Document 850LQb7vKg6Vp6ZZKNEQ2rEgB

SCIENTIFIC REVIEW PANEL VERSION TECHNICAL SUPPORT DOCUMENT PART C IIMTffl(FOATrOrci IF PERCHLOROETHYLENE A &m (HTTAffiflOIKlMrT MAY 1991 State of California Air Resources Board SL 038664 This report has been reviewed and approved by the staffs of the California Air Resources Board and the Department of Health Services. The contents do not necessarily reflect the views and policies of the Air Resources Board or the Department of Health Services, nor does mention of trade names of commercial products constitute endorsement or recommendation for use. >>5 PART C STAFF RESPONSES TO PUBLIC COMMENTS ON THE PERCHLOROETHYLENE TECHNICAL SUPPORT DOCUMENT Prepared by the Staffs of the Air Resources Board and the Department of Health Services May 1991 SL 038666 PART C TABLE OF CONTENTS I. COMMENT LETTERS RECEIVED A. COMMENTS FROM CITIZENS FOR A BETTER ENVIRONMENT B. COMMENTS FROM HALOGENATED SOLVENT INDUSTRY ALLIANCE C. COMMENTS FROM INTERNATIONAL FABRICARE INSTITUTE D. COMMENTS FROM SAN DIEGO AIR POLLUTION CONTROL DISTRICT Page 1 2 5 88 98 II. AIR RESOURCES BOARD STAFF RESPONSES TO COMMENTS ON PART A 100 A. RESPONSES TO COMMENTS FROM CITIZENS FOR A BETTER ENVIRONMENT B. RESPONSES TO COMMENTS FROM INTERNATIONAL FABRICARE INSTITUTE C. RESPONSES TO COMMENTS FROM SAN DIEGO AIR POLLUTION CONTROL DISTRICT 101 104 106 III. DEPARTMENT OF HEALTH SERVICES STAFF RESPONSES TO COMMENTS ON PART B 109 A. RESPONSES TO GENERAL COMMENTS B. RESPONSES TO COMMENTS ON TOPIC: HAZARD IDENTIFICATION C. RESPONSES TO COMMENTS ON TOPIC: DOSE-RESPONSE ASSESSMENT 110 110 112 SL 038667 I. COMMENT LETTERS RECEIVED Si 0J668 GCOOOI i'<7 i0 ^ /<jC Donald J. Ames Assistant :;n ry Source 1 visit-..* ;ssojrcss Scar; Attn: Percnloraethylene PO Sox 2315 Sacramento, C A $5312 P\>< 9 Ifc / 32-^ - 6003 ** C?* , *''*' - +SW 1 - ^ - 6' ...^*"' Chief <^,` * .CITIZENS * FOR A BETTER ENVIRONMENT Dear M r. Ames , The following are our comments on t.re Draft Report on "erchloroetr.ylsne Part A, cf Decemoer 1989. _ Additional sources of Indoor exposure mustbe assessed Tnere r,as seen a failure in Part A of trie report to aaecuately assess one total public exposure to Parcr.loro ethylene { P C E from all i n d o c " environments as required by state star .The report only assesses exposure from indoor air in tne nome, anc neglects to consider exposure in sucn places as hospitals* schools, offices, and the work place. "or instance, an employes at a dry-cleaner car. Jace exposure at work anc in the home to potentially high caily lev els of ACE, anc needs to be considered. Exposure to a consumer at a cry cleaning counter shoulo also be assessed. Information must be adced to the final report to adequately assess the multiple routes of exposure from the different sources of ?CH in indoor air. California Health and Safety Code Article 2, Section 399S0.5 (a) states that "In evaluating the level of potential r.uman exposure to toxic air contaminants, the state board shall assess that exposure in indoor environments as well as ambient air conditions." (c) "When the state board identifies toxic air pollutants that nave found in any Indoor environment, the state Doard shall refer all available data on that exposure and the suspected source cf the pollutant to the state Department of Health Services..." (emphasis aaded } seen (d) "In assessing human exposure tc toxic air contaminants in indoor environments pursuant to this section, tne state board shall identify the relative contribution to total exposure to the contaminant from inaoor concentrations, taking into account both ambient and indoor air environments." (emphasis added) {cont) SL 038669 Need further data on PCE in food supply * CITIZENS FOR 1A : BDECTI TICERn NVinONMENT adsorption onto fats in foods through contact with sirecrn PCE, then date camoarlnc concentrations of airoern PCE in California ana Europe intake * i g u r e s , associated cancer n s k figures should be presentee ralatsa to ingestion. Include worst case numbers for assessment of exposure worst case exposure can be assessed. Information or, ir,dividual cancer risk associated with worst case a no overage concentrations should also be included In saditior, maximum daily and maximum chronic exposure cor peoole using several consumer products containing PCE, who also usa cry-cleaners frequently, who work; in environments with nigh levels of PCE, who live of - C E . should be considered, and total worst case daily intake and cancer risk data should be presented. Append Lo report xlaLo from federal SARA III TRI database It is highly appropriate for the purooses of tne Toxic Air Contaminant {TAC) identification process to include in the report in formation on specific sources of PCE emissions. The Mnal report should append tne 1987 , 1988 , and 1989 data identifying PCE emissions -'rom all sources in the federal SARA Title III database, which are readily available. The pudic nas the -ight to know of anc review such data, and the ARB TAC documents should provide this information on a ceogrsor,ic basis ( c o n t) HALOGEN ATED SOL V ENTS NDUSTRV 4 L !_ A N C E 1225 I9rn S:ree: vi W Sjice 300. Wdsmnaon DC 22036-24! i -2C2I 2I3-559C February 1, 1990 Mr. Donald 0. Ames Assistant Chief Stationary Source Division Air Resources Board Attn: Perchloroethylene F. 0. Box 2815 Sacramento, CA 95812 Stationary L-.rc: Division fpBttou-- - ~ Dear Mr. Ames: The Halogenated Solvents Industry Alliance (HSIA) offers the enclosed comments on the draft Technical Support Document: Proposed Identification of Perchloroethylene as a Toxic Air Contaminant (Part B: Health Effects of Perchloroethylene). HSIA is an association of producers, distributors, importers, and users of halogenated solvents, including perchioroerhylene. Our members, as well as other users of per chloroethylene have a vital interest in the accuracy and scientific validity of the Report. Sincerely, (X Paul A. Cammer, Ph.D. President Enclosure 03867^ V . SL BEFORE THE CALIFORNIA AIR RESOURCES BOARD COMMENTS OF THE HALOGENATED SOLVENTS INDUSTRY ALLIANCE ON THE DRAFT TECHNICAL SUPPORT DOCUMENT: PROPOSED IDENTIFICATION OF PERCHLOROETHYLENE AS A TOXIC AIR CONTAMINANT (Part B: Health Effects of Perchloroethylene) Halogenated Solvents Industry Alliance 1225 19th Street, N.W. Suite 300 Washington, D. C. 20036 202-223-5890 February 1, 1990 Paul A. Cammer, Ph.D. President SL 038673 c,, Vj C i i \J BEFORE THE CALIFORNIA AIR RESOURCES BOARD COMMENTS OF THE HALOGENATED SOLVENTS INDUSTRY ALLIANCE ON THE DRAFT TECHNICAL SUPPORT DOCUMENT: PROPOSED IDENTIFICATION OF PERCHLOROETHYLENE AS A TOXIC AIR CONTAMINANT (Part B: Health Effects of Perchloroethylene) Tntr.pfrjction The Halogenated Solvents Industry Alliance (HSIA) offers these comments to the Air Resource Board (ARB) cn Part B (Health Effects of Perchloroethylene) of the draft Technical Support Document for the Proposed Identification of Perchloroethylene as a Toxic Air Contaminant. Our comments include a summary of the r`f ; w SL 038674 2 pertinent literature on the carcinogenic potential of perchloroethylene in animals and humans, a discussion of the importance of species differences in metabolism of this chemical, and a recommendation to develop a more plausible estimate of potential risk that incorporates pharmacokinetic ir.f::n:.:r.. H5IA is an association of users, distributors, and producers of chlorinated solvents, including perchloroethylene. Our members, as well as other users of perchloroethylene, have a vital interest in the accuracy and scientific validity of the Technical Support Documents which serve as the basis for the proposal to identify perchloroethylene as a toxic air contaminant. Decisions made by the ARB on the basis of the Technical Support Documents will have a significant effect on actions taken by local air districts in California to regulate perchloroethylene, on possible future proposals from Cal-OSHA to change worker exposure levels, and on other risk assessmentrelated legislation and regulation within the state. As a consequence of those actions, a large number of industrial and commercial users of perchloroethylene will be affected, as will the public that benefits from the applications of the chemical. SL 038675 Cr -' W V-- 1/ , J 3 Executive Summary The overall weight of the scientific evidence for perchlcrcethylsr.e suggests that it is unlikely t: pose a carcinogenic risk to humans at ambient environmental or occupational exposure levels. The health effects of perchloroethylene have been studied extensively. Long-term bioassays of perchloroethylene have shown that it produces liver cancer in certain species of mice, but not in rats. The proximal carcinogen appears to be trichloroacetic acid, a metabolite of perchloroethylene, which induces proliferation of peroxisomes in liver cells. Humans produce less trichloroacetic acid than mice and rats, and do not exhibit the critical biological response of peroxisome proliferation, which is responsible for the formation of liver tumors in rodents. These documented species differences in response to perchloroethylene exposure between mice and rats, and between rodents and humans, provide strong support for the conclusion that the chemical does not pose a carcinogenic risk to humans. Adding support to this conclusion are the findings of epidemiologic studies in workers exposed to perchloroethylene. The overall results of epidemiology studies carried out on drycleaner workers to date do not provide support for the conclusion that perchloroethylene poses a cancer risk to humans. Op ^ p f Wt k SL 038676 The draft report must be revised to reflect more clearly the significant qualitative differences in metabolism of perchloroethylene between mice and rats, and between rodents and humans, and the negative evidence for carcinogenicity in human studies. The weight of the available Scientific evidence, as reviewed by the International Agency for Research on Cancer (IARC) and the Science Advisory Board of the U.S. Environmental Protection Agency (EPA), does not support the conclusion that the chemical is a probable human carcinogen. If the final report includes an estimate of potential risk, HSIA strongly recommends that it incorporate available pharma cokinetic information. In the past year, both ARB and its Scientific Review Panel (SRP) have recommended that the Department of Health Services go further to incorporate pharmacokinetic information into its risk assessments. Although the pharmacokinetic data for perchloroethylene are discussed in the draft report, the final risk assessment range (5-21 x 1Q~6 for a l ug/m3 lifetime exposure) was calculated without incorporating pharmacokinetic information, since this is the range that will be used for regulatory purposes, pharmacokinetic information has for all practical purposes not been reflected. This is flatly inconsistent with the California carcinogen Guidelines ("Guidelines'1) which provide (A-16) that "[p]harmacokinetic data on metabolism of dosed substances, SL 038677 a Vy '**> 4; ..! 