will be period was 34 years. The control group was composed of described here. The cohort exposed to TRI was composed 190 workers who worked at the same factory in the same of 1,698 males (31,552 person-years) and 1,391 females period without being exposed to TRI. The SMR was (28,353 person-years). Each worker in the TRI cohort calculated compared with the local population. By the closing underwent urinalysis for TCA 2.5 times on the average. The day of the study (December 31,1992), 50 members of the median urinary TCA level was 48 mmolll for males and 60 exposed cohort had died (SMR 0.79), 15 of the 50 from mmolll for females (malesfemales). The incidence of cancers (total, liver, these workers, 15 died of malignant neoplasms (1.16), but biliary tract, gallbladder, pancreas, kidney, bladder, none of them died of kidney cancer. Three workers of the lymphohematopoietic tissue, brain and nervous system, control cohort died of brain tumors (9.38; 95% confidence genital organs, and tissues likely to be exposed to TRI [oral interval 1.93-27.37). As for the incidence of cancer, the cavity, pharynx, larynx, nasal cavity, trachea, and lung]) was diagnosis of kidney cancer was made in five workers of the investigated by using the cancer registry in Finland (1967- exposed cohort: four with renal cell cancers and one with a 1992). Tumors developed in 208 workers in the TRI cohort. urothelial cancer of the renal pelvis. The time interval For workers exposed to TRI for 20 years or longer, the SIR between the TRI exposure and diagnosis was 18 years or

Industrial Health 1997, 35, 301-324 HEALTH EFFECTS OF TRICHLOROETHYLENE 305 longer. None of the control cohort was diagnosed as having evaluation is based mainly on the epidemiological findings. kidney cancer. The SIR calculated with the data of the Danish In this connection, placing great stress on the cohort studies, cancer registry was 7.97 (2.59-18.59). The SIR for kidney the IARC cited the reports of Garabrant et al.5), Spirtas et cancer, as calculated with the data of cancer registry in the a1.6), Axelson et al.'), and Anttila et al.8), and attached former West Germany, was 9.66 (3.14-22.55). After the end particular importance to the reports of Spirtas et al., Axelson of the observation period, two additional kidney tumors (one et al., and Anttila et al. (when revising its classification of renal cell and one urothelial cancer) were diagnosed among TRI carcinogenicity,the IARC did not take into consideration the members of the exposed cohort. Based on the results of the study of Henschler et al.9) for the reason that the study this study, Henschler et al. concluded that the incidence of was initiated after the observation of a cluster of renal cancer kidney cancer was significantly elevated among workers cases). The results of the four studies are summarized by exposed to TRI. Nevertheless, none of the epidemiological the IARC13)as shown in Table 2. The agency put particular studies concerning TRI-related cancer have reported a stress on the finding that the SIR or SMR for hepatobiliary relationship between TRI and kidney cancer2-4, 6, x,10-12) cancer and non-Hodgkin lymphoma exceeded 1.0 in the Henschler et al. have emphasized that their exposed cohort studies of Spirtas et al., Axelson et al., and Anttila et al. was exposed to TRI at high levels for prolonged periods of However, the lower confidence of the 95% confidence time and that their study differed from other studies in that interval of the SIR or SMR for such tumors was below 1.0. the incidence (SIR) was used for evaluation in place of the Anttila et al. affirmed the carcinogenicity of TRI, whereas mortality (SMR). However, no relationship between Spirtas et al. and Axelson et al., unlike the IARC, did not exposure to TRI and kidney cancer was found in the studies reach any affirmative conclusion. The JSOH thus judged of Axelson et al.') or Anttila et al.g) who also used the SIR that it is too early to classify the TRI carcinogenicity as as an evaluation parameter. Group 2A and that it is appropriate for the moment to promote In summary, a negative view is predominant exposure control under a classification as Group 2B." epidemiologically with respect to the carcinogenicity of TRI. However, no unanimous conclusion has been reached (2) Animal experiments concerning the carcinogenicity of TRI in humans because The U.S. National Cancer Institute (NCI)16)first published epidemiological studies conducted to date have the following the results of a carcinogenicity experiment in 1976. It was drawbacks: the cohort sizes were small, the durations of found that TRI caused hepatocellular carcinoma in B6C3F1 observation were short, and the assessments of the exposure mice. However, since the TRI used in the experiment to TRI were insufficient. contained the stabilizer epichlorohydrin, a known mutagen In 1995, the International Agency for Research on Cancer and carcinogen, the findings of the experiment were open (IARC)13) changed the carcinogenicity classification of TRI to question. In this connection, the U.S. National Toxicology from Group 3 (not classifiable as to carcinogenicity to Program (NTP)17) conducted new carcinogenicity tests of humans) to Group 2A (probably carcinogenic to humans), TRI. TRI not containing stabilizers was dissolved in corn stating that there is limited evidence to support the oil and given orally to F344/N rats (50 of each sex) and carcinogenicity of TRI to humans. The Committee for B6C3F1 mice (50 of each sex), 5 days/week for up to 103 Occupational Exposure Limits of the Japan Society for weeks. Based on the results of a preliminary experiment, Occupational Health (JSOH)14), commenting on the IARC's the dosage was set at 0, 500 or 1,000 mg/kg for rats and 0 or carcinogenicity classification of TRI, issued its own proposal, 1,000 mg/kg for mice. A group of 50 male and 50 female which is summarized as follows: "In the evaluation of IARC rats were used as untreated control rats. concerning the carcinogenicity of TRI, the description given The survival of the TRI-treated F344/N rats was in 198715) is that while evidence for the carcinogenicity to significantly reduced (p<0.005) in comparison with the humans is inadequate, there is limited evidence for the untreated controls. An increased incidence of renal carcinogenicity to animals. On the other hand, the description adenocarcinomas was seen in males treated at 1,000 mg/kg given in 199513)is that while evidence for the carcinogenicity dose (p<0.05). Two males that received the 500 mg/kg dose to humans is limited, there is sufficient evidence for the developed renal adenomas. The incidence of tumors in carcinogenicity to animals. TRI was classified as Group 3 female rats was not increased at any site. The fact that the and Group 2A on the basis of overall evaluation in 1987 survival rate of treated rats was low (with 20% of deaths and 1995, respectively. This indicates that the overall due to failure in forced administration) makes the 306 T KANEKO et al.

Table 2. Summary of data from four cohort studies of trichloroethylene (IARC13))

interpretation of the results obtained with rats difficult. rats. The TRI used by Fukuda et al. contained Histopathological examination revealed increased incidences epichlorohydrin as a stabilizer. However, its concentration of hepatocellular carcinomas in the TRI-treated B6C3F, mice: was low; 0.04 and 0.12 ppm at TRI concentrations of 150 in the males, hepatocellular carcinomas were found in 8/48 and 450 ppm, respectively. Although the possibility of a controls and 31/50 treated animals (p<0.001), and in females, combined effect of epichlorohydrin with TRI cannot be ruled the carcinomas occurred in 2/48 controls and 13/49 treated out entirely, the lung adenocarcinomas in mice in this animals (p<0.05). The increase of hepatocellular adenomas experiment are likely to have been induced by TRI. This in TRI-treated mice was also noted (7148 vs. 14150 in males, report is the first that indicated a correlation between inhaled p<0.05; 4/48 vs. 16/49 in females, p=0.001). TRI and lung cancer in animals. Fukuda et al.'8 exposed female ICR mice and female The experimental findings described above can be Sprague-Dawley rats to air containing TRI at 0, 50,150, or summarized as follows: they provide ample evidence to 450 ppm 7 hr/day, 5 days/week for up to 104 weeks, and indicate that TRI promotes the development of liver cancer performed necropsies at the 107th week. The mean number in mice (B6C3F1 mice in particular) but no evidence of this of lung tumors per animal was 3--4 times higher in mice compound causing the cancer in rats. There is no report exposed to TRI at the concentration of 150 or 450 ppm than that TRI caused kidney tumors in mice, but a possibility in control mice. The incidence of lung adenocarcinomas that this compound causes such tumors in male rats is was significantly higher in these exposed mice than in the suggested. All of the TRI carcinogenicity tests in animals control mice (p<0.05);1/49 for the 0 ppm, 8/50 for the 150 revealed distinct species differences (between mice and rats). ppm, and 7146 for the 450 ppm groups. The tumor incidence However, the IARC13~has concluded that there is sufficient was not increased in any organs or tissues of the TRI-exposed evidence for the carcinogenicity of TRI to animals.