5 effective d se at target site, or species differences b tween laboratory test animals and humans should be considered in dose-response assessments wnen they are available." The DHS risk estimate is more than ar. order of ngr.it-ods higher than the most recent range under consideration by EPA (2.9-9.5 x IQ-7) , which was calculated using pharmacokinetic data. Incorporating metabolic and pharmacokinetic data into the risk calculations will reduce the uncertainties inherent in the risk assessment process and will result in a more plausible estimate of risk to humans. In light of the availability of extensive pharmacokinetic data (as reflected in the draft) and the Guidelines requirement that they be taken into account, rejection of a physiologically-based pharmacokinetic model in favor of an approach based on default assumptions appears to be an abuse of the Agency's discretion. Carcinogenic Potential of Perchloroethvlene I. Experimental Animal Data The principal animal carcinogenicity studies reviewed in the draft are summarized below. A. 1978 Rampy Study In this inhalation study, groups of male and female Sprague- *5 SL 038678 6- Dawl y rats were exposed to 0, 300, and 600 ppm of the chemical for 6 hours per day, 5 days per week for 12 months. They were then observed for an additional 18 months. No increase in tumors in the exposed animals was observed. B. ' NCI Bioassav An increased incidence of mouse liver tumors was observed in a National Cancer Institute (NCI) gavage bioassay reported in 1977. There are a number of controversies surrounding the use of this study to determine human cancer risk. The National R search Council, in a review of the NCI study, stated that "the quantities of TCE [tetrachloroethylene] given were so large that marked, dose~dependent mortality in both species occurred throughout the study period'* (NRC, 1980) . It concluded that "the findings of this study should be interpreted with caution, recognizing the limitations of the experimental design fa.a., massive doses of TCE, large volumes of oil vehicle, marked nephrotoxicity, diminished lifespan)." We question whether this is "properly conducted bioassay," as required by the Guidelines, for the evaluation of potential human hazard from perchloroethylene. SL 038679 IwT w ^ **. c. trrE-Bipflgsay The National Toxicology Program (NTP, 1986a) conducted an inhalation tioassay and concluded that the study shewed clear evidence of carcinogenicity in male Fischer 344 rats, based cn an increase in the spontaneous incidence of mononuclear cell leukemias and a non-statistically significant increase in renal adenomas and carcinomas; some evidence of carcinogenicity in female rats- based on an increase in the spontaneous incidence cf mononuclear cell leukemias; and clear evidence of carcinogenicity in male and female mice based on an increase in the spontaneous incidence of hepatocellular adenomas and carcinomas. II- Interpretation and Significance of Animal Bioassav A. _Rat Mononuclear Cell Leukemia EPA/s Science Advisory Board determined that the high spontaneous incidence of rat leukemias observed in the NTP inhalation bioassay was not related to perchloroethylene exposure. Slight increases in mononuclear cell leukemia, which occurs spontaneously at a high and variable incidence in the Fischer 344 rat, were observed in male and female rats exposed to 200 or 400 ppm tetrachloroethylene for two years. Incidences increased from 36 percent in control male rats to approximately r\ ^ SL 038680 8 60 percent in exposed males and from a control value of 56 percent to approximately 80 percent in exposed female rats. Importantly, the incidence of mononuclear cell leukemia m the exposed rats was not dose-related. The increased incidence observed is unconvincing with respect to human risk since ' i questions were raised involving whether the proper staging criteria were used, (ii) it is uncertain whether there is actually a human counterpart for this type of leukemia, and (iii) the control incidences exceed those previously seen in the testing laboratory, and also exceed the NTP historical control value. In this regard, it should be noted that incidence rates in the tetrachloroethylene-treated rats were 37/50 (male 200 ppm), 37/50 (male 400 ppm), 37/50 (female 200 ppm), and 29/50 (female 400 ppm). Comparison to a study conducted at the same laboratory at the same time shows leukemia rates in untreated rats of 34/50, (NTP, 1986b). The etiology and pathogenesis of mononuclear cell leukemia in the rat are unclear, which has to this point precluded mechanistic study of this response. Be that as it may, sev ral associations in the response render the biological significance of this observation for humans doubtful. As noted above, it is exceedingly common in the Fischer 344 rat. Importantly, leukemia was not observed in the 1977 NCI study (Osborne-Mendel rats) nor by Rampy (Sprague-Dawley rats) in the study described above. Nor has tetrachloroethylene induced leukemia in mice. These data, coupled with the apparent lack of genotoxicity of SL 038681 /"> ^ * 9 tetrachloroethylene, lead us to conclude that the observed Fischer 344 rat mononuclear cell leukemia is a strain-specific phenomenon and, as the Science Advisory Board stated, is not of relevance to humans. B. Male Rat Kidney Tumors As to the other result in rats, a marginal, non-statistically significant increase in kidney tumors observed in the same study, the Board stated that the mechanism responsible "appears to be unique to male rats", and that recent research "indicates that for many halogenated organics, probably including perchloroethylene, the mechanism producing these types of tumors is probably not operative in humans and, therefore, may not be relevant for human risk assessment" (SAB, 1988) . Two renal tubular cell adenocarcinomas were observed in male rats in the NTP study at 400 ppm, while none were observed at 200 ppm or in the control group. Two renal tubular cell adenomas were also observed in the 400 ppm group of male rats versus three at 200 ppm and one in the control group (NTP, 1986a) . This sex-specific observation is not without precedence -- small increases in male rat kidney tumors have been occasionally noted across the class of short-chain aliphatic hydrocarbons. Renal tubular cell neoplasms have been shown to be produced by unleaded gasoline in male rats only. 6ft'2' f> r* f~ - 10 - An legant series of experiments conducted on unleaded gasoline at the Chemical Industry Institute of Toxicology (CUT) has shown the male rat kidney tumor response to be related to th following events: complexing of trimethylpentane with an alpha-2u-glctulir., renal tubular absorption of tbs oor.plax. renal tubular hyaline droplet formation, renal tubular injury, enhanced renal tubular regeneration, and subsequent tumor development secondary to ongoing injury. Perchloroethylene, under the conditions of the NTP study, has been shown to be nephrotoxic and has been shown, like gasoline, also to elicit this hyaline droplet nephropathy in exposed male, but not female, rats. In recent experiments conducted at CIIT (Goldsworthy et al., 1988) and at the Central Toxicology Laboratory at ICI, Ltd., oral gavage administration of 1000 mg/kg and inhalation of 1000 ppm perchloroethylene 6 hours/day for 10 days resulted in hyaline droplet formation in the P2 segment of the proximal tubule. The work at CIIT has further confirmed deposition of alpha-2uglobulin in renal proximal tubule cells, resulting in cellular toxicity, and an increase in cellular regeneration in the P2 segment of the proximal tubule. Thus, perchloroethylene, in addition to its generalized nephrotoxicity at doses used in the NTP study, has been shown to elicit the male rat-specific hyaline droplet nephropathy. This is a qualitatively different response (male-rat specific) which would not be expected to occur in humans. SL 038683 Cr\ r\ / v r* u w w-* ) It should be mentioned that data from the laboratory cf Or. Henschler in Germany suggest the existence of a second, very minor metabolic pathway involving perchloroethylene conjugation with glutathione (Dekant et al., 1986). This conjugate represents approximately 0.1 percent of the administered dose in the mouse and approximately 0.01 percent in the rat based on pooled 72-hour urine. Dr. Henschler has suggested that this minor metabolite may be a substrate for renal beta-lyase, producing an electrophilic metabolite that could potentially react with renal tissue macromolecules including DNA. The existence of a perchloroethylene-glutathione conjugate in mice and rats has been identified at ICI. The minor metabolic pathway, GSH-transferase, has been shown to be approximately six times more active in rats than in mice, and renal, beta-lyase activity to be approximately five to six times higher in rats than in mice. Within its limits of detection, the ICI laboratory has found no evidence for conjugation of perchloroethylene with GSH in human tissue. Thus, while this minor pathway could conceivably have played a role, in concert with renal cytotoxicity as well as male rat-specific hyaline droplet formation, humans appear qualitatively different from rats due to the apparent absence of rormation of the initial GSH conjugate. Moreover, the absence of a direct conjugate-mediated genotoxic effect was demonstrated in SL 038684 ^ r > f >i . v, w 4 rats at CUT by the failure of perchloroethylene to induce DNA repair (UDS) in the rat kidney using in vivo/in vitro techniques (Goldsworthy et al., 1988). C. Mouse Liver Tumors Some scientists believe that liver tumor findings in the B6C3F1 mouse, in the absence of demonstrable direct genotoxic activity, but in the presence of other epigenetic (promotional) events, are not relevant to humans. The high spontaneous incidence of these tumors complicates both the statistical and biological evaluation of the weight of the evidence. Two recent papers show that DNA from liver tumor tissue taken from untreated B6C3F1 mice that have spontaneously developed the tumors expressed the active H-ras oncogene (Fox and Watanabe, 1985; Fox et al., 1987). These studies indicate that the B6C3F^ mouse may be genetically predisposed to liver tumors, making it an inappropriate model for direct comparison to the human. Perchloroethylene, in common with many nongenotoxic promoters of carcinogenesis, induces hepatic peroxisome proliferation and cellular replication in B6C3F1 mice (but not rats) at levels used in chronic bioassays. Moolgavkar describes how a multi-stage process consisting of initiation (i.e., by a direct genotoxic agent; by indirect effects on the genome secondary to the pharmacologic/toxicologic effects of an agent; or by oncogene SL 038685 O nop/1 oC c; activation via a spontaneous or chemically induced mechanism) followed by cellular replication induced by a nonqenotoxic agent would lead to an increased incidence of tumors (Moolgavxar ana Knudson, 1981). This theoretical explanation suggests that perchloroethylene, a nongenotoxic agent, would be markedly less likely to induce tumors in species that are not genetically predisposed. In sum, there is widespread scientific agreement that an increased incidence of mouse liver tumors, in the absence of other significant carcinogenic effects, is of questionable significance in assessing cancer risk to humans (International Expert Advisory Committee to the Nutrition Foundation, 1983; Schach von Wittenau and Estes, 1983; Butler and Newberne, 1975; Tomatis et al., 1973; Grasso and Crampton, 1972). One reason is the very high spontaneous incidence of liver tumors in mice. The r cent identification of an oncogene in BgC3F^ mouse liver tumors casts further doubt on the value of mouse liver tumors as an end-point is assessing human risk. III. Species Differences The increased incidence of liver cancer in rice exposed to perchloroethylene appears to have resulted from peroxisome proliferation, a mechanism to which rodents are extremely sensitive relative to primates and humans. In addition, perchloroethylene is biotransformed to trichloroacetic acid, the 03B66 ^ /> ** `W '*/** 14 ** proximat peroxisom -prolif rating agent, to a greater extent m mice that in rats. Humans, in turn, bioactivate even less perchloroethyiene than do rats. Trichloroacetic acid has itself been shewn to cause peroxisome profileration in mouse liver cells and to induce liver tumors in mice when given alone (Herren-Freund et al., 1986). The level of peroxisome proliferation in perchloroethyleneexposed mice corresponds closely to the level of trichloroacetic acid production (Odum et al., 1988). Significantly, research has shown that peroxisome proliferation does not occur in human liver cells following in vitro exposure to trichloroacetic acid (Elcombe, 1985). Specifically, mechanistic studies published by scientists at CIIT and Id's Central Toxicology Laboratory have shown peroxisome proliferation to be induced in mice, but not rats, after repeated gavage dosing of 1000 mg perchloroethyiene/kg/day for 10 days, and after inhalation of 200 or 400 ppm perchloro ethyiene for up to 28 days (Odum et al., 1988; Goldsworthy and Popp, 1987). The induction of peroxisomes has been shown to be directly related to the metabolism of perchloroethyiene to trichloroacetic acid. After exposure to 400 ppm perchloroethlyene for 6 hours, peak blood levels of trichloroacetic acid were 13 times greater, and the area under the plasma