Industrial Health 1997, 35, 301-324 HEALTH EFFECTS OF TRICHLOROETHYLENE 307

Fig. Metabolic pathways of TRI (adapted from the IARC13~ with modifications)

(3) Mechanism of species differences in TRIcarcinogenicity with GSH, into either cytotoxic, genotoxic, or mutagenic with special reference to its metabolism metabolites23~.According to Dekant24~,TRI is metabolized A major portion of TRI absorbed into living bodies is to S-(1,2-dichlorovinyl) glutathione in the liver by GSH S- metabolized by cytochrome P450 systems (probably P450 transferase present in the microsomal or soluble fraction. 2E119))to chloral hydrate (CH), which is then oxidized to The conjugate is converted to S-(1,2-dichlorovinyl)-L- trichloroaceticacid (TCA) or reduced to trichloroethanol(TCE) cysteine, a cysteine S-conjugate, by the removal of glutamic (Fig.). TCA and TCE are excreted in the urine either directly acid and glycine. This conjugate is transported to the kidney (TCA) or after conjugationreaction (TCE). An epoxide (1,2,2- and activated by f3-lyase to produce highly electrophilic trichlorooxyrane) was once postulated as an intermediate chlorothioketenef which can acylate the macromolecules, during the oxidation of TRI to CH; however, later studies producing nephrotoxicity (Fig.). Since mercapturic acids on the biotransformation of TRI and other chlorinated olefines N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S- suggest a stepwise oxidation of TRI to CH, in which the (2,2-dichlorovinyl)-L-cysteine have been identified in the epoxide is not an obligatory intermediate2o,21> urine of workers exposed to TRI25~,humans may have the Reduced glutathione (GSH), which is present in high capacity to form GSH conjugates. concentrations in most living cells and participates in a variety It is clear that TRI orally administered at a high dose of vital cellular reactions, protects cells from potentially toxic induces hepatocellular carcinomas in mice. However, the electrophiles formed via the metabolism of xenobiotics. Such carcinogenicity of TRI in rats has not been demonstrated in reactions involving GSH have long been associated with any carcinogenicity tests. Many attempts have been made the process of detoxification. However, in recent years, to elucidate the underlying mechanism of the species evidence has accumulated indicating that GSH conjugation differences in TRI carcinogenicity, focusing mainly on the plays an important role in the formation of toxic metabolites differences in TRI metabolism between mice and rats. from a variety of chemicals including TRI22>.A minor portion Stott et a1.26>exposed mice (B6C3F1) and rats (Osborne- of absorbed TRI is thought to be converted, via conjugation Mendel) to TRI by inhalation for 6 hr at a concentration of 308 T KANEKO et al.

10 or 600 ppm. Although there was no species difference expired air in the first 24 hr. At 2,000 mg/kg, however, between the mice and rats at the exposure concentration of 78% of the dose was eliminated unchanged in the rats, but 10 ppm, at the exposure level of 600 ppm, the amount of only 14% in the mice. The area under the concentration- TRI metabolized was 262% larger in the mice than in the time curve (AUC) of TRI and its metabolites in the blood rats with the binding of TRI to macromolecules in the liver calculated at a dose of 1,000 mg/kg was greater in mice being 322% greater in the mice than in the rats. In the same than in rats: the ratio of AUC in the mice relative to that in report, the oral administration of TRI caused hepatocellular the rats was 7.9 for TRI, 3.9 for trichloroethanol (TCE), toxicity in repeated dosing trials in mice. The hepatic effects and 6.3 for trichloroacetic acid (TCA). This finding indicates observed in mice treated with a maximum tolerated dose of that the mice were exposed to significantly higher 2,400 mg/kg/day of TRI for three days were primarily a concentrations of TRI metabolites than were the rats at high centrilobular hepatocellular swelling with focal dosages. In particular, the blood TCA levels in mice remained hepatocellular necrosis. These effects led to an enhanced high for over 30 hr. A high blood concentration of TCA is regenerative process as indicated by the increased hepatic known to induce hepatic peroxisome proliferation in mice DNA synthesis activity (220% of control) and the incidence but is insufficient to induce this response in rats28~. The of mitotic cells. In contrast to the mouse data, rats appeared results of this study suggest a link between TCA-induced to be less sensitive to a maximum tolerated dose level of proliferation of peroxisomes and liver cancer28~,which forms TRI (1,100 mg/kg/day), displaying enhanced hepatic DNA a basis of the species difference in response to TCA. synthesis levels (175% of control) with no histopathology. According to the study conducted by Elcombe et al.28>, From these results, Stott et al. concluded that an epigenetic TRI administered to B6C3F1 and Alderley Park mice by mechanism of tumor formation is operative in B6C3F1 mice, gavage for 10 consecutive days at doses of 500 to 1,500 implying that a tumorigenic response to TRI exposure in mg/kg increased both the liver weight (175% of control) this species would only be evident upon chronic and the synthesis of DNA (500% of control) in the liver. In administration of high, cytotoxic dose levels of TRI. contrast, similar treatment of Osborne-Mendel rats and The genetic mechanism of carcinogenesis is characterized Alderley Park rats resulted in a smaller increase in the liver by a direct interaction (alkylation or intercalation) of a weight (130% of control) with no effect of TRI on the DNA chemical with DNA, while epigenetic mechanisms of synthesis in either strain of rats. The increased DNA synthesis carcinogenesis do not involve a direct interaction of a in mice was probably not due to regenerative hyperplasia, chemical with DNA. An epigenetic mechanism that since no signs of necrosis were seen in these animals. While characterizes the tumorigenic action of TRI in B6C3F1 mice the increased liver weight in rats appeared to be due to liver may be that of recurrent cytotoxicity. This mechanism of cell enlargement (hypertrophy), both hypertrophy and tumorigenicity involves the enhancement of the normal hyperplasia (cell proliferation) were operative in the increase cellular mutation rate during regenerative activity (increased in mice; an increased number of mitotic cells (10 times the DNA synthesis) in response to a cytotoxic challenge by TRI. mitotic index of control) was observed in parallel with the Thus, a tumorigenic response of animals to TRI is observed increase in DNA synthesis in the TRI-treated mice. An only after prolonged exposure to toxic dose levels. Another important observation was that TRI induced the peroxisomal possible epigenetic mechanism is that TRI causes a enzyme activities, catalase and cyanide-insensitive palmitoyl- hypertrophic response in the liver and only enhances CoA oxidation (147 and 786% of control, respectively), in preexisting oncogenic changes present in B6C3F1 mice. mice but not in rats. Furthermore, increases in peroxisome Prout et a1.27~gave [14C]TRI orally to rats (Osborne-Mendel volume density (up to 1,110% of control) were observed in and Alderley Park Wistar) and mice (B6C3F1 and Swiss- the mice that received TRI. These observations suggest that Webster) at doses of 10-2,000 mg/kg and observed the the species difference in the hepatocarcinogenicity of TRI toxicokinetics of TRI and its metabolites. The metabolism is due to a species difference in peroxisome proliferation of TRI in the mice was linear over the range of doses used, and cell proliferation, the peroxisome proliferation leading whereas in the rats it became constant independent of dose to increased reactive oxygen species and DNA damage, and at 1,000 mg/kg and above (metabolism saturation). TRI given the cell proliferation then acting to promote this lesion. at a dose of 10 mg/kg was almost completely metabolized Nelson and Bu1129~evaluated the ability of TRI and its in both rats and mice, 60% of the dose being excreted in metabolites to induce single-strand breaks in the hepatic DNA urine with only 1 to 4% being eliminated unchanged in of male B6C3F1 mice and Sprague-Dawley rats in vivo using

Industrial Health 1997, 35, 301-324 HEALTH EFFECTS OF TRICHLOROETHYLENE 309 an alkaline unwinding assay. Doses of TM of 22-30 mmol/ suggests that a metabolic pathway from TRI to DCA may kg were required to produce strand breaks in DNA in rats, exist only in mice. The blood DCA concentrations observed whereas a dose of 11.4 mmol/kg was sufficient to increase in the mice given a carcinogenic dose of TRI (15 mmol/kg) the rate of alkaline unwinding in mice. The TRI metabolites were of the same magnitude as those observed with TCA, dichloroacetic acid (DCA) and chloral hydrate (CH) carcinogenic doses of DCA. In contrast, when TCA was induced strand breaks in hepatic DNA in a dose-dependent given to animals, DCA was detected in the blood of both manner in both species. The slopes of the dose-response species and the blood DCA level was higher in the rats than curves and the order of potency of these metabolites differed in the mice. Based on these findings, Larson and Bull significantly between rats and mice, suggesting that different suggested that it is highly likely that both TCA and DCA mechanisms of single-strandbreak induction may be involved play a role in the induction of hepatic tumors in B6C3F1 in the two species. In addition, the dose of metabolites mice by TRI. required to induce strand breaks was lower in mice than in The hepatocarcinogenicity of TRI is likely due to an rats. In particular, a 100-fold difference was noted between epigenetic mechanism (promoter) rather than a genotoxic the two species in the minimum dose of TCA required to mechanism (initiator). Liver cancer develops after TM has induce the breaks. This finding coincides with the report that been given over extended periods of time at doses high TCA is liable to cause peroxisomal proliferation in mice28'30) enough to produce cytotoxicity. However, the view that The study of Nelson and Bull is another explanation for the the cancer is caused by a genetic mechanism cannot be ruled different sensitivityof mice and rats to the hepatocarcinogemc out entirely since the binding of TRI to DNA was effects of TRI. demonstrated in vitro in the presence of a metabolism- Intercellular gap junctions are comprised of proteinaceous activating system21~,and since an increased induction of plasma membrane channels that link the interiors of adjacent micronuclei36~and DNA single-strandbreaks29> were observed cells and permit cells to directly exchange small (<1,000 in vivo (mice and rats). Daltons) molecules and ions. One cellular effect exhibited Why does the site of tumor development by TRI depend by a number of nongenotoxic carcinogens and tumor on the route of administration? The oral administration of promoters is the inhibition of gap-junction mediated TRI caused liver cancer in mice17~,whereas the main tumors intercellular communication31~.Klauning et al.32~assessed caused in mice exposed to TRI by inhalation were pulmonary the effects of TRI and its metabolites (TCA, TCE, and CH) tumors18~.The carcinogenicity of TRI is mediated not by on gap-junction mediated intercellular communication in TRI itself but by its metabolites, DCA or TCA. The activity cultured B6C3F1 mouse and F344 rat hepatocytes. TRI and of cytochrome P450 enzymes that catalyze the metabolism TCA inhibited intercellular communication in the mouse is greatest in the liver, with only weak activity in the lung hepatocytes but not in the rat hepatocytes. TCE and CH and kidney. After oral administration, all of the amount of had no effect on hepatocyte intercellular communication in TRI absorbed passes through the liver where TRI is processed either rat or mouse cells. The inhibitory action of TCA was (first-pass metabolism), and the liver cells come into contact stronger than that of TRI. In addition, TRI appeared to require with active metabolites at high concentrations. In contrast, cytochrome P450 metabolism by the mouse hepatocytes to TRI exposed to animals by inhalation is first metabolized exhibit its inhibitory effect on the communication. Thus, in the pulmonary tissues, and it may thus cause injuries in the inhibitory action of TRI was likely to be produced by the tissues if sufficient amounts are metabolized. the metabolite TCA. A difference in the metabolic capacity In summary, the most probable explanation for the reason between mice and rats (TRI metabolism, mice>rat25~)may why TRI-induced hepatocellular carcinoma occurs in mice also be involved in the difference in the inhibitory action but not in rats is that the activity of cytochrome P450 enzymes between both species. The findings of Klauning et al. also in the liver of mice is higher than in rats. The occurrence of provide an explanation for the species differences in liver cancer depends on the enzyme activity, especially when susceptibility to TRI-induced liver carcinogenesis. TRI is given to animals at a high dosage. This is because There are reports that DCA, a metabolite of TRI, shows the metabolism in the liver is rate-limited by the enzyme carcinogenicity in B6C3F1 mice33'34). In experiments with activity (capacity-limited metabolism) at such high doses37~. rats (Sprague-Dawley)and mice (B6C3Fr),Larson and Bull35> Another reason is that the B6C3F1 mice used in many demonstrated that DCA was detected in the blood of mice carcinogenicity tests have spontaneously occurring liver but not in rats following TRI administration, a finding which cancer with high frequency. 310 T KANEKO et al.

(4) Mutagenicity to an unusually high MN frequency in the controls. A After examining short-term mutagenicity test data in the subsequent replication of the 1-day 5,000 ppm TCE exposure published literature, Jackson et al.38~summarized that TRI with rats again showed a highly significant increase in MN containing no mutagenic stabilizers (e.g., epichlorohydrin, frequencies compared to controls. 1,2-epoxybutane, etc.) is unlikely to induce gene or Konietzko et al.43~reported a pathological rate of chromosomal mutations or aneuploidy. hypodiploid cells in the lymphocytes of male workers Metabolites of TRI were demonstrated to bind to DNA exposed to a high level (206 ppm) of TRI. There was a in in vitro systems21' 39,40)According to Miller and significant correlation between the rate of hypodiploid cells Guengerich20' 21>,TRI oxide is probably not the TRI metabolite and TRI exposure concentration (r=0.46, p<0.05). The rate responsible for the irreversible binding to DNA. Concerning was 10.9% for the exposed group (27 workers) and 6.5% the binding in a living system, while one report suggested for the unexposed group (10 workers). In addition, the rate slight binding to hepatic DNA41>,the others were unable to of chromosomal breaks in mitotic cells was 5 times higher give direct evidence of the formation of TRI-DNA adducts in the exposed group than in the unexposed group. However, in the liver39,40~. a major drawback of this report is that the effect of smoking As was discussed earlier, Nelson and Bull29>demonstrated was not taken into consideration. the ability of TRI to induce single-strand breaks in the hepatic Nagaya et al.44~compared the frequency of SCE in the DNA of male B6C3F1 mice and Sprague-Dawley rats in vivo, peripheral lymphocytes between 22 workers occupationally using an alkaline unwinding assay. The strand breaks were exposed to TRI (mean TCA concentration in urine: 183.6 observed at doses that produced no observable hepatotoxic mg/l) and 22 control workers matched by age, sex and effects as measured by serum aspartate aminotransferase smoking history. Although the smokers had a high frequency and alanine aminotransferase levels. However, since strand of SCE, no difference in the smoking-adjusted frequency breaks are not always induced by direct interaction between was found between the two groups. Seiji et al.45~examined chemical compounds and DNA42~,the finding of Nelson and the frequency of SCE in peripheral lymphocytes from 38 Bull does not necessarily mean that TRI acts as an initiator workers exposed to TRI at 7 ppm and 19 workers exposed of the carcinogenicity. to a mixture of TRI and tetrachloroethylene (TETRA) (8 Kligerman et a1.36~performed inhalation studies using male and 17 ppm, respectively) (TRI + TETRA). The results C57BL/6 mice and CD rats to determine whether TRI can were compared with the findings in control subjects matched induce cytogenetic damage in vivo. Animals were exposed by age, sex, smoking habits and place of residence. No to target concentrations of 0, 5, 500, or 5,000 ppm TRI significant increase in SCE frequencies was observed in (reagent grade) for 6 hr. Tissue samples were taken between association with the exposure to TRI or TRI + TETRA. 18 and 19 hr after exposure. Cultured peripheral blood Cysteine S-conjugates, TRI metabolites via the GSH lymphocytes (PBL) in rats and splenocytes in mice were pathway of TRI metabolism, have long been known to bind analyzed for the induction of sister chromatid exchanges to DNA, and the conjugate from TRI is mutagenic in the (SCE), chromosomal aberrations, and micronuclei (MN) in Ames bacterial mutation assay47~.However, according to cytochalasin B-blocked binucleated cells. Bone marrow Green et al., kidney f3-lyaseactivity is far lower in humans polychromatic erythrocytes (PCE) were analyzed for MN. than in mice and rats. It is thus uncertain whether the GSH The only positive response observed was for MN in rat bone pathway in the metabolism of TRI has any relevance to TRI marrow PCE. TRI caused a significant increase in MN at carcinogenicity in humans. all concentrations, inducing an approximate four-fold increase over the control levels at 5,000 ppm. TRI was also cytotoxic Toxicity Other than Carcinogenicity in rats, causing a significant concentration-related decrease in the ratio of PCE/normochromatic erythrocytes. In follow- The acute toxicity of TRI originates from its skin- and up studies, CD rats were exposed for 6 hr/day over 4 mucous membrane-irritating action and anesthetic action. consecutive days to 0, 5, 50 or 500 ppm TRI. No significant As is clear from the fact that TRI was formerly used as an concentration-related increases in cytogenetic damage were inhalational anesthetic, it is a strong central nervous system observed. While the MN frequencies in the 4-day study depressant. As the exposure level is increased, cognitive were comparable to those at the equivalent concentrations ability and associated movement are affected together with in the 1-day study, they were not significantly elevated due eye irritation, sleepiness, headache, and malaise. When a