concentration vs. time curve 6.7 times greater, in mice than in rats. Statistically significant increases in the peroxisomal 038687 15 t marker nzyme -- CN insensitive palmitoyl CoA activity -- as well as increased numbers of hepatic peroxisomes, were also observed in the mice. Consistent with the marked relative insensitivity of higher mammalian species, including humans, to peroxisome proliferating agents, trichloroacetic acid did not stimulate peroxisome enzymes in human cells in culture. These data strongly suggest that perchloroethylene would not cause liver tumors in humans (Stott, 1988) . Even if peroxisome proliferation is only a marker of liver cell involvement in the carcinogen process, it is clear that human cells have a qualita tively different reaction to the rodent proximal carcinogen. Thus, humans are doubly unlikely to show a carcinogenic response to perchloroethylene due to (i) significantly lower production of trichloroacetic acid, and (ii) the relative insensitivity of humans to peroxisome proliferation, the critical biochemical response. Indeed, the SAB has indicated that a mechanistic model such as peroxisome proliferation is likely to be important for perchloroethylene (SAB, 1988). Induction of peroxisomes is not thought to be a linear function of trichloroacetic acid concentration in the liver and, in the case of perchloroethylene, is accomplished by the induction of DNA synthesis in the BgC3F1 mouse liver. The data suggest that peroxisome proliferation and induction of hepatic DNA synthesis, along with the genetic predisposition of the BgC3F1 mouse liver to liver tumor induction, act in C TC r'V. SL 038688 16 concert to nhance tumor formation in perchloroethylen -exposed B6C3Fi mice. By analogy to trichloroethylene, it appears that genetic predisposition is a critical factor. Henschler has reported that, after oral exposure to purified, amine-stabilized trichloroethylene, Swiss r.ioe do not appear to de%'eicp r.ecatic tumors (Henschler et al., 1984). Bg^^l ^ice do develop hepatic tumors, on the other hand, although both strains are thought to metabolize trichloroethylene in a similar fashion quantitatively. The foregoing data, taken together, overwhelmingly support the concept that perchloroethylene appears to be enhancing spontaneous liver tumors in B6C3mice by a secondary (promotional) mechanism, which conceptually embodies the principle of a practical threshold. If a substance acts as a promoter, rather than an initiator, the dose-response relationship would be expected to exhibit a practical threshold (Ames et al., 1987). Much of the scientific research referenced above is neither discussed nor referenced in the draft report. Copies of the relevant articles are attached. It is very important that the final report take into account these recent scientific findings showing a clear species difference in response to perchloroethylene. In this regard, we would be pleased to arrange for a meeting with the Air Resources Board or Department of Health Services staff to discuss this scientific information. Si 3s 69 ^ /"> - 17 IV. Epidemiology Overall, the draft report's evaluation of the epidemiology data base is consistent with that of B?A'3 Science Advisory Beard (SAB) which stated in'1985 that the data available from the six epidemiology studies on dry cleaning workers exposed to perchloroethylene and other solvents as of the time of the Board's review "do not substantiate the hypothesis that the chemical is carcinogenic to humans" (SAB), 1985). Interpretation of the majority of these studies is complicated by the existence of one or more of the following confounding variables: simultaneous exposures to petroleum distillates and/or other solvents; absence of worker smoking histories; and an absence of distinction between laundry and dry cleaning workers which complicates characterization of types and levels of exposure. Since the time of the Board's review, results have become available from the most complete epidemiology study of the industry conducted to date (Brown and Kaplan, 1987) . In this retrospective cohort mortality study of the dry cleaning industry sponsored by the National Institute for Occupational safety and Health (NIOSH), the authors examined the vital status of 1,690 workers employed for at least one year prior to 1960 at shops where perchloroethylene was the primary solvent. The vital status of cohort members was determined as of December 31, 1932. (f> V/ ^ *1 SL 038690 - 18 The auth rs found no increased risk of cancer in a subcoh rt of 615 dry cleaning workers exposed only to perchiorcethvler.e. In the cohort of workers exposed to other solvents as well, the overall cancer mortality rate was higher than, but not significantly different from, that predicted using V.3. mortality rates. The relative number of cancer deaths among the workers studied was reduced when compared to the higher cancer mortality rates in metropolitan areas investigated. No deaths due to liver cancer were observed. Among the site-specific cancer mortalities, urinary tract cancer (particularly the bladder) was the only one found to have a significant increase. Within the subcohort of workers exposed only to perchloroethylene, the incidence of mortality from urinary tract cancer (and from cancer in general) was lower than that expected based upon overall U.S. mortality rates, in sum, this study shows nn increased risk of cancer in dry cleaning workers exposed only to perchloroethylene. Genotoxicitv The draft report mentions (page 3-22) in vitro studies that provide mixed results as to the genotoxicity of synthesized tetrachloroethylene oxide, an epoxide of perchloroethylene. Such results must be interpreted cautiously. Tetrachloroethylene oxide was administered directly to cells in culture at excessive (non-physiological) concentrations that probably overwhelmed the capacity of the cells to detoxify the purported metabolite. Si 369j Cr* t, \_ \j.~\r 19 Thus, the reported positive results were generated under artificial physiological conditions vith respect to evaluating the likelihood of activity of tetrachloroethylene epoxide. The extensive genotoxicity data base fcr perrhlcrcethy1 a'must supercede these in vitro results. Perchloroethylene has consistently shown negative activity in a wide variety of generic toxicity tests under normal physiologic conditions of metabolite formation. Thus, perchloroethylene is considered to be nongenotoxic by NTP (1986a). Moreover, the Halogenated Organics Subcommittee of the EPA Science Advisory Board specifically addressed the tetrachloroethylene epoxide question (1987): The Subcommittee disagrees_with the statement in the draft Addendum that perchloroethylene is oenotoxic bv implications because a metabolite of perchloroethylene is genotoxic. Tetrachloroethylene oxide, the metabolite in question is not a demonstrated metabolite of perchloroethylene but a postulated metabolite, although the assumed pathway is reasonable. The hypothetical conversion of perchloroethylene to tetrachloroethylene oxide does not Cr~ r - r*. -- w < ' - ---* SL 038692 appear to account for the carcinogenic properties f perchloroethylene, because perchloroethylene is not mutagenic and because tetrachloroethylene oxide is apparently not carcinogenic. VI. Weight of__the Evidence for Carcinogenicity It would be scientifically inappropriate not to take all available scientific evidence into consideration in assessing the carcinogenic potential of perchloroethylene. The Executive Summary of the draft report states that EPA is likely to classify perchloroethylene in category B2, but it is our understanding from recent discussions with EPA staff that the Agency has not reached a final decision as to how to classify the chemical. The conclusion in the 1986 draft EPA Addendum to the Health Assessment Document for Perchloroethylene was not consistent with the Science Advisory Board's review of that document, or with its most recent statements (1988) that the weight of the evidence for perchloroethylene "lies on the continuum between the categories B2 and C of EPA's risk assessment guidelines." The Board's conclusion should be reflected in the Executive Summary of the draft report and throughout the draft report wherever EPA's classification of perchloroethylene is mentioned. SL 038693 r* ^ U ^ ` .J 21 In its most recent review of perchloroethylene, IARC has concluded that there is not sufficient evidence to warrant a determination that perchloroethylene is probably carcinogenic to humans. Applying the same criteria as IARC results in a determination that perchloroethylene is doss ib Iv carcinogenic to humans; it does not justify hazard identification or regulatory action premised on a determination that it is more likely than not that perchloroethylene poses a human cancer risk. Indeed, the change in terminology describing IARC Group 2B was made in part because of the "misuse, or ... exaggeration ... in the use and interpretation of experimental animal results" (Tomatis, 1987). There are other significant reasons why perchloroethylene should not be identified as posing a cancer hazard to humans. The Guidelines recognize (A-14) that when there is conflicting evidence in several animal bioassays, the positive and negative results should be weighted by the adequacy of the study design, the appropriateness of the species tested, the pharmacokinetics of the species, and the statistical power of the test. The Guidelines further state (A-15) that final conclusions concerning the carcinogenicity of a chemical should be drawn from evaluation of the total body of relevant evidence. Because the nature, extent, and the quality of data concerning carcinogenicity vary widely among different compounds, the evidence of carcinogenicity also varies among them. The final evaluation of a specific chemical must, under the Guidelines, contain an assessment of the V ^ v# 4 SL 03869* 22 strength of the evidence as to its carcinogenicity and should also contain a description of the uncertainties underlying the assessment. The available data on metabolic, r.ecr.anist ic, and genetic factors, summarized above, has been regarded as highly significant by EPA's Science Advisory Board and others in the scientific community. The Air Resources Board and the Department of Health Services must make a full and fair evaluation of all these data, as part of an overall weight-of-evidence determination as to the potential carcinogenic hazard of perchloroethylene. VII. Available Pharmacokinetic Information The risk estimates presented in the draft report range from 5 to 21 x 10~6 for lifetime exposure to 1 ug/m3 of perchloroethylene. In comparison, EPA has developed unit risk estimates ranging from 2.9 to 9.5 x 10~7 (1986 draft Addendum to the Health Assessment Document for Perchloroethylene). There is, in our view, no good scientific basis for the presentation of 20-fold higher risk estimates, based on default assumptions, than those being considered by EPA. The draft report should incorporate physiologically-based pharmacokinetic (PB-PK) information in order to develop a more plausible estimate of potential risk. At a public hearing of the SL 038695 *"* * s iy t ** * 1 23 California Air Resources Board concerning the propos d identification of methylene chloride as a toxic air contaminant, Chairwoman Jananne Sharpless stated that such an estimate would 'help this Board try to interpret the information on how we go about controlling it" ;(T-ly 13, 1989);. Sharplass also alluded to the upcoming reviews of other chlorinated solvents (i.e., perchloroethylene and others), and expressed her desire on behalf of the Board that a "most plausible" risk estimate be developed. HSIA urges the development of such an estimate by reflecting pharmacokinetic information. Use of body-surface area correction factors are not appropriate in the case of perchloroethylene. Surface area scaling assumes that humans are more sensitive than rodents, despite the fact that carcinogenic responses in rodents after exposure to perchloroethylene are unlikely to be observed in humans. Body weight provides a better basis for dose adjustment. In a previous draft assessment of perchloroethylene, the Department of Health Services recognized that "[t]here is extensive information available on the metabolism and uptake ;of perchloroethylene] in mammals," and the "[t]he product(s) of this metabolism, rather than PCE molecule per se ... are thought to be responsible ... for the carcinogenicity in laboratory animals." The draft report mentions some of the older pharmacokinetic r *-- WUv\ >v 24 information, but does not address several more r cently published works applying a physiologically-based phamarcokir.etic (PB-PK) model. While section 5 contains several calculated dose adjustments, the draft report concludes (page 18) that "it appears premature to use the metabolized icse in current estimations of human risk." The failure to incorporate available published information seems inexplicable, in light of the requirement in the Guidelines (A-16) that "[p]harmarcokinetic data on metabolism of dosed substances, effective dose at target site, or species differences between laboratory test animals and humans shall be considered in dose-response assessments when they are available." If the report continues to provide an estimate of potential cancer risk associated with exposure to perchloroethylene in the environment, all available scientific data should be incorporated into the estimate. Several scientific articles published in the last two years apply PB-PK models to perchloroethylene. These include Ward, et al-, Pharmacokinetics of Tetrachloethylene, Tox. App. Pharmacol. 93: 108-117 (1988); Travis al., A Physiologically Based Pharmacokinetic Approach for Assessing the Cancer Risk of Tetrachloroethylene, in The Risk Assessment of Environment and Human Health Hazards: A Textbook of Case Studies (Paustenbach, ed), J. Wiley & Sons (1989); and Chen and Blancato, Role of Pharmacokinetic Modeling in Risk Assessment, Perchloroethylene as SL 038697 Cr> o a ^ L \J J 25 an Example, in National Research council, Pharamacokinetics and Risk Assessment, Drinking Water and Health, Vol. 8, National Academy Press (1987). Copies of these articles are attached. We urge that the final report make use of the available pharmacokinetic information. VIII. Flaws in Estimates in Draft Report The range of unit risk estimates presented in the draft report is entirely inconsistent with past human experience. In order to test the predictive value of the risk estimates in the draft report, we have calculated below the risk to individuals occupationally exposed, assuming exposures to have been at or below 200 parts per million (the AGGIH TLV for 1948 to 1981 -- the relevant latency period). Using the upper end of the range presented in the draft report (144 per million at 1 ppb), the risk at 200 ppm is: 1-exp(-2 00x1000x144x10(-6) x10 10 cubic meters x 5 days x 49 weeks x 30 years) -------------------------------------------------------------------------------------------- =0.58 20 cubic meters x 7 days x 52 weeks x 70 years) SL 038698 f> ^ ^ 26 CORRECTED PAGE This calculation shows, in other words, a potential risk of 980,000 in 1,000,000. Needless to say, this is a very large and very detectable potential risk. The example of a 30-vear exposure at the TLV may be unrealistic, however. Thus, the table presented at the end of our comments shows similar calculations for different fractions of the TLV and for the lower and upper bound of the risk estimates in the draft report. It can be seen from this table that even the lower bound on the risk estimate range shows extreme (e.g., 7,000 in 1,000,000) potential lifetime risks. Assuming that the exposure scenarios are realistic, either the calculated potential risk is real, does exist, and has gone undetected, or the calculated unit risk estimates are wrong. Past manufacturing plant experience would indicate that particularily the low end of the ppm-year scenarios are very consistent with past practices and may even be under estimates. For example, drycleaning workers, who have a geometric mean exposure of 22 ppm with a skewed distribution, may have an average exposure generally exceeding 25 ppm. Yet the dryclearning industry has been the subject of numerous epidemiology studies, none of which have detected a risk of the magnitude shown in the table. This is true as well for other industries that use perchlorethylene. Thus, we are left with the conclusion that the unit risks estimates in the draft report vastly overestimate the potential risk to humans. Si <>38699 27 IX. Other Comments The statement in the Executive Summary char cercniorcethylene is used for coffee decaffeination is incorrect. We are not aware that perchloroethylene has ever been used for coffee decaffeination; it is not being used for that purpose currently. The Executive Summary states that the OSHA PEL for perchloroethylene is 50 ppm for an 8-hour TWA. The OSHA PEL was recently changed and is now 25 ppm for an 8-hour TWA. The statement on page 1-9 that the EPA range of risks includes the DHS range in the draft report is arithmetically incorrect. The EPA range is 2.9 to 110 x 10"7, whereas the range in the draft report is 50 to 210 x 10"7. The interval 50 to 210 is not included in the interval 2.9 to 110. The statement near the bottom of page 5-2 that metabolized doses lead to higher unit risks than administered doses is mathematically correct, but misleading and irrelevant. Metabolized doses will lead to higher unit risk estimates, but equivalent doses will be smaller, thus risk estimates themselves can be higher or lower, depending upon the metabolism and distribution of the particular compound. SL 038700 r'- *<> 28 The Q* correcti n on the bottom of pag 5-8 should be clarified to differentiate between exposure period and observation period. The correction is used as a policy decision when the observation period of an experiment is less than lifetime, for example in the first butadiene mouse st-dy. if the dosing is for one year, and the animals are observed for an additional year, as in many of Maltoni/s studies, the correction factor is 104/Te raised to the unity power, not the third power. The comment that inter-individual variability is not accounted for in the PB-PK models is correct, but the same comment could be made for other models, including the LMS model, unless the surface area factor is viewed as an individual tissue s nsitivity factor. SL 038701 ^ - :9 TABLE Exposure Sc nario 200 ppm 30 years 150 ppm 30 years 100 ppm 30 years 60 ppm 30 years 100 ppm 20 years 100 ppm 10 years 50 ppm 10 years 25 ppm 20 years UPPER BOUND RISK 144/MILLION per ppb 0.98 0.96 0.87 0.71 0.75 0.50 0.29 0.29 LOWER BOUND RISK per ppb 1 0.59 ; 0.45 i 0.36 0.24 1 0.26 0.14 0.07 0.07 ^ W ** SL 038702 PSPCHIflBQSTHYLEre EEIE&SNCSS Ames, B.N., Morgan, R., Gold, L.S., Ranking Possible Carcinogenic Hazards, Science 236: 271-280 (1987). Andersen, M.E., Tissue Dosimetry in Risk Assessment, or what's the Problem Here Anyway? In: Pharmacokentics in Risk Assessment. Drinking Water and Health. Volume 8, National Academy Press, Washington, D. C. (1987). Brown, D.P., and Kaplan, S.D., Retrospective Cohort Mortality Study of Dry Cleaner Workers Using Perchloroethylene, J. Occup. Med. 29: 535-541 (1987). Butler, W.H., and Newberne, P.M. (Eds.), Mouse Hepatic Neoplasia. Elsevier, Asterdam (1975). Chen, C.W., and Blancato, J.N., Role of Pharmacokinetic Modeling in Risk Assessment: Perchloroethylene as an Example, In: Pharmacokinetics, in. Risk Assessment. Drinking Water and Health. Volume 8, National Academy Press, Washington, 0. c. (1987). Dekant, W., Metzler, M., and Henschler, D., Identification of S-l, 2,2-Trichlorovinyl-N-acetylcysteine as a Urinary Metabolite of Tetrachloroethylene: Bioactivation through Conjugations as a Possible Explanation of its Nephrocarcinogenicity, J. Biochem. Toxicol, l: 57-72 (1986). Elcombe, C.R., Species Differences in Carcinogenicity and Peroxisome Proliferation Due to Trichloroethylene: A Biochemical Human Hazard Assessment, Arch. Toxicol. (Suppl.) 8: 6-17 (1985). Fox, T.R., Schumann, A.M., and Watanabe, P.G., Activation of a Cellular Proto-Oncogene in Spontaneous Liver Tumor Tissue of the B6C3F1 Mouse, Arch. Toxicol. 10: 217-227 (1987) . SL 038703 2 Fox, T.R., and Watanbe, P.G., Detection f a Cellular Oncogene in Spontaneous Liver Tumors in B6C3F1 Mice, science 228: 596-597 (1985). Goldsworthy, T.L, Lyght, 0., Burnett, V.L., and Popp, j.a., Potential Role of Alpha-2u-Globulin, Protein Droplet Accumulation, and cell Replication in the Renal Carcinogenicity of Rats Exposed to Trichloroethylene, Perchloroethylene, and Pentachlcrcethane, Toxicol. Ate*. Pharmacol. 96: 367-379 (1988). Goldsworthy, T.L., and Popp, J.A., Chlorinated HydrocarbonInduced Peroxisomal Enzyme Activity in Relation to Species and Organ Carcinogenicity, Toxicol. Appl. Pharmacol. 88: 225-233 (1987). Goldsworthy, T.L., Smith-OLiver, P., Loury, D.J., Popp, J.A., Butterworth, B., Assessment of Chlorinated HydrocarbonInduced Genotoxicity and Cell Replication in Rat Kidney cells, Env. Mol. Muta. 11 (Suppl. 11): 39 (1988). and Grasso, P., and crampton, R.F., The Value of the Mouse in Carcinogenicity Testing, Food Cosmet. Toxicol. 10: 418-426 (1972). Hazard Evaluation Section, Proposed Maximum Contaminant Level for Tetrachloroethylene in Drinking Water (R-75-87), at 17-23. Henschler, D., Elsasser, H., Romer, W., and Eden, E., Carcincogenicity Study of Trichloroethylene, With and without Epoxide Stablizers, in Mice, J. cancer Res. Clin. Oncol. 107: 149-156 (1984). Herren-Freund, S.L., Pereira, M.A., Olsen, G., and DeAngelo, A.B., The Carcinogenicity of Trichloroethylene and Its Metabolites, Trichloracetic Acid and Dichloroacetic Acid, in Mouse Liver, Proc. Amer. Assoc. Cancer Res. 27: 91 (1986). International Agency for Research on Cancer (IARC), Monographs, Evaluation of the Carcinogenic Risk to Humans, Supplement 7, Lyon (1987). International Expert Advisory Committee to the Nutrition Foundation, The Relevance of Mouse Liver Hepatoma to Human Carcinogenic Risk (9183). Moloney, W.C., Boschett, A.E., and King, V.P., Spontaneous Leukemia in Fischer Rats, Cancer Res. 30: 41-43 (1970). Moolgavkar, S.H., and Knudson, A.G., Mutation and Cancer: A Model for Human Carcinogenesis, J. Natl. Cancer Inst. 66: 1037-1052 (1981). v> SI* 038704 National Cancer Institute (NCI), Bioassay of Tetrachlor thylene for Posaibl Carcinogenesis, U.S. Departs nt of Health, Education, and Welfare, Bethesda, MD, NIH Publication No. 77-813 (1977). National Research Council (NRC), Safe Drinking Water Committee, Drinking Water and Health. Vol. 3, National Academy Press, (1980). National Toxicology Program (NTP), Technical Report on the Toxicology and Carcinogenicity Studies of Tetrachloroethylene in F344/N Rats and B6C3F1 Mice (Inhalation Studies), NTP TR 311, NIH Publication No. 85-2567 (1986a). National Toxicology Program (NTP), Technical Report on the Toxicology and Carcinogenesis Studies of Dichloromethane (Methylene Chloride), NTP TR 306 (1986b). Odum, J., Green, T., Foster, J.R., and Hext, P.M., The Role of Trichloroacetic Acid and Peroxisome Proliferation in the Differences in Carcinogenicity of Perchloroethylene in th Mouse and Rat, Toxicol. Appl. Pharmacol. 92: 103-112 (1988). Rampy, L.W., Quast, J.F., Leong, and Gehring, P.J., Results of Long-Term Inhalation Studies on Rats of l,1,1-Trichloroethane and Perchloroethylene Formulation, In: PEP.ceedings .of the First International Congress on TPXidPloqy, Plaa, G.L., Duncan, W.A.M. (Eds.), Academic Press, New York (1978). Schach von wittenau, M., and Estes, P.C., The Redundancy of the Mouse Carcinogenicity Bioassay, Fund. Appl. Toxicol. 3: 631-639 (1983). Science Advisory Board (SAB), U.S. Environmental Protection Agency, Letter and Report to William D. Ruckelshaus, EPA Administrator (January 4, 1985). Science Advisory Board (SAB), U.S. Environmental Protection Agency, Letter and Report to Lee M. Thomas, EPA Administrator (January 27, 1987). Science Advisory Board (SAB), U.S. Environmental Protection Agency, Letter and Report to Lee M. Thomas, EPA Administrator, SAB-EHC-88-011 (March 9, 1988). Stott, W., Chemically Induced Proliferation of Peroxisomes: Implications for Risk Assessment, Reg. Toxicol. Pharmacol. 