Industrial Health 1997, 35, 301-324 HEALTH EFFECTS OF TRICHLOROETHYLENE 311 human is exposed to an extremely high level of TRI, its latency of the masseter reflex (mean 10.4 ms) had increased anesthetic action may result in death. However, when a (0.4 ms). With respect to the blink reflex, no prolongation person is acclimated to an odor specific to TRI, such was found. No impairment was found in the functions of symptoms appear only rarely during exposure at a motor and autonomic nerves. Based on these results, Ruijten concentration of 50 ppm or lower. et al. concluded that long-term exposure to TRI at 35 ppm Regarding the health effects of chlorinated hydrocarbons, may slightly affect the trigeminal and sural nerves. This hepatotoxicity has been a matter of great concern. However, study also suggests that the refractory period is a sensitive TRI is weakly hepatotoxic relative to such hepatotoxicants indicator of preclinical toxic neuropathies. as carbon tetrachloride and chloroform49~Nakajima et al.5o~ Feldman et al.56>evaluated the use of the electrophysiologic exposed rats to TRI by inhalation for 2 hr at a concentration blink reflex as an indicator of neurotoxic effects of TCE in of 500-8,000 ppm and found that the alanine occupationally exposed workers. The blink reflex was tested aminotransferase activity in the plasma, an indicator of liver in individual cases with documented histories of exposure function disorder, was not significantly increased until the to TRI (n=18). When compared with the nonexposed concentration was raised to 4,000 ppm. laboratory control values (n=30), the subjects with a The health effects of long-term exposure to TRI are chiefly significant history of TRI exposure demonstrated the most manifested as neuropathy. Most of the individuals chronically prolonged latencies (greater than or equal to 3 SD above exposed to a certain level of TRI present subjective symptoms the nonexposed group mean) in the R 1 component of the related to the nervous system such as headache, sleepiness, blink reflex measurement. Considering a latency value above dizziness, malaise, nervousness, finger tremor, nausea, and the mean ± 3 SD of the control group to be positive, the anorexia, for example. These symptoms appear in workers blink reflex latency test allowed to detect the effect of TRI exposed to TRI at 50 ppm or higher for extended periods of on the trigeminal nerve with 50% sensitivity and 90% timed-53~, specificity. The results of this study indicate that the electrophysiological blink reflex has application in assessing (1) Neurotoxicity TRI exposure and in documenting the neurotoxic effects of Rasmussen et al.54~examined the clinical neurological its exposure on trigeminal nerve functions in humans. manifestations after long-term exposure to degreasing Liu et al.53~examined 103 workers exposed to TRI (mostly solvents, mainly TRI. Seventy metal degreasers exposed at less than 50 ppm) and 111 non-exposed control workers to TRI at a relatively constant level (mean exposure for subjective symptoms, hematology, serum biochemistry, concentration in terms of urinary TCA level: recent years, and glucose, protein and occult blood in the urine. The 7.7 mg/l; 1950s-1970s, 40-60 mgll) were divided into three clinico-laboratory test results in the TRI-exposed group were groups according to the length of working years, i.e., low- essentially normal, and no clinically significant effects of exposure group (mean 0.5 year), mid-exposure group (2.1 TRI exposure were found in the blood or liver functions years) and high-exposure group (11.0 years). The most among the exposed workers as compared with the controls. marked finding was a highly significant dose-response The prevalence of the subjective symptoms was, however, relation between solvent exposure and motor significantly higher in the exposed group than in the controls, dyscoordination, a finding that was retained after a and a dose-response relationship was established in some multivariate control of relevant confounders. Vibration selected symptoms such as nausea, heavy feeling in the head, thresholds increased by solvent exposure at a bivariate level, forgetfulness, tremor in extremities, cramps in extremities but the multivariate analysis showed that age explained most and dry mouth. These findings warrant further attention to of the increase. No significant trigeminal nerve dysfunction the effects of TRI, especially on the central and autonomic was found. nervous systems at concentrations lower than 50 ppm. Ruijten et a1.55~performed neurological tests in 31 printing Taken together, the findings described above indicate that workers exposed to TRI (mean age, 44 years; mean duration the chronic effects of TRI exposure at low concentrations of exposure, 16 years; cumulative exposure level, 704 ± 583 present as neurological changes. In addition to neurological ppm-year) and 28 controls (mean age, 45 years). A slight tests such as the measurement of nerve conduction velocity reduction (-1.1 mis) in the sural nerve conduction velocity and its refractory period, subjective symptoms are useful and a prolongation (0.4 ms) of the sural nerve refractory for detecting such neurological effects. period (mean of the controls 1.95 ms) were found. The 312 T KANEKO et al.

(2) Hepatotoxicity of y glutamyltransferase in the serum was found to be TRI-induced liver injuries are discussed in the above elevated concomitantly with increasing solvent exposure. section, "Mechanism of species differences in TRI The significance of this relationship, however, was not able carcinogenicity with special reference to its metabolism." to withstand a multiple regression analysis, with age and Hence, only the hepatic effect of occupational exposure to alcohol abuse as confounding variables. The study indicates TRI will be discussed here. no significant association between occupational TRI exposure Takamatsu divided degreasing work with TRI into three at levels lower than the current permissible exposure level categories: high-exposure (150-250 ppm), mid-exposure and the results of tests screening for early liver dysfunction. (50-100 ppm), and low-exposure (<50 ppm)50).TRI-related To examine the early hepatic effects of chronic exposure liver function disorders were found in the workers engaged to low-level TRI, Nagaya et al.58> determined the total in the high- and mid-exposure works, but not in the workers cholesterol (T C) and high-density-lipoprotein cholesterol engaged in the low-exposure work. (HDL-C) contents and the activities of AST, ALT and y- Liu et al.53~examined 101 workers exposed to TRI (<50 GTP in the sera of 148 workers (a cross-sectional study) ppm) and 111 non-exposed control workers for serum and of 13 workers (a 2-year follow-up study) occupationally biochemistry, i.e., the activities of alanine aminotransferase exposed to TRI. In the cross-sectional study, three exposure (ALT), aspartate aminotransferase (AST), y- groups were defined by urinary total trichloro-compounds glutamyltranspeptidase(y GTP), alkaline phosphatase (ALP), (TTC) levels; a low-exposure group (TTC<10 mg/g creatinine, lactate dehydrogenase (LDH) and leucine aminopeptidase n=49), a moderate-exposure group (10<-TTC<100, n=56), (LAP) in the serum, the thymol turbidity test (TTT), the and a high-exposure group (TTC>100, n=43). With zinc turbidity test (ZTT), and the total bilirubin content in increasing exposure levels, the T-C and HDL-C levels were the serum. Significant differences (in either the mean of slightly but not significantly (p=0.143 and 0.080 by ANOVA, values or the rate of abnormal/total cases) were observed respectively) increased. The exposure, however, had no effect only in the activities of ALT, LAP and LDH. The deviations on the activities of the three serum enzymes. In the follow- from the normal ranges of liver function indicators were all up study, the fluctuations in TTC were well reflected in slight and of subclinical levels, except for two cases in which subclinical changes in HDL-C, AST, and y-GTP, but not in both ALT and AST were elevated to a degree of clinical T -C or ALT. Based on these results, the authors suggested significance (ALT 115; AST 75 Karmen units) in a 25-year that exposure to low-level TRI influences hepatic functions, old nondrinking exposed man, and to subclinical levels (ALT affecting cholesterol metabolism rather than causing hepatic 24; AST 32 Karmen units) in a nondrinking exposed man at cell damage, and that these influences are subclinical and the age of 48 years. Since all the values in the other liver reversible. function tests were essentially within the corresponding normal ranges and there was no significant difference in (3) Nephrotoxicity either the mean or the prevalence of abnormal values between In the TRI carcinogenicity test conducted by the U.S. NTP the exposed and control groups, the authors concluded that with B6C3F1 mice and F344/N rats17~,renal adenocarcinoma the slight deviation from normal ranges were not associated developed solely in male rats. In the study of Maltoni et with TRI exposure. al.S9~,TRI was administered by inhalation to rats (Sprague- Rasmussen et al.57~examined the dose-response Dawley) and mice (Swiss and B6C3F1) 7 hr/day, 5 days/ relationships between long-term exposure to TRI and a week, for 8 or 104 weeks at various concentrations. The battery of liver function tests among 99 metal degreasers. animals were kept under observation until spontaneous death. The study group was divided into four groups with nearly The most relevant finding was the dose-related increased equal numbers of workers accordingto a cumulative exposure incidence of Leydig cell tumors in male rats, and the onset index calculated using the number of hours of degreasing of few renal adenocarcinomas at the highest dose (600 ppm), work, multiplied by the number of years of exposure, always in male rats. The renal adenocarcinomas were multiplied by 45 working weeks per year, i.e., group I preceded by and associated with a characteristic lesion of (reference group) less than one year, group II 1-2.8 years, the kidney: tubular cell karyomegaly (megalonucleocytosis). group III 2.9-6.7 years, and group IV 6.8-35.6 years. The Protein (hyaline) droplet nephropathy has been linked to mean urinary TCA level in the highest exposure group was the a2-microglobulin specifically occurring in male rats60~. 7.7 mg/1with a maximum TCA level of 26 mg/l. The activity Goldsworthy et al.61~ attempted to determine whether

Industrial Health 1997, 35, 301-324 HEALTH EFFECTS OF TRICHLOROETHYLENE 313 chlorinated hydrocarbon-induced protein droplet controls (50.6 ± 17.0 mglg creatinine; n=20). These results accumulation was related to the male rat specific protein, suggest that the adverse effects of occupational exposure to a2-microglobulin. Male and female F-344 rats were gavaged TRI on the kidney, if any, are mild and glomerular rather for 10 days with TRI (1,000 mg/kg), tetrachloroethylene than tubular. (TETRA, 1,000 mg/kg) or pentachloroethane (PENTA, 150 mg/kg). Protein droplet accumulation in the cytoplasm of (4) Pulmonary toxicity (including association with lung the P2 segment of the proximal tubule was evident in the cancer) male rats administered TETRA and PENTA, whereas the Forkert and Birch64>examined the time course of TRI- droplet accumulation in the male rats treated with TRI did induced pulmonary injuries, focusing on the morphological not differ from that in the control rats. Immunohistochemical changes and the covalent binding of [14C]TRIsoon after staining for a2-microglobulin revealed a marked correlation administration of a single dose of TRI (2,000 mg/kg) to CD- between the presence of a2-microglobulin and the protein 1 male mice. At one hour after treatment, Clara cells of the droplets. In addition, cell replication rates increased bronchiolar epithelium exhibited necrotic changes involving specifically in the histologically damaged P2 segments of the mitochondria and endoplasmic reticulum. Dilatation of the male rats treated with TETRA or PENTA, a fmding which the endoplasmic reticulum became more severe at two hours suggests that the increase in cell replication may be linked after TRI administration, and by 4 hr, distended cisternae to the tubular damage caused by male rat-specific a2- coalesced to form small vacuoles within the cytoplasmic microglobulin. Since compensatorycell divisionis postulated matrix of the Clara cells. The severity of cellular damage to affect all of the stages of the carcinogenic process, the increased between 8 and 12 hr, and by 24 hr, the majority of increased incidence of renal tumors in male rats after TETRA Clara cells within the airway were severely vacuolated. The or PENTA treatment may be related to the protein droplet covalent binding of [14C]TRIto lung macromolecules was nephropathy specifically occurring in male rats. The results evident at one hour and peaked at four hours. The peak of the study of Goldsworthy et al. suggest that the binding (142.6 ± 31.8 nmollg of wet weight) represented mechanisms involved in TCE-induced renal carcinogenicity, approximately 20% of [14C]TRI distributed to the lung. if any, may differ from TETRA- and PENTA-induced renal Although the levels of binding in the liver were at all times carcinogenicity. greater than those in the lung, the liver injuries were relatively Case reports on nephropathy induced by TRI have been insignificant. The results demonstrated a positive correlation published sporadically. For example, David et al.62~reported between the onset of Clara cell injury and the formation of a 34-year-old patient with acute renal failure that developed reactive metabolites, as assessed by the covalent binding of after the completion of an 8-hr computer ribbon-cleaning [14C]TRI. The selective damage to Clara cells by TRI job using TRI. On admission (3 weeks after the exposure), suggests that TRI undergoes metabolic activation by the patient presented a remarkable foot edema, proteinuria, cytochrome P450 existing in the cells. and a conspicuous degree of hematuria and pyuria. Renal When animals are exposed to TRI by inhalation, Clara cells tubular necrosis secondary to allergic interstitial nephritis are directly in contact with TRI in the inhaled air together was histologically established by biopsy. The solvent used with TRI carried there by the bloodstream. It is unclearwhether for the work was TRI of 99.5% purity, but the details of pulmonarytumors, believed to be caused by TRI' 8'59>, originate TRI exposure were unknown (David et al. estimated the from Clara cells. One reason why lung tumors are frequently exposure concentration to be 166-3,700 ppm). caused in mice may lie in the high frequency of spontaneous A comparison was made concerning the urine levels of adenomatosis in some strains of mice. It must be borne in total protein and f3 -microglobulin between 104 male workers mind that ICR mice used by Fukuda et al.18>and Swiss mice occupationally exposed to TRI (15 ppm) and 102 male used by Maltoni et al.59~have lung adenomas with relatively nonexposed controls63~.The urinary total protein levels of high frequency. However,no data are availablefor comparing the exposed group, rather than the urinary fl-microglobulin TRI metabolism in the lung between rats and mice. level, was slightly higher than those of the controls. However, Nichols et al.6S~examined the susceptibility of pulmonary the difference in total protein level between the two groups cells and the mechanisms of cytotoxicity of TRI using was not significant. The mean total protein level for the enriched subpopulations of isolated rabbit (New Zealand 35- to 44-year-old workers (62.5 ± 21.3 mg/g creatinine; white) lung cells incubated with TRI. The ability of TRI to n=23) was significantly higher than that for corresponding induce selective pneumotoxicity was evaluated by its 314 T KANEKO et al. bioactivation in three cell types, i.e., Clara cells, alveolar the order of mice>rats>humans, and human Clara cells lack type II cells, and alveolar macrophages. TRI at concentrations smooth endoplasmic reticulum, where P450 is localized. of 5 and 10 mM was nonselectively and dose-dependently Thus, the response of humans to TRI is likely to differ from cytotoxic to Clara cells, type II cells and alveolar that of mice. macrophages. Since the cytotoxicity of TRI was not ameliorated by 1-aminobenzotriazole, an inhibitor of (5) Fetal toxicity (including teratogenicity) cytochrome P450, Nichols et al. ruled out the bioactivation In 1981, the groundwater for a small area in the Tucson of TRI by P450. However, such a conclusion may be Valley, Arizona, U.S. was found to be contaminated with premature because the concentration employed in this study TRI. Goldberg et al.68)compared the prevalence of congenital was too high (5 or 10 mM) to make a conclusion about the heart disease in children between parents who were exposed metabolism inhibition. to TRI through TRI-contaminated wells and parents who In the study of Odum et a1.66),female CD-1 mice exposed were never exposed to the contaminated water. The to TRI (6 hr/day) at concentrations of 20-2,000 ppm proportion of infants with congenital heart disease as developed a highly specific lung lesion after a single compared with the live births was significantly higher for exposure, characterized by vacuolation of the Clara cells, mothers in the contaminated water area than for mothers the number of cells affected increasing with increasing dose with no exposure. The odds ratio for parents of case children level. At the highest dose levels, pyknosis of the Clara cells was three times that for parents of control children (p<0.005). was apparent. After 5 days of repeated exposure, the lesion Goldberg et al. concluded that their data suggest a significant had resolved but the exposure of mice following a 2-day association but not a cause and effect relation between break resulted in recurrence of the lesion. The changes in congenital heart disease and parental exposure to water mouse lung Clara cells were accompanied by a marked loss contaminated with TRI. of cytochrome P450 activities. In contrast, no morphological To experimentally verify the epidemiologic phenomenon changes were seen in the lungs of rats (female A1pk:APfSD) suggesting a greater than expected number of pediatric exposed to 500 or 1,000 ppm TRI. Isolated mouse lung patients with congenital heart disease in areas where the Clara cells metabolized TRI to CH, TCE and TCA. CH drinking water was contaminated by TRI, Dawson et al.69) was the major metabolite. TCE glucuronide was not detected. infused TRI as a saline solution (15 or 1,500 mg/l) through The activity of UDP-glucuronosyltransferase was compared a catheter into the gravid uterus by an intraperitoneal osmotic in mouse lung Clara cells and hepatocytes using two phenolic pump to the developing rat fetus in utero during the period substrates and TCE. Hepatocytes readily formed of organ differentiation and development (7th gestation day). glucuronides from all three substrates, whereas Clara cells Although only a very small number of congenital heart were only active with the two phenolic substrates. When anomalies (3%) were found in the control group (saline the three major metabolites of TRI, CH (inhalation), TCE solution only), the incidence of anomalies was found to be (inhalation) and TCA (intraperitoneal administration) were 9% and 14% in the lower and the higher dose groups, each administered to mice, only CH had an effect on mouse respectively (p<0.05). While a variety of cardiac defects lung, causing a lesion (Clara cell) identical to that seen with appeared, only a single noncardiac anomaly was found. From TRI. The lesions produced by CH at a concentration of 100 these findings, Dawson et al. concluded that TRI appeared ppm were equivalent to the changes caused by TRI at 1,000 to be a specific cardiac teratogen. From the experiment ppm. Based on these results, Odum et al. proposed that the simultaneously conducted, Dawson et al. reported that 1,1- failure of Clara cells to conjugate TCE leads to an dichloroethylene was a much stronger heart teratogen: the accumulation of CH, resulting in cytotoxicity. The known chemical caused congenital heart anomalies almost to the genotoxicity of CH suggests that this lesion may be related same extent as TRI at 1/10 the dose of TRI. In this to the development of lung tumors in mice exposed to TRI experiment, however, neither the TRI- nor the 1,1- by inhalation. dichloroethylene-treated group differed from the control Since TRI entering the body by inhalation comes into group in the number of resorbed, dead or live fetuses. It direct contact with the bronchioli where Clara cells containing may be true that a high dose of TRI causes congenital heart cytochrome P450 exist most abundantly in the pulmonary anomalies in rats. However, the animal experiment of tissues, cytotoxicity is likely to develop at the same sites. Dawson et al. does not provide sufficient evidence to establish According to Smith et al.67),the number of Clara cells is in a relationship between congenital heart disease and