8: 125-159 (1988), Tomatis, L., Remarks at the Annual Winter Toxicology Forum (February 1987). SL 038705 HtaS U ^ - J 'm. 4 Tomatis, L., Partensky, C., and Montesano, R., The Predictive Value of Mouse Liver Tumor Induction in Carcingenicity Testing -- A Literature Survey, Int. J. Cancer 12: 1-20 (1973). Travis, C.C., White, R.K., and Arms, A.D., A Physiologically Based Pharmacokentic Approach for Assessing the Cancer Risk of Tetrachloroethylene, In: The Risk Assessment of Environmental and Human Health Hazards: A Textboox of Case Studies. Paustenbach, D.J. (Ed.)/ J* Wiley & Sons, New York (1989). Ward, R.C., Travis, C.C., Hetrick, D.M., Andersen, M.E., and Gargas, M.L. , Pharmocokinetics of Tetrachloroethylene, Toxicol. Appl. Pharmacol. 93: 108-117 (1988). f* v y w, r*\ SL 038706 January 27, 1987 Honorable Use M. Theras Administrator U. S. F-.nvironmentai Protection Agency 401 M Street, s. w. Washington, D. C. 20460 SAB-2S:-87-0ia Dear Mr. Thcras: The Science Advisory Board's Environmental Health Committee has completed its review off a draft Addendum to the Health Assessment Document for Percnloroethyiene. The Ccmittee previously reviewed the draft Health Assessment Document on May 9-10, 1984. An Addendum is desirable because of newly available data, primarily an inhalation bioassav of rodents by the National Toxicology Program. The Comittee has conducted its review primarily througn the Halogenatod Organics Subctroittee, wnese report is attached. The Subcorrutcee believes it is reasonable to describe the weight of the epidemiological evieence in humans as conforming to the EPA guidelin for carcinogen ris* assessment definition of "inadequate." The SuDcarnittee concludes that the animal evidence of carcinogenicis "limited" because of positive results m only one strain of mouse of ' type of tumor that is cerron and difficult to interpret. Thus, the S--ccrciitt.ee concludes that perchloroethyiene belongs in the overall weigr -.-of-the--evidence category C (possible human carcinogen).' Given the current evidence, the Subccmittee hynothesizes that, operationally, perchloroethylene may be an indirect acting carcinogen or carcinogenic promoter of la* potency. By pranoter, the Subccranitt-^e means that perchloroethylene alone does not induce tumors. Instead, perchioroetnyle appears to act in concert with other substances, endogenous processes, viruses oncogenes, or radiation, whicn can initiate cancer in the absence of promoters ve appreciate the opportunity to current on this important public health issue ana request that EPA formally respond to our report. Sincerely. Norton Nelson Chair, Executive Ccmittee Richard A. Griesemer Chair, Enviromental Health Ccmittee SL 038707 X 'J N \ T z 3 Octcnvc 26, 1936 Dr. Richard A. Griesemer Chair, Environmental Healt.n CsraittaScience Advisor/ Board U.S. Environmental Protection Agency 401 M Street. SM Wasningtcn, DC 20460 Dear Dr. Griesemer: The Halogenated Organics Suhcopmtr.ee of the Environmental Health Conr'.ittee has corpleteo its review of a draft Addendum to the Health Assessment Document for Tetrachloroethylerw (Permloroethylene: Updated Carcinogenicity Assessment: EPA-600/S-82/005FA: March, 1986). The Environ mental Health Ccnr.ittee previously reviewed the draft Health Assessment Document for Perchloroethyiene on May 9-10, 1934, and transmitted a report on this craft to the Agency on January 4. 1985. The craft Addendum is based on a National Toxicology Program inhalation bioassay of perchloroetnylene in rodents. The S . conmttee finds that the bioassay is of reasonably good quality, and that ..setul results for ris* assessment can be obtained front it. The Subcoirr.ttee disagrees with the Agency's interpretation of the data that increase in either renal tubular cell neoplasia or mononuclear call leuhetnias in -334 rats were associated with perchloroethylene exposure. The Subcrrruttee agrees with the conclusion in the document that perchloroethylene inhalation is associated with a significant increase m the frequency of liver carcinana in B6C3FI mice. This result provides experimental verification of an assumed extrapolation between routes of administration frcn a gavage study, as described in the Health Assessment Document. The Suoocmittie believes it Is reasonable to describe the weight of the epidemiological evidence in humans as conforming to the EPA guideline for carcinogen risx assessment definition of "inadequate." The Subccrmittee concludes that the animal evidence of carcinogenicity is "limited- because of positive results in only one strain of mouse of a type of tumor that is cciairon and difficult to interpret. Thus, the Subccmittee concludes that perchloroethylene belongs in the overall v*eight-cf-the-vidence category C (passible human carcinogen). /N ^ -4 U m w < <e In the opinion of seme members of the Subccnnittee, a quantitative assessment of perchloroathylene is desirable, and the mouse oata are adequate for this purpose. Treatment of this assessment as a "what-if" calculation, as presented in the original Health Assessment Document, is desirable. Such a quantitative assessment probably will srew that an increase in cancer would not be detected in the groups most exposed to percnloroethylene at currant exposure levels. This inference deserves mention in cne executive sumary. The analysis of phacaacoxi.net:cs in tr.e draft Addendum is commendable ana will support further estimates of the possibility of cancer in exposed populations. The Subcommittee requests that the Environmental Health Ccraittee address the question of whether one-tailed or two-tailed statistical tests of significance are appropriate for the routine analysis of bioassay data. Agency staff reported at the meeting that one-tailed tests are routinely usee. This use assumes that chemical substances can only increase the frequency of cancer, but this assumption seems contrary to ersirioal observations. Resolution cf this issue will influence the conclusions regarding perchloroethyiene. In addition, the Subcommittee requests that the Environmental Health Committee arrange foe a detailed review of th physiolcgical-pnamacokinetic model used in the analysis of percr.loroethylene. The Subcomittee reviewed sene results of the rodel but not the model per se. EPA is likely to use the model in the future to assess the risks of other suostances. In support of the review of perchloroethyler.-, the Subccmittee requests that the Agency provice the members with analyses ~>f (1) human carcinogens and their effects in the rat ana mouse, (2) hunar '-.epatotoxins and their effects in the rat arc mouse, and (3) human renal toxins and their effects in the rat and mouse. The Subcommittee believes that the final Addendum will ennar.ee th value of the Health Assessment Document and that, contingent on the correction of the issues discussed in the attached report, the document will be scientific ally adequate to meet its stated purposes. We appreciate the opportunity to errent on this public health issue and request a Comal response to our advice. Sincerely SL 038709 John Oouli,"'^C., Ph.D,. \ Chair, Halogenated Organics Subcommittee r // Seynour Abrahamson, Ph.D. Vice-Chair, Halogenated Organics Subcamitte / v* REPORT OF THE HALOGENATED ORGANICS SUBCOMMITTEE OF THE ENVIROtflENTAL HEALTH COMMITTEE ON A DRAFT ADOENDOM TO THE HEALTH ASSESSMENT DOCUMENT FOR TETRACHLGROETHYLENE (PERCHLOROETKY1ENE) Introduction The Halagenated Organics Subcartittee of the Environmental Health Ccrr.ittee of EPA's Science Advisory Board net on May IS. 1986 in Madison, Wisconsin, tc review a craft Addendum to the Health Assessment Document for Tetrachloroethyler.e (Per chlore*thyiene; Updated Carcinogenicity Assessment; EPA-60Q/8-32/005FA; March, 1986). The Environmental Health Committee previously reviewed the draft Health - Assessment Document for perchloroethylene on May 9-10, 1984. A report on this draft was sent to the Agency on January 4, 1985. The draft Addendum primarily analyzes the results of a 1985 inhalational bioassay by the National Toxicology Program performed at Battelle Pacific Northwest Laboratories. The Subcamitze tharws the National Toxicology Program for sending a representative, Dr. Jonn Mmnesr, to contribute to the discussion at the May 15th meeting. The Subcornu tree concludes that the Addendum improves the scientific foundation of the existing Health Assessment Document and further improves the Agency's ability to perform a risx assessment for this empouna. The Agency intends for tne Health Assessment Document to serve as a multimedia source document. The Subcsmittee believes that, contingent on the correction of the issues discussed belew, tne draft Addendum will also be scientifically adequate for this purpose. Quality Assurance of the Bioassav The Subcamitr.ee reviewed the available quality assurv ze information of the bioassay. It concludes that these audits were properly carried out and generally are consistent with each other. The quality assurance pror.-ss identified discrepancies during the collection of in-life toxicology data, verification of analytical chenistry results, cage injuries, brief overexposure of the high dose group of rats, auto lysis of seme specimens, and the failure to section all lesions. Sane animal escapes and confusion over animal identification -were reported. How ever. there is little possibility that animals raved from control to test cages, or between test groups, because the animal housing in the Battelle inhalation chamber is well controlled, and the chambers are in rooms within a barrier facility. The Suoccmittee also ooes not believe that resorting a few animals would lixely create a positive result as an artifact with a rare tumor. The randan movement of a few animals between cages is more likely to obscure the detection of a statistically positive finding of an infrequent lesion. These problems are fairly cannon in bioassay work and do not impede the inter pretation of clear, distinctive findings. The Subcommittee believes that the bioassay provided data that are adequate for risk assessment, unless EFA attempts to interpret small differences between groups of animals. The Subcocmittee reccrrenrts that the Agency summarize and assess the imp Lic tions of the quality assurance information in the final Addendum. Intersretation cf the Carcinogenicity a suits The draft Addendum describes two Lesions of potential interest that occursec m Fisner 334/N rats--renal tubular neoplasia and mononuclear cell leukemias. The Subcommittee concludes that the National Toxicology Program bioassay does not provide a scientific Oasis to associate either lesion with inhalational exposure to perchloroethylene. 3oth findings would result frcn small differences between control and treated groups, and they conflict with other bioassays of percr.loroethylene in the rat and which are problematic in relation to the quality assurance conclusions, fiotn findings have multiple proolems, any one of which overwhelms the interpretation. These pronLems include: a) Percnloroethylere neither appeared to induce an increase in rare renal tubular neoplasia in male rats, nor was the trend in these tumors among tne male rats dose-related. No renal tubular neoplasia 'were observed in fenale rats. The reported numbers of adencmas in male rats were 1/49 (control), 3/49 (200 ppm) and 2/50 (400 ppn). Renal tubular carcinanas occurred in 0/49 (control), 0/49 (200 ppm) and 2/50 (400 ppn) of the male rats. 'When results of both tumors are reported, the nuroers of animals affected were 1/49 (control), 3/49 (200 ppn) and 4/50 (400 ppm). To attribute statistical significance to the findings in male rats, the analysis aggregated adencmas and carcincnas. However, tne analysis of numcers of animals with adenomas or carcinanas as a group is not an obvious biological procedure. The pathology cf these tiroes is not well-understood, and little background information is available in the literature. The dia-visis of renal tubular neoplasia in the rat is not a clearly understood pro:--lure among experts. Whether or not conversion frcn adenena to carcinoma ':curs is not known, and tne draft Addendum does not review this subject. In addition, the statistical analyses supporting conclusions m the text are in error. The Fisher exact test has been miscalculated, and trend has not been analyzed. The Subccmittee recommends that the Agency develop better descriptions cf (1) tne patholojy of the renal tubular neoplasia in rats (including speculations about progression or conversion), and (2) the rationale for aggregating the numbers of animals with adenomas or carcincnas. At a minimum, it should assess each data set independently before evaluating tne aggregated data, and the results of statistical tests for any trends. For the benefit of potential non-expert readers, the final Addendum needs to clarify that the enumeration of rats with carcinoma or adenana is subject to debate. To analyze the renal tubular neoplasia results, the Agency has to address several competing hypotheses, such as an unusual occurence within: the specific group of F334 rats used in the bioassay, aberant housing conditions, histori cal undemetection of renal tubular neoplasia, induction of tvanors by perehloroetnylene. and so forth. Eight (8) out of 148 (5.4%) male rats had findings of adenoma or carcinoma. Either this frequency or the frequency for all un treated rats, male and female (8/296 ; 2.7%) can be compared to the reported historical frequency of 4/1,720 or 0.23%. (The note in the draft Addendum does not clearly state the basis of the historical observations.) The Addendum also needs to address the credibility of this number in the light of the probably variable search for lesions in the absence of cross observations that suggest a neoplastic response. Historically, renal tubular neoplasia in control rats tend to be under-reported. SL 038711 V ^' V, v, %- <' Questions can oe asked and then ans-ered, aocut possible biological or statis tical reasons Cor the differences in control ana overall incidence. Th Sudconmitcee suggests that the staff calculate the prior livelihood of the frequency of renal tubular neoplasia under different hypotheses aDout the average rate of occurrence# using the Poisson distribution. The staff can corpare these results to each other and to tne biological interpretations of each hypot.-.esis. b) ?ercr.loroetnylene did not appear to induce a marginal increase in mono nuclear cell leukemia in rats. At tne present time, the scientific ujtimnicy has a poor understanding of the pathology of mononuclear cell leukemia. The high frequency in all groups# including controls is not usual for F304 rat*. The results suggest faulty pathological diagnoses or some unusual circumstances in tne rat colony at the turn. The extant of characterizing mononuclear cell leukemias was histopathological examination. Other means of characterization, which are necessary to distinguish neoplasia from leukocytic hyperplasias that may develop in older rats# were not used. The results of the statistical analysis are not convincing. The Addendum presents the mononuclear cell leukemia data in terms of a progressive three stage classification which appears to be preliminary and ad hoc. The staging of diagnoses does not represent a consensus effort of the ccmunity of experienced pathologists. Hcwevcr, the draft Addendum states that the strength of th evidence for carcinogenicity in the F334 rat rests on the resolution of issues regarding tne uncertainty m the assignment of frequency within the stages of mononuclear cell leukemia. No human analogue is known for mononuclear cell leukemia of the rat. This absence is not important for EPA's policies on carcinogenicity# although a lack of correspondence drvs concern sane biologists. However# the aosence of a hunan analogue is important, when staging is considered, since staging refers to the usually more extensive i.-.'irmation on leukemic progression in h^nans. It a two-tailed test is used, the most striking observation in the results occurred at 200 ppm, in which 13 of 50 female rats in the control group were reportedly diagnosed as having mononuclear cell leukemia in one of the three stages versus 30 of 50 of the perchloroethylene treated female rats. This comparison leads to a confidence limit of about p * 0.03 by the Fisher exact test (two-tailed). Regardless of the statistic, 13 versus 30 is not a striking ooservation and, given the generally high frequency of mononuclear cell leukemia diagnoses in ail groups, it is worth inquiring what the chance is of finding such a result if tve of six groups are drawn at randan, each group being subject to the same hign, random frequency of diagnoses (reported as of 300 or about 603). In oral statements at the ineetireg, Agency staff reported that a statistically significant difference between untreated and perchloroethylene treated rats could be observed for mononuclear cell leukemia, if the time-to-tumor was an alyzed. This may be the case, but these oral statements contradict the written statements in the draft Addendum regarding cine-to-tumor. Sane Subcamittee members have attempted to evaluate whether or not diagnosable mononuclear cell leukemia occurred earlier in perchloroethylene exposed rats than in unexposed. r /s. ,* /\ W /h -.A J SL 038712 -4- The presentation of data in th draft Addendum is sucn that trus carparison cannot be made becaus neither the actual data, nor th dependent pronamlities. are presented. (See "statistical analyses," belew.) However, the draft Ad dendum states anectodally at several points that this canparison is not worth naming because of analyses contained in the National Toxicology Program report. The draft .Addendum does not review the analyses, and does not present the supporting data. However, if the oral carments are correct, then either the National Toxicology Program analysis, or the interpretation cf this analysis in the draft Addendum is in error. The Subcamittee suggests that the Agency will experience difficulty in gaining scientific support for tne conclusion t,hat percnlocoethylene exposure is associates with increased frequency of mononuclear cell leukemia m rats, based on the National Toxicology Program bioassay data. Any effort to do so should begin with an explanation of why the frequency of this rumor did not increase aft r perchloroetnylene exposure of rats in the bioassays performed by the National Cancer Institute and by Rampy and co-worxers. The Subcommittee agrees with the statement in the draft Addendum that first generation hybrid mice of C57BI6 and C3H parental orgin (36C3F1) exhibit statistically significant increases in carcinoma of the liver associated with e^osure to per ch loroe thy lene by inhalation. These results confirm the findings of a National Cancer Institute study with the sane strain of mouse and administration of per ch loroethylene by gavage. The Environmental Health Corwittee and its Subccnroittees have consistently urged the Agency to calculate the potency of a carcinogen for ail routes of arministration when data are available fo- only one route (using the best general information aoout uptake, absorption, netabolism, distribution, elimination and mechanism). Once data has existed for >ne route, it has advised E?A to use the empirical evidence as the basis for dec. r.orrmaKing and t forgo the hypothetical calculation. Perchloroethylene provv-.-s an example of experimental validation, both qualitatively and quantitatively, of the hypothetical extrapolation. However, this validation does not change the interpretation on which a decision might be based. No new, aispositive information has been gained. Although the possibility exists that carcinomas arise de nova, the availabl vidence strongly supports.the idea that the adenanas and carcinomas represent a single disease process to which scientists have applied an arbitrary division into two diagnostic terms. Since we usually don't know the rate at which the various lesions progress after exposure to* a given test chemical, and because nistologic evidence alone is not entirely a satisfying indicator of biological behavior, the Subcaimittee recatronds analyzing the lesions both separately and combined. It should be rememoered that many mice with hepatic carcinomas also have adenomas that have not been includec in the smeary tallies. Other Data from the National Toxicology Program Bioassav The Subcommittee requests that Agency staff fully assess all of the information available frext the National Toxicology Program study. The draft Addendum notes the occurrence of squamous cell metaplasia of the nasal cavity in male rats but does not provide statistical analysis of significance or trend with dose. Th draft Addendum refers to a finding of renal tuoular cell hyperplasia in rats, but no data are provided. Renal tubular karvanegaly is noted in rats and mice of both sexes, but no data are provided. 03873.3 Si* t v* V- ` > > The Subcarruttee also recannends that Agency staff thoroughly assess and Interpret the significance of mortality outcomes for rodents chronically exposed to perchloroethylene in the National Toxicology Program bioassay. These data can -- be important in setting standards for drinking water. One interesting possibility is that the kidney also is a target organ. The draft Addendum notes excess mortality in mice at 100 pjw and 200 pjn but suggests that this result is caused by hepatic cancer. .An appropriate statistical analysis of mortality will correct for this effect by correctng for deaths from hepatic cancer (a time-to-not-tumr calculation). Statistical Analysis of the Bioassay Results The display of data and statistical analysis of these data in the draft Addendum needs revision. The Subcommittee found sane critical instances of misquotation anc error. Mule the statistical analyses reported in the Addendum can be reproduced by the Subcommittee, this can only be done if a one-tailed Fisher exact test is used. The use of a one-tailed test is appropriate, if perchloroethylene only can increase the frequency of cancer. This assumption is dubious when the background in the control group is hign, and it is contrary to the general knowledge about the effects of chemicals on tumor frequency in rodents.* Instead, a two-tailed test seems appropriate. The Agency should state whether an analysis is one-tailed or two-tailed in the text. :*etabolisn and Pharmacokinetics The Suncommittee believes that the draft Addendum <nd the final Health Assessment document provide a thoughtful response to t~? comments regarding pharmacokinetics made during the previous review. The :ata in the draft Addendum are adequate for evaluating potential metabolic mechanisms which pertain to possible carcinogenic effects perchloroethylene. Further, the Subcommittee cornnencs the Agency for the discussion of the different mechanistic implications of perchloroethylene metabolites in the induction of cancer. At present, the Subcommittee has only reviewed sane results of the model used by Agency staff to analyse cata for perchloroethylene. Because of the potential importance of suer, models for SPA risk assessments, the Subcommittee recommends that the Environmental Health Committee undertake a review of the general approach. However, the Subcommittee has developed a consensus regarding one issue that was subject to contention during the public meeting. EPA has not double counted the factor for interspecies extrapolation of metabolized dose. Because staff have modeled the absolute amount per unit volune (tissue specific concentration), same extrapolation between species is required. However, the Agency loses sore of the power of the physiological-pharmacokinetic models when this approach is taken. * See, for'exanple, J.K. HASEMAN, "Patterns of Timor Incidence in Two-year Cancer Bioassay Feeding Studies in Fisner 334 Rats," Fundamental and Applied Toxicology 3 (1983), pp. 1-9. r*' SI* 038714 Because of the ir^lication that tetrad loroethylene oxid is a carcmcg rue intemediate, discussion of the reactivity of various haloethylene oxides should he included. Agency staff should search for studies which correlate the cnemicai reactivity, hepatotoxicity and carcinogenicity of haloethylene oxides, such as those by Henschler cr Van Duuren. Most studies have attributed the metabolism of perchloroethylene to a proposed reactive metabolite, tetrachloroethylene oxiae, which is converted by rearrangement to trichloroacetyl chloride. The latter will acylate rather than alkylate macro molecules. The acylation reaction