Industrial Health 1997, 35, 301-324 HEALTH EFFECTS OF TRICHLOROETHYLENE 315 groundwater contaminated with TRI in humans. TRI was associated with SAB, nor did it appear to confound the injected directly into the gravid uterus at a high dose level association seen with occupational use. Windham et al. in this experiment. In humans; the concentration of TRI in concluded from this study that occupational exposure to at drinking water is about 300 ppb at the highest, and the least some solvents appeared to be associated with SAB. compound absorbed by, pregnant mothers reaches the fetuses In the experimental study of Cosby et al.73~,female B6D2F1 after passing through the =mothers'liver where the first-pass mice were gavaged from days 1 to 5, 6 to 10, or 11 to 15 metabolism, :prevails. (day 1=vaginal plug) with TRI in corn oil at 0, 1/100, and Another epidemiologic study was conducted by Deane 1110 of the oral LD50 (2,402 mg/kg). Litters were counted, et al.70~to investigate a suspected cluster of adverse outcomes sexed, weighed, and measured for crown-rump length until of pregnancies conceived in 1980-1981 among women who weaning on day 21, and some offspring were allowed to resided in a census tract in Santa Clara County, California develop to 6 weeks of age. At this time, a minimum of two that was thought to be exposed to drinking water from a litters from each dose group were killed and the gonads were well contaminated by TRI. A comparison census tract that removed, weighed, and preserved in Bouin's fixative. Litters received water from a different source was selected on the were also assessed for developmental abnormalities. No basis of demographic comparability. The cluster was maternal or reproductive effect of TRI was seen at either confirmed; the odds ratio for spontaneous abortion was 2.3 dose level. In a second series of studies, TRI and its (95% CI 1.3-4.2) after adjustment by multiple logistic metabolites DCA, TCA and TCE were added to culture media regression for maternal risk factors, including maternal age, to assess the toxic effects on in vitro fertilization (IVF) in alcohol consumption, smoking, and prior fetal loss. The mice. TRI at a concentration of 1,000 mg/l did not affect relative risk for congenital malformations was 3.1(1.1-10.4). the success rate of IVF, whereas the metabolites decreased Because of the lack of precise information on the timing the rate in a concentration-dependent manner. and extent of contamination, the pattern of spontaneous Hayasaka et al.74)gave oral TRI to S1c:ICR mice at a dose abortion rates throughout the study period cannot be used of 2 mUkg once or 1.25 mUkg twice at a 4-hr interval on to support or refute a causal inference. Subsequently, Deane gestation days 10-13, and observed its fetotoxicity. Fetal et al.71 investigated the relation between spontaneous malformations (mainly cleft palate) developed at both dose abortions and consumption of tapwater in the same cohort levels. Moreover, the weights of fetuses from mice given as used in the previous study70~.The results suggested an TRI twice at 1.25 mUkg were significantly lower than those association between spontaneous abortions and reported cold from control mice. However, the body weights of pregnant tapwater consumption. The observed effect was not due to mice were concomitantly decreased by the Till administration maternal risk factors, nor was it°a function of consumption at either dose. Accordingly, it can be said from this study of bottled water. After controlling for bottled water, the that such a high dose of TRI as to cause general toxicity in odds ratio for consumption of tap water was 3.4(0.6-19.4). dams (weight loss) can produce a teratogenic effect as well Windham et al.72~conducted a large case-control study as developmental toxicity. (n=1,926) in Santa Clara, California concerning the In the study of Coberly et al.75~,pregnant Swiss ICR mice relationship between spontaneous abortion (SAB) and were treated with TRI, orally (0.1 and 1.0 µg/kg and 48.3 exposure to organic solvents. Exposure to solvents was and 483 mg/kg) or intraperitoneally (0.01, 0.02 and 10 µg/ ascertained by a telephone interview that asked about the kg), when embryos were traversing the pronuclear stages occupational use of 18 specific solvents or products, as well of development. Pregnant animals treated with the vehicle as an open-ended `other' solvent category. The adjusted (water or olive oil) served as the control. No treatment- odds ratio for the use of any solvent was 1.1(95% CI 0.8- related effect was seen on the total number of embryos 1.5). Solvents for which at least a doubled crude risk of recovered from oviducts at the 2- to 4-cell stage of SAB was found included tetrachloroethylene(4.7;1.1-21.1), development. The 4-cell embryos were tested for cellular TRI (3.1; 0.9-10.4), and paint thinners (2.3;1.0-5.1). The proliferative disadvantage by the embryo chimera assay in odds ratio for TRI was 7.7 (1.3-47.4) when low levels of which embryos from treated mothers (experimental embryos) exposure (<0.5 hr/week) were excluded. Comparing were paired with embryos from nontreated mothers (control exposure greater than 10 hr per week versus less did not embryos) to form aggregation chimeras. TRI did not affect show consistent dose-response effects. The household use the development of embryos regardless of the route of of solvent-containing products was generally not strongly administration across all the doses examined. Since the 316 T KANEKO et al. embryo chimera assay is believed to be a sensitive technique a massive dose of TRI. for detecting the reproductive toxicity of chemical Anicteric hepatitis and uveitis are reported to have occurred compounds, Coberly et al. concluded that the reproductive in a 48-year-old woman engaged in degreasing work with toxicity of TRI is almost negligible. TRI78).According to this report, the patient had been exposed Smith et al.76~evaluated the developmental effects of TCA, to TRI at concentrations of 40-800 ppm for 3 hr daily. She a metabolite of TRI, in pregnant Long-Evans rats. Animals showed a 4- to 5-fold increase in serum alkaline phosphatase were administered TCA by oral intubation on gestation days (ALP) activity and a 3-fold increase in serum transaminase 6-15 (plug=0) at 0, 330, 800, or 1,800 mg/kg/day. activity, with an elevation of body temperature to 38.5°C. The control rats were treated with distilled water. Maternal Serologic tests ruled out hepatitis A and B, and serologic parameters included clinical signs, weight change, and gross tests for EB virus and cytomegalovirus were also negative. evaluation of organ weights and uterine contents at necropsy While the acute hepatitis slowly resolved about 6 weeks (day 20). There were no maternal deaths associated with later, pain in the eyes and impaired vision then appeared. A toxicity prior to sacrifice. Weight gain during treatment diagnosis of bilateral anterior uveitis was made. These ocular was reduced at 800 mg/kg or higher. Spleen and kidney symptoms were improved with corticosteroid treatment. weights were increased in a dose-related manner. The mean Because all of the symptoms disappeared with the test values percent of resorbed implants per litter was 34, 62 and 90 at being restored to normal 6 months after the onset of hepatitis, 800, and 1,800 mg/kg, respectively. Live fetuses the patient returned to work. Two weeks after resuming showed dose-dependent reductions in weight and length. work (TRI level 550 ppm), a malaise appeared again with a The mean frequency of soft tissue malformations in the live transient increase in serum ALP activity. For this reason, fetuses ranged from 9% at 330 mg/kg to 97% at 1,800 mg/ the worker was relocated to an office work section. She kg. These were principally in the cardiovascular system was followed for more than one year thereafter, and no (interventricular septal defect and levocardia). Skeletal abnormality was noted. According to the authors, no cause malformations were found only at 1,200 and 1,800 mg/kg other than an occupational exposure to TRI was found and were mainly in the orbit. The results of this study indicate concerning the development of hepatitis and uveitis. The that TCA is developmentally toxic in the pregnant rat at hepatic injury owing to high levels of TRI exposure is doses of 330 mg/kg and above. acknowledged, but the relationship between the uveitis and Taken altogether, the findings above suggest that whereas TRI exposure remains to be clarified. However, since not a the incidence of spontaneous abortion and the birth rate of few cases of TRI-related scleroderma-like disorders have infants with congenital heart disease were reported to be been reported7~85~,it is of interest that a worker exposed to higher in areas where the drinking water was contaminated TRI developed uveitis, a disorder regarded as one of the with TRI than in uncontaminated areas, the fetal toxicity of autoimmune diseases. TM (teratogenicity or developmental toxicity) has not been The inhabitants of Tucson Valley,Arizona, U.S. had used, confirmed at a likely dose in human exposure. as living water, ground water containingTRI at concentrations of 5-500 ppb or higher, for 25 years. Kilburn and Warshaw86> (6) Immunotoxicity applied the criteria of systemic lupus erythematosus (SLE) The long-term oral administration of TRI at high doses (arthritis, Raynaud's phenomenon, sore mouth-repeated induced liver cancer in mice16'17) . Such a large dose is likely ulcers, anemia, malar rash, skin lesions, pleurisy signs, hair to diminish the function of the immune surveillance system, loss, protein in urine, and seizure or convulsion) in 362 which may play a role in the development of tumors. Wright subjects who had used the contaminated water (exposed et al.77~reported a decrease in the activities of natural killer group) and 158 inhabitants matched to the exposed group (NK), natural cytotoxic (NC) and natural P815 killer (NPK) members by sex, age and educational level, serving as the cells following a 3-day administration of TRI at a massive control group. The frequencies of arthritis, Raynaud's dose (Sprague-Dawley rats, 5 mmol/kg/day; B6C3F1 mice, phenomenon, malar rash with sunlight, skin lesions, and 10 mmol/kg/day). This high TRI dose decreased the seizure or convulsion were significantly higher in the exposed splenocyte count in the rats and the relative spleen weights group than in the control group. The number of subjects in the mice. The results of this study suggest that who complained of four or more symptoms in the exposed compromised immune function may play a part in the group was also 2.3 times larger than that in the control group. carcinogenic responses in experimental animals exposed to These symptoms predominated in females in the exposed

Industrial Health 1997, 35, 301-324 HEALTH EFFECTS OF TRICHLOROETHYLENE 317 group. Furthermore, the titers of antinuclear autoantibodies (20121),locally involving the sigmoid colon in the majority measured by fluorescence (FANA) in serum greater than (14) of the patients. Most of the patients with primary PCI 1:80 were approximately 2.3 times higher in these females. (16121) were factory workers, of whom 15 (71.4%) were The acute rheumatic arthritis (ARA) score and FANA rank engaged in the degreasing of manufacturing products with were correlated with a coefficient of 0.1251, r2=0.0205 TRI. From the high percentage of TRI workers among the (p<0.036) in females. The authors concluded that long-term patients with primary PCI, Sato et al. concluded that low-dose exposure to TRI in contaminated well water occupational exposure to this agent constitutes an etiological significantly increased the symptoms of lupus erythematosus factor in the development of this disease. However, since as perceived by the ARA score and the increased FANA reports of TRI-associated PCI have so far been limited to titers. Japan, the evidence is still not enough to conclude that there Reports suggesting the development of pneumatosis is a causal relationshipbetween primary PCI and occupational cystoides intestinalis (PCI), a relatively uncommon disease TRI exposure. characterized by the multiple occurrence of gas-filled cysts Several studies have thus referred to TRI-related within the wall of the intestine, following occupational TRI autoimmune diseases (e.g., multiple sclerosis). However, exposure have been made by Japanese investigators87,88>. much more evidence is needed to establish a causal Yamaguchi et al.87~carried out a case-control study to assess relationshipbetween autoimmune diseases and TRI exposure. the association between PCI and working conditions, including occupationalexposure to organic solvents. Thirteen Health Risk Assessment of TRI Toxicity patients with primary PCI were individually matched with 5 controls by sex, age, and admission year. It was found When, as in the case of TRI carcinogenicity, no that there was a close association between the development epidemiologicaldata are available, carcinogenicity to humans of primary PCI and occupational exposure to TRI. While is estimated by extrapolating animal data to humans. In only 5 of 65 controls (7.7%) were exposed to TRI, 12 of the such a case, route-route and high dose-low dose differences 13 patients with PCI (92.3%) were found to have been must be taken into account, in addition to the species exposed occupationally to TRI, resulting in the odds ratio difference in toxicokinetics. The greatest barrier to animal- of 144. Moreover, the healing and recurrence of PCI in human extrapolation underlies the difference in lifespan these patients substantially paralleled the profile of their between both species. Eight hours a day is not biologically occupational exposure to TRI. Two pairs of patients with equivalent between rodents and humans. The same length PCI had been working in the same factory, where they had of time occupying the lifespan of each species (mice and degreased camera lenses with TRI. Since this study was rats, 1,000 days; humans, 80 years) is a 29-fold difference. conducted in one area of Japan, it is premature to conclude TRI must undergo metabolic activation to exhibit that a causal relationship between PCI and TRI has been carcinogenicity. The bioactivation may be mediated by either established on the basis of this study. However, the exposure the cytochrome P450 pathway or GSH conjugation pathway. odds ratio of 144 is too high to consider the relationship to However, it is not clear which pathway is detoxification or be accidental. Sato et al.88)collected the cases of PCI on a activation. Moreover, the concentration of an ultimate national basis during a 5-year-period from 1979-1983, and metabolite in the target tissue is not always proportional to found that TRI exposure-associated primary PCI cases were the amount of total metabolites. When the dose is increased, distributed almost throughout Japan. The relationship the relative rate for each metabolic pathway is changed, between PCI and occupation was studied with the 66 patients thereby changing the relationship between activation and thus collected. Information concerning their occupation was detoxification. At present, for convenience only, the amount obtained from 37 (56.1%) patients; 16 patients had secondary of a certain TRI metabolite is used as the effective dose PCI and 21 had primary PCI. The secondary PCI was (EFD) for carcinogenicity by assuming that the EFD is associated with collagen diseases such as progressive proportional to body surface area. systemic sclerosis in 6 patients (37.5%) who were all females TRI induced hepatocellular carcinomas in B6C3F1 and were affected in the small intestine. No particular mice'7.89). The U.S. Environmental Protection Agency characteristicswere noted in the occupations of the secondary (EPA)90)extrapolated this animal data (gavage test) to humans PCI patients. Primary PCI, occurring more frequently in using a linearized multistage model in order to estimate the females (15121), affected predominantly the large intestine human risk of TRI exposure. The same agency later estimated 318 T KANEKO et al. the risk of TRI exposure by inhalation using the EFD Proposal for Air Quality Standard of TRI calculated by a compartment mode191~. Computer-assisted physiologically-based toxicokinetic (1) Occupational exposure limit for workers (PBTK) models are now widely accepted as a useful tool Since TRI has not been confirmed to be a human for extrapolating animal data to humans, using parameters carcinogen, and since the carcinogenicity (liver cancer) of such as body weight, Vmax and Km. Cronin et a1.92>and TRI to B6C3F1 mice may be based on an epigenetic rather Fisher and Allen93> used a PBTK model coupled with a than genotoxic mechanism, it is not appropriate to establish linearized multistage model to derive the human TRI the occupational exposure limit of TRI in occupational carcinogenic risk extrapolation from animal data. This settings presupposing that TRI is a carcinogen. TRI technique is certainly useful if the differences between carcinogenicity, if any, would be induced only after this animals (B6C3F1 mice) and humans are only quantitative. chemical has been inhaled for a long period of time at such However, if, as in the case of TRI carcinogenicity, a high concentrations as to cause cytotoxicity. qualitative difference that cannot be overcome is present, The health effects of long-term exposure to TRI are chiefly such a technique cannot be considered to significantly manifested as neuropathy, and thus the occupational exposure improve the reliability of risk assessment. As in the risk limit for TRI could be established on the basis of its assessment by the U.S. EPA, the assessment performed by neurotoxicity. The current exposure limit proposed by the the PBTK modeling does not have reliable implications in American Conference Governmental Industrial Hygienists that both risk assessments were based on the same (ACGIH) (threshold limit value, TLV) and the JSOH carcinogenicity test where B6C3F1 mice were used" s9),as (occupational exposure limit, OEL) is 50 ppm (270 mg/m3). discussed below. Evidence has accumulated that a long-term exposure to TRI Abelson94~ has severely criticized the risk assessment at 50 ppm will cause neurotoxic effects to industrial workers, performed by the U.S. EPA9o,9" as follows: "In calculating suggesting that the value should be lowered. the risks of human cancer and establishing regulations, the In this connection, the study of Ahlmark and Forssman9S~ U.S. EPA makes a series of conservative assumptions that is of utmost importance because they examined 122 workers have no sound scientific basis. Exaggerations of risks are for the neurotoxic effects (subjective symptoms) of TRI in inherent in the procedures by which rodents are employed relation to the urinary levels of TCA. They found that the as stand-ins for humans. Dependence on results of maximum workers who excreted more than 30 mg of TCA per liter of tolerated doses in the most sensitive strain or species of rodent urine manifested certain symptoms to a greater extent than is questionable. Important examples involve B6C3F1 mice, others. The symptoms most commonly complained of were which in contrast to humans in developed countries, have a abnormal fatigue and increased need of sleep. Diffuse gastric high control incidence of liver cancer. Those mice often symptoms such as tympanism (meteorism), a sensation of respond with liver tumors when exposed to large doses of a dryness in the mouth, a feeling of sickness, and intolerance substance. Other rodents are less often affected. Questionable to alcoholic beverages were also frequently complained of. assessments involving liver cancer in B6C3F1 mice include Ahlmark and Forssman summarized their own work as the risks posed by TRI, TETRA, methylene chloride, follows: "One may say that a constant exposure to TRI which butadiene, and phenobarbital." Steinberg and DeSessol~ have gives rise to an excretion of TCA of less than 20 mg per also questioned the EPA's risk assessment based on a linear liter of urine does not entail, or entails only in exceptional extrapolation from the effects of high doses in rodents to cases, any definite TRI effects, whereas at the values of over risks for humans at low doses, stressing that, instead of a 20 mg per liter of urine one sees such effects in an increasing straight-line extrapolation model, a threshold model may percentage of cases directly proportional to the increasing be more appropriate in the case of risk assessment of TRI excretion." However, the readers should pay attention to carcinogenicity. In other words, since the carcinogenicity the finding that among 21 workers who excreted 11-20 mg/ of TRI is preceded by cytotoxicity (necrosis), there may 1 of TCA, 7 were judged to have a `possible effect' and 5 an `actual effect exist a threshold for the carcinogenicity. .' This indicates that the TCA level of 20 mg/ 1 corresponds to a LOAEL (least observed adverse effect level) rather than a NOAEL (no-observed adverse effect level). Ahlmark and Forssman used `morning urine' for the urinalysis for TCA. It was not defined when the `morning

Industrial Health 1997, 35, 301-324 HEALTH EFFECTS OF TRICHLOROETHYLENE 319 urine' was collected, in the morning before the beginning the general, population can also be established on the basis of work or during the work in the forenoon. Although the of the reference value employed in the occupational settings. exposure concentration of TRI corresponding to 20 mg/l of The ACGIH defined the TLUs as "airborne concentrations TCA in `morningurine' cannot have been exactly determined, under which it is believed that nearly all workers may be it can well be said that the concentration may be higher than repeatedly exposed day after day without adverse health 10 ppm but lower than 30 ppm. Indeed, Ahlmark and effects." Furthermore, the ACGIH states that "because of Friberg96~proposed 30 ppm as the maximum allowable wide variation in individual susceptibility, a small percentage concentration of TRI on the basis of urinalysis for TCA. of workers may experience discomfort from some substances According to Liu et al.53~,the prevalence of subjective at concentrations at or below the threshold limit; a smaller symptoms in workers exposed to TRI around 50 ppm was percentage may be affected more seriously by aggravation significantly high in comparison to control workers. A dose- of a pre-existing condition or by development of an response relationship could be established in some selected occupational illness.... Individuals may also be symptoms such as nausea, a heavy-headed feeling, hypersusceptible or otherwise unusually more responsive forgetfulness, tremor in extremities, and dry mouth. These to some industrial chemicals because of genetic factors, age, findings suggest that the effects of TRI on the central and personal habits (smoking, alcohol, or other drugs), autonomic nervous systems appear at concentrations lower medication, or previous exposures. Such workers may not than 50 ppm. be adequately protected from certain chemicals at According to Ruijten et al.55~,occupational exposure to concentrations at or below the threshold limits." This TRI (cumulative exposure level, 704 ± 583 ppm-year) caused indicates that the reference value in the industrial workplace a slight reduction (- I .1 m/s) in the sural nerve conduction has been established on the condition that it carries some velocity and a prolongation (0.4 ms) of the sural nerve risk a priori. The OELs of the JSOH are essentially the refractory period (mean of the controls 1.95 ms). Ruijten same as the TLUs of the ACGIH. et al. concluded that long-term exposure to TRI at 35 ppm To establish an EAS for the general population on the may slightly affect the trigeminal and sural nerves. basis of the reference value for healthy workers engaged in From the findings described above, the present authors work with a definite sense of duty, a wide margin of consider that the reference value of TRI in the work uncertainty factor must be taken into consideration. With environment should be lowered to below 50 ppm, presumably these factors in mind, the EAS can be calculated as a guide to 25 ppm (135 mg/m3). Incidentally, the U.S. National from the following equation: Institute for Occupational Safety and Health (NIOSH) recommends 25 ppm as the recommended exposure limit EAS = (Reference value for workers) x (8124) x (2401365) x (40180) x 10-2, (REL) for TRI. (1) (2) (3) (4) (2) Environmental air quality standard for the general where population (1) conversion from occupational exposure (8 hr) to The contamination of air or water with TRI in the general exposure of the general population (24 hr), environment is related to industrial activities, with the (2) conversion from the number of yearly workdays (240 compound thereby incurring health effects not only on days) to the number of days a year (365 days), industrial workers but also on the general population. (3) conversion of occupational exposure (40 years) to According to Brown et al.97~,TRI exposure in the general lifelong exposure of the general population (80 years), environment allows a great margin of safety concerning TRI- and induced hepatic injuries and subsequent liver cancer (4) a total uncertainty factor for conversion from development. Hence, it is reasonable to establish the occupational exposure to exposure of the general environmental air standard (EAS) for TRI from health effects population. other than its carcinogenicity. Among such health effects The total of (1) x (2) x (3) x (4) is approximately 10-3. caused by long-term exposure to TRI at low doses, its That is, 10-3 of the reference value for occupational TRI neurological effect may be the most appropriate one for exposure will be a guide for the EAS for the general consideration. Since the reference value of TRI for industrial population. The present authors propose a value falling in workers is based on relatively sufficient data, an EAS for the range of 25-50 ppb (135-270 ,ug/m3) as an EAS of TRI. 320 T KANEKO et al.

Values within this range may be scientifically sound as the trichloroethylene exposure and cancer mortality. J EAS, since a wide range of safety (uncertainty factor=1,000) Occup Med 20,194-6. is already taken into account. What value is adopted as the 3) Tola S, Vilhunen R, Jarvinen E, Korkala M-L (1980) EAS of TRI is a question of risk management. A cohort study on workers exposed to trichloroethylene. The above equation simplyproposes a procedure for setting J Occup Med 22, 737-40. up the EAS of TRI on the basis of the reference value for 4) Shindell S, Ulrich S (1985) A cohort study of employees occupational TRI exposure. The only reason for adopting of a manufacturing plant using trichloroethylene. J 40 years as the duration of occupational exposure is that Occup Med 27, 577-9. the occupational lives of the Japanese are considered to be 5) Garabrant DH, Held J, Langholz B, Bernstein L (1988) at 20-60 years of age (a spread of 40 years). Although it is Mortality of aircraft manufacturing workers in southern true that very few workers are engaged in the same job for California. Am J Ind Med 13, 683-93. 40 years, a sufficient margin of safety will be ensured as 6) Spirtas R, Stewart PA, Lee JS, Marano DE, Forbes CD, the result of the assumption that the duration of exposure is Grauman DJ, Pettigrew HM, Blair A, Hoover RN, 40 years. There is no precise reason for the uncertainty Cohen JL (1991) Retrospective cohort study of workers factor of 102. One `10' is an uncertainty factor in at an aircraft maintenance facility. I. Epidemiological consideration of the differences in susceptibility between studies. Br J Ind Med 48, 515-30. industrial workers (healthy adults) and the general population 7) Axelson 0, Selden A, Andersson'K, Hogstead C (1994) including infants, elderly persons and highly susceptible Updated and expanded cohort study on trichloroethylene persons. In addition, while a recovery period of 16 hr exists and cancer risk. J Occup Med 36, 556-62. in occupationally exposed workers, the exposure of the 8) Anttila A, Pukkala E, Sallmen M, Hernberg S, general population lasts for 24 hr without intermission. This Hemminki K (1995) Cancer incidence among Finnish is taken into consideration in this uncertainty factor. The workers exposed to halogenated hydrocarbons. J Occup other ` 10' is included from the personal view of the present Environ Med 37, 797-806. authors that the reference value for occupational exposure 9) Henschler D, Vamvakas S, Lammert M, Dekant W, is near to the LOAEL rather than to the NOAEL. The Kraus B, Thomas B, Ulm K (1995) Increased incidence uncertainty factor converting from LOAEL to NOAEL is of renal cell tumors in a cohort of cardboard workers normally considered to be `10.' Lastly, it should be borne exposed to trichloroethylene. Arch Toxicol 69, 291-9. in mind that the above equation is applicable to only TRI 10) Blair A (1980) Mortality among workers in the metal on the condition that the compound is not classified as a polishing and plating industry, 1951-1969. J Occup human carcinogen. However, the IARC has changed the Med 22,158-62. carcinogenicity classification of TRI from Group 3 to Group 11) NovotnaE, DavidA, Malek B (1979) An epidemiological 2A. A new uncertainty factor may be needed to be taken study on hepatic tumor incidence in subjects working into account in the future, if this compound is identified as with trichloroethylene. 1. Negative result of a human carcinogen based on a genotoxic mechanism. retrospective investigations in subjects with primary liver carcinoma. Parcovni Lekarstvi 4,121-3. Acknowledgment 12) Paddle GM (1983) Incidence of liver cancer and trichloroethylene manufacture: joint study by industry This work was financially supported in part by the Japan and a cancer registry. Br Med J 286 (6386), 846. Environment Agency. 13) International Agency for Research on Cancer (IARC) (1995) IARC monographs on the evaluation of References carcinogenic risks to humans. 63, 75-158, IARC, Lyon. 14) Occupational Exposure Limit Committee of the Japan 1) Steinberg AD, DeSesso JM (1993) Have animal data Society for Occupational Health (1996) been used inappropriately to estimate risks to humans Recommendation of Occupational Exposure Limits from environmental trichloroethylene? Regul Toxicol (1996-1997). San Ei Shi 38,134-47 (in Japanese). Pharmacol 18,137-53. 15) International Agency for Research on Cancer (IARC) 2) Axelson 0, Andersson K, Hogstedt C, Holmberg B, (1987) IARC monographs on the evaluation of Molina G, de Verdier A (1978) A cohort study on carcinogenic risks to humans. Suppl 7, 364-6, IARC,

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