could be followed by spontaneous hydrolysis and regeneration of the free macromolecules. Thus, no genetic effect may be observed. Indeed, Van Duuren and coworkers have concluded from their studies of the carcinogenicity of various halo-substituted ethylene oxides that tetrachlor oethylene oxide is not carcinogenic when administered to rats by any of several routes. The discussion in the addencun suggests that tetrachloroethylene oxide is the only reactive, carcinogenic metabolite formed following perchloroethylene ad ministration. The Subcommittee reccnnends that ocher putative carcinogenic metabolites be described. For example, glutathione conjugation products should also be included. The role of these potential metabolites in eliciting effects, such as renal damage or carcinogenicity, should be discussed. Henschler has suggested glutathione conjugates of various haloethylene expounds as th prcxinal initiators of renal toxicity, particularly after hydrolysis in the kidney renal t'JDule. Several authors have described the covalent binain; of radioactive perchloroetr.ylene to tissues after mstadolic activation. This "nding may be partially due to the formation of acyl derivatives after the formation of trichloroacyl chloride as an intermediate, as suggested by studies ir. which trichloroacetic acid was fexind after acidic hydrolysis of labelled macromolecules. The sig nificance of the acylation reaction in genotoxicity is not clear. However, no covalent binding to deoxyribonucleic acid has been demonstrated, which is in dicative of a protective or hydrolytic mechanism, perhaps accelerating the decomposition of tetrachloroethylene oxide to trichloroacetyl chloride before the oxice can gain access to deoxyribonucleic acia. Trichloroacetyl chlorid can react with macromolecules to form various tricnloroacetic acid esters which may unoergo rapid enzymatic hydrolysis to yield trichloroacetic acid and re generate the macromolecules. This hypothesis merits investigation. Genotoxicity The Subcatmittee disagrees with the statenent in the draft Addendum that perchloroetnylene is genotoxic by implication because a metabolite of perchloro ethylene is genotoxic. Tetrachloroethylene oxide, the metabolite in question, is not a demonstrated netabolite of perchloroethylene but a postulated metabolite, although the assumed pathway is reasonable. The hypothetical conversion of perchloroethylene to tetrachloroethylene oxide does not appear to account for the carcinogenic properties of perchloroethylene, because -perchloroethylene is not mutagenic and because tetrachloroethylene oxide is apparently not care mag nic. Percnloroethylene has been tested in many mutagenicity bioassays, a few of which show positive activity, but on balance the weight-of-the-evidence is borderlin and not conclusive. 3S7i5 f~\ C / ' % .. i Mechanist Given the current evidence, che Subcanmittee hypothesizes that, operational!;^^ percr.loroethylen may be an indirect acting carcinogen or carcinogenic promoter (fl of lew potency. By promoter, the Subcaimi ttee means that perchloroetnylene alone ^ does not induce tumors. Instead, perchloroethylene appears to act m concert with other substances, endogenous processes, viruses, oncogenes, or radiation, which can initiate cancer m the aDsence of pranoters. Initiators are usually thought to he genotoxic substances, binding to deoxyribonucleic acid in order to cause initiating events. When perchloroethylene is present, however, tumors are observed when they would not otherwise be, even when the initiator is not Known. Although definitive evicence is lacxing, perchloroethylene appears to act at a later stage in the carcinogenic process. The evidence which leads to the Subcomtattee's hypothesis that perchloro etnylene may act as an indirect acting carcinogen or a promoter is that perchloro- thylene: (1) probably is not rutajenic; (2) does not bind to deoxyribonucleic acid; (3) increases the frequency of liver carcinanas in B6C3F1 mice when thes tumors are commonly seen in the same strain not exposed to perchloroethylene; (4) induces liver carcincna in a species and strain specific manner; (5) induces peroxisanes in the livers of 36C3F1 mice, which provides an alternative mechanism; and (6) acts consistently in ccnparative studies of halo-substituted ethylenes which indicate that asymmetrically substituted canpounds generally are carcinogenic, whereas symmetrically susstitutec generally are not. Spidemolocy The Environmental Health Comitr.ee has previously reviewed the epidemiological evidence as it was discussea in the Health Assessment "^oument. The Subccrmtt e finds no reason to alter the Ccrmttee's previous findi-MjS at this time. The M National Cancer Institute may publish a new epidemiology study of perchloroethyleneTM The Subcommittee recannends that the Agency evaluate tr.ase results in the Add ndun, if they are available in a timely and satisfactory for-. Meicht-of-the-evidence Category Based on the National Toxicology Program bioassay results and the Agency's guidelines for carcinogen risx assessment, the Subcommittee concludes that "limited" evidence exists for the carcinogenicity of perchloroethylene in animals because the evidence arises only fren a single strain of mouse and because the Kind of tumor associated with perchloroethylene exposure in this mouse strain maxes it difficult to create an inference regarding human carcinogenicity, the epidemiological evidence is described in the Health Assessment Document as "in adequate." WorKing frem EPA's proposed guidelines, the Subccmiuttee concludes tnat the overall weight-of-the-evidence category is C ("possible human carcinogen'*). The Subcanmittee has carefully considered and rejected the position of sane staff that positive evidence of liver carcinoma in the B6C3F1 mouse associated with xpesure to perchloroethylene by two different routes of administration should change the weight-of-tne-evidence category to B ("probable human carcinogen"). See A. BLAIR, P. TOLBERT, T. THOMAS and D. GRAUMAN, (Abstract) Mortality among Dry Cleaners. Fourth International Symposium on Epidemiology in Occupational Health (September 10-12, 1985). SL 038716 p /h Retrospective Cohort Mortality Study of Dry Cleaner Workers Using Perchloroethylene David P. Brown, MPH. and Samuel D. Kaplan, MD To tverjsie the carcmogtaic potential from occupeoaoel Unitod Stotoo are potentially exposed to PCE.1 The exposure to perchloroethylege (PCE). s rvcraspectirv cohort poootbility thot PCE could pooo a significant occupa mortality icuay at' worsen employed in the dry ciesmnt industry woo conducted among J.SSO mrnn from bar tabor irtmnt The majority at' the cohort had potoaual txpaaure to potroteum solvents aa well no to PCE while woraag ta the dry clnaninr industry. Mortality Cram primary cmacer of the liver wme of particular internet, duo to the /ladings of vxcem Ueer eaaeor ta mice exposed to PCS. Other wtteo efoaacer were alee of interest. tional hoslth risk to thooo workers was rauod after a National Canoor Institute (NCT) bioessay indicated that PCE induced Uver rumors in exposed mice.* In edditioo. tome tubular nephropathy vu observed in the treated mice. la an unpublished study conducted by The Dow Chem ical Company is 1977. there was so evidence of a eu- A total of 493 deaths were adeemed, whereas 37X3 were moropamc reepoaae ia rats reposed by inhalation to expected hosed oa OS mortality rosea. Mortality Orem all cancers oomotaed woe greater than expected fl4B theemtii r 130-9 expected). Ho deaths due to Uver aameer were eheervad. Urinary erect cancer wee the only spemOe ate where there woe a statistically sigmOeant enoeee ta utosi-rto deaths (13 observed r 4.7 expected). There was same ooasisttacy in these lladings across the four individual unions and across race)sex groups. A suheohort at worSers whs were employed eaty in dry cleaning maps that seed PCE as their primary solvent PCE. There was increased mortality among the rate in tha high-doae gmup. Tho National Toxicology Procram (NTP)* recently completed a study where F344/N ram and BGC3F. mice were exposed to PCE by inhalation PCE produced renal tubular cell karyomecaly. which is an abnormal enlargoment of the cell nucleus, and renal tubular call hyper plasia in rata It also increased the incidence of renal was ideaaflsd from the union records. There wee only one tubular cell srieoomas or adenocaronomaa in male rats. death from urinary tract mincer, whereas deaths were Both iow and bich doses of PCE were associated with an expected ia this suheohort. increased incidence of mononuclear ceil leukemia in male rata and in female rata the lew doae increased the Perehloreethyleae (PCS'). also known as tetrschloroetbylene. is a solvent used commonly (tor el--nine insideace of leukemia. In mice there was a doswreiated increase in the incidence of hepateceUular neoplasms. fisbric (dry cleaning) end tar degreasing metals. It has PCE also produced renal tubular cell karyomecaly in boos estimated that at loose 1.6 million workers is tho fr to* tatowtfywiO* iMlo Inaa. 0M> vf Tn i !! i Sum tnluuu eaS run St--1m. Ms M--I lasatete be Otoe* Otnnnoi Safety u4 HML <v----OB (Or Brews. Muon Chief ): u4 theStutor* Soon* lesUtute.------------ 1 (Or Kreleo; to to eurteutlj tmeieyoa ty tto CaUtorwa OmutMi vf Bseitn Sarwioaai. Due to the potential careinoceaio effect of PCE as demonstrated by the animal stadias and due to the widespread use of PCE in the workpiece, aa epidenuolocie retrospective cohort mortality stody was con ducted to examine the effects on sxpossd workers, par ticularly tha risk of mortality from cancer. After evaiuasinc tha numerous occupational cronps potentially exposed to PCE. tho dry cleaning industry was chosen for tho epidomiolocie ready, because PCE bad been used aa a cleaning solvent ia ** industry for at least 30 years. PCE was introduced into the dry cleaning industry in the late 1930b but did not replace ether synthetic sol* Joumai of Occupational Medicine/Volume 29 No. 6/June 1987 38?i7 O r--' " > e., \e / . t 535 vasu juca as carbon tetrachloride until shortly tittr world War Q. Sunny this seme period petroleum deriv ative* (primarily venous ijpw of Stoddard solvents) wore the predominant solvent* uaod in dry cleaning. A gradual shift from petroleum derivatives to PCE began in the late 1940a. This shift la solvents increased in the 1950s and early 1960a. However, in the period bofore 1960. petroleum denvattves were still the dominant solvents. By 1977. the indusay estimated that approx* imately 74% of commercial dry cleaning shope used PCS. about 24% uaod petroleum solvents, and the re mainder used fluorocarbons (personal communication from William Fisher. International Fabncarv. 1985). An exposure evuluatiee survey, which included a random sample of tho famlitiee (still in business) in the epidemiologic study, was conducted from 1977 to 1979.* Thors were 44 commercial dry cleaning shope included in the survey. Tlme*weighted average (TWA) and peak exposures to PCE were determined by collecting per sonal air samploo. Other solvents used Car spot removal were also sampled. The operator or dry cleaner had exposures significantly higher than the other workers (geometric mean TWA of 22 ppm v approximately 3.0 ppm. respectively). Based on historical exposure data some of which date back to 1956. the levels of exposure to PCS in commercial dry cleaning shops have remained fairly constant sines its introduction into the industry. Tho survey also revealed that there was consistency in the level of expoeurse by geographic location. Tho only substance demoted m the air samples during tho survey was PCE. Therefore, eves though other solvents wore used for spot removal, their airborne concentrations were nondetectahie. Methods The study cohort was defined to include workers exposed to PCE for a """" of 1 year prior to 1960. and with so riiown previous occupational exposure (la the dry cleaning industry) to carbon tetrachloride or trichloroethylene. Records maintained by four local unions were used to identify dry eieaaer workers who mot tho definition of tho study cohort. Tho majority, if not ail. of the dry cleaning shops wore commeraal as opposed to industrial cleaners. Workers were chosen only whsn thers was doeuasotatloa that they were employed for at least 1 year prior u 1960 at a shop where PCE was tho pnmary solvent. Soma and/or a complete solvent history was available for approximately half of the shope employing union members. If no solvent history was available for a particular shop, employment in that shop was not considered in determining tho eligibility of union mem* bers. For each eligible worker n history of employment in PCE as well as non-PCE dry eieaaer shope was coded. If solvent history was untiMwo prior to 1960, it was assumed to bo non-PCE inssmilf h ns most shops used petroleum solvents prior to this date. In an attempt to restrict an analysis to a cohort of workers primarily exposed to PCS with no confounding expoeure to petroleum solvents, a sutococort of wferxen who were known to bo employed only m shape daerv PCE was the primary solvent was identified. The vital status of cohort members was deter as of Dec 31. 1962. For those idecofled as dec^l copies of thetr death certificates were obtained and tbv underlying cause of death was coded by a trainee aosoi* ogist according to the Revision of tho International Classification of Diseases (ICD) in effect at the time of death. Those lost to follow-up (unknown vital status) and those who died subsequent to the closing date of the study, ie. Dec 31. 1988. were considered alive for pur poses of analysis. Person-years at nak (PYAR) were calculated for eacn worker starting after 1 year of employment in a PCE shop(s) and ending nt the date of death or the closing date of the study, whichever occurred first. Using a life- table analysts system.* the PYAR for each worker were combined into 5-year (miaudar time periods and 5-year age groups* PYAR wore additionally distributed by length of employment sod by time since first employ ment in PCE shops (latency). Employment in unknown or petroleum solvent shops was cot used in the calcula tions of length of employment or Latency. The PYAR stratified into age and calendar time pe riods were multiplied by the corresponding US morcalitv rates to yield expected numbers of deaths. At the time of this study, the life-table analysis system only main tained US mortality rasas through 1978. tho end of the eighth revision of tho ICD. To calculate expected deaths through 1988 for this study the death rmtaa for the interval 1975 to 1979 were based on US doathiss,ourux- nng through 1978. and tho death rates for thoi ll^^al I960 to 1988 ware eeenmod to bo identical to u^fre- i time period (1978 to 1979). i deaths were were tested *"'"t the distribution-* Tho risk is reported as a stand ardised mortality ratio (SMR). defined as observed/ X 100. ) each of the unions included in the study war* located la large metropolitan cities where the mortaiitj rates frem cancer are generally higher than those o the total US. state mortality rates corresponding to th< location of each union wore also used in calcuiatinc expected cancer deaths. Tho state mortality rates mon closely estimate tho rates of the cities. Rasufls The cohort totaled 1.690 workers with 493 deaths an< contributed 11287 PYAR to the analysts. The vito status follow-up through Doc 31. 1988. was successh. for 93% (ie. 7% loot to follow-up) of the cohort. Th follow-up for females (98%) was much less complet than for males (97%). This is primarily duo to cam changes and woman dropping out of the work fores an earlier age. Therefore, they are lees likely to be list* is the Security Administration records. Tho subeohort of workers employed only i^shhic- 536 Dry Cleaner Workers and PefcMoronttiyteng/Brown Akxapia SL 038718 where PCS was the primary solvent totaled SIS: 113 white males. 94 non-waite malae. 199 whit* females and 313 nonwtut* females. Thsrs were a total of 137 dssths identified in this suOcodort. Although all litas of caaear and particularly caacar of the liver ware the causes of greatest interest, multiple causae of death ware also examined. Table 1 lists the observed and expected deaths for most major dlsaasaa and for specific sites of cancer. As seen in most cohort studies of workers, the observed number of deaths for all causes is less than expected (493 observed v 973.3 expected: S2d * 36) with statistically tiguiflcant def icits in of the circulatory system and diseases of the nervous system (primarily stroke), demonstrating the healthy worker effect. The number of observed deaths for all neoplasms is higher than expected (143 observed v 122.9 expected: SMS. " 116). No deaths from Liver cancer were observed, whereas 3.9 were expected. Only urinary tract cancer showed a statistically signif icant excess in observed deaths (12 observed r 4.7 expected: SMB " 239). Within the urinary tract cancer category, both kidney and bladder cancer were elevated with bladder cancer being statistically significant (8 observed v 2.7 expected: SMB " 296). Other interesting results among ths malignant neo plasms include cancer of the cervix uteri which shows an elevated SMS (10 observed r 3.1 expected: SMB " 196) and cancer of the breast which is slightly lower than expected (12 cbeerved v 13.3 expected: SMB m 37). These results probably reflect the ininmiuwnn status of ths cohort which is gsssrally lower inooms. This type of pattsrn has been shown in other studies.T In tbe analysis of cancer mortality based on death ratos of tbe state where each union was located (rather than ths OS death rates), there are 140J2 expected deaths from all cancer sites which is closer to the 142 deaths observed in the study population. However, ex cept for pancreatic cancer (11 cbeerved v 10.7 ex pected). there was very Uttle change in the expected number of deaths from the specific sites of interest. For urinary tract cancer ths expected aumoer of deaths based on stats rates is 4,9. r 4.6 based on US rates. Therefore, all remaining results in this manuscript are based on expected deaths calculated using US daatn rates. Mortality from ail major organ systems (other than oanear) was lower than expected, except for a slight excess in diisasss of ths digestive system (22 observed v 18.3 expected: SMB 117). Mortality from ail acci dents is significantly lower than expected (3 observed v 23.9 expected: (SMB * 13). Almost ail categories of accidents showed a deficit in mortality, with transpor tation accidents accounting for the largest portion of the overall category (0 observed v 10.7 expected). Mortality by race and sex group was examined sep arately. For ail groups, overall mortality was lower than expected, and cancer mortality was higher than expected. In nonwhite males the excess in cancer mor tality was statistically significant. In three out of the (bur race/sex groups mortality from bladder and kidney cancer was elevated, although these were baaed on small numbers of obeerved end expected deaths. For bladder cancer a statistically significant sxesss was observed in aeawhite melee (3 obeerved r 0.6 expected: SMB = 900). Breast caacar was lower in both rmmal groups and cervix uteri cancer was higher than expected. In all (Our groups mortality from semdsats was significantly lower than expected. In white males there was an un- TA6LK 1 Cauae-memie Maser* Orv Qsarwr Wmi Aon AS tar mens mas Canon (AS Maos n Set boas' Caw <Snm lew. SMS ss%a At masgnant neoaumns (MN) MN at ouocw esvey sno oneryrx* MN of ogesuve organs and pwnoneum MN of imssisw esem maun MN of Ivor MNof osneruu MN of resorstory system MN of brssst MN of female gervtai orgm MN of csrvot utsn MN of maw portal organs MN of urinary organs MN oftaoney MN of ttaooer mn or otrwr ano unsoaMtoo aas MN of tymonsuc anc hamamoowue msus Oiseases at mo nervous system Owesses of Via mmasnry system Oiseases of me resonny system Diseases ot me agesme systam Crmosis ot me uvar Oisesses Of me gervroimary systsrn Cacus of me usury sysam teairm Vkssncs as oaems 14* 3 38 16 0 11 29 12 16 10 8 12 4 8 20 4 44 163 23 22 14 10 2 3 10 493 122.9 2.9 33.3 11.8 X5 6.4 29.4 1X8 1X7 XI SJ 4.7 XO X7 lil 9.3 aofi' 23X0 28.7 18.8 1X9 102 0-3 2X9 1X4 573.3 116 97-136 103 21-302 107 78-147 138 78-220 172 86-310 114 76-164 67 46-132 126 72-204 196 99-363 143 63-286 235 132-130 200 35-317 296 128-586 133 93-236 43 12-110 73 93-96 70 60-62 60 31-120 117 73-177 109 60-183 98 47-180 667 73-2186 13 3-37 81 39-149 68 78-94 * itwisms used are: SAM. stanasraxsd mortawy nso. CL oaroosnee eitervw. Journal of Occupational Modidno/Volumo 29 No. 8/June 1987 537 SL 038719 ri r,` i finding within lit-------of tbo genitourinary sy- **b: & jtatuticaiiy txcsas lb mortality from w^iiti of tho urinary system won found (2 ooaervsd r 0.09 ozpoetod). Mortality wan aloe examined aeroas individual unions which represent totally independent cohorts. Consistent with the (ladings of the combined eonorc. a deficit in overall mortality and an excess in eaacsr mortality was noted sense ell four unions. There was also a deficit in accidents in all four la three of the four unions there was an excess in bladder cancer, two of which were statistically significant. Two of the four unions had an excess in kidney cancer. All unions had an (nrreate in cancer of the intestine- The SMSs by race and sax groups and by union fbr seloctod cause# of doatb are summarised in Tablee 2 and 3. Analyses for cancsr of tho intestlns sad bladdsr by loogth of omploymoat in PCS shops (a sumgate of oxposurs) and by timo sines first oxpeaurs (latency) ware conducted to aid in the interpretation of tho re sults. These anniysea are given in Tablee 4 and S. There appears to be a positive trend of increasing risk with an increase in latency and exposure for both cancer sites. Separata analyses for kidney cancer are not presented because of'the small numbers. In the subeohort of workers who were employed only in shops where PCS wee the primary solvent, there was only one death from urinary tract cancer, whereas 1.3 were expected. Both of the deaths from renal otlculi were included in this subeohort. whereas only 0.09 deaths were expected. An attempt was made to confirm the cause of death fbr each of the bladder and kidney cancer oases. Medical histones and pathology reports were requested from the hospital where the death occurred. Information waa obtained for six of tho eight bladder cancers and one of the four kidney cancers. The cause of death from the death certificate was confirmed in each ease where information was available from the hospital. exposed to PCE developed liver tumors. The NTP *tud\ also demonstrated the PCE may be ssaninatsri with rsna carcinogenicity and leukemia in rats. Because clear bow an animal carcinogen might express a; a human carcinogen, all other sites of cancer were o interest as well. Mortality from all causae was found to bo leas that expected, which is probably duo to the "healthy worts: effect.~ Mortality from all cancers waa higher tha: expected, almost reaching staastfoBl tigniflrenrw 93? confidence interval for the SMS was 97 to 136. Ti_ axesm in all cancers was a consistent finding across s. race and sex groups and all unions: however, this exeea waa reduced when state mortality rates were used fo calculating expected deaths. Urinary tract cancer was the only specific site fount to have a statistically significant excess in observe, deaths. This excess was primarily due to bladder cancer however, kidney cancer was also found in excam. Thar was some consistency in this finding in that bladdo cancer was elevated is three out of the four race/a* groupe. and in three out of the four unions, aithoug TABLE 3 Caw at Bteei ------- At mmgrarn nsootexos MNPMMnt MNof panoeas HM of kidney Mtcromoosr lineal 71 132 1ST 167 --r 1.000* lineal S3 124 IIS 214 222 41* 10* OyLtion UmaaS Utsaa * 84 96 103 106 138 156 83 167 -- 333 200 *P< JJS. t ---no TABLE 4 Time! a WrsriwwmwwaAMncil arenasuwmww Discussion The diseases of concern in this study were ail sites of cancer, especially cancer of the liver. Liver cancer was of particular interest because of the NCI bieassay and the recent NTP study which demonstrated that mice <10 10-19 2D-29 >30 Tmi 0/1.8 -- 8/3.9(1541 10/4.9 (204) 0/1-2 -- 16/11.8 (138) 0/0.4 -- 0/0.9 -- 5/1.1 (4551 3/0.3 <1.0001 8/2.7(286) * Abamesmns used are: mn. msagnant naocusrc SAM. sanoa tzsa monasty rsoa Can-- it o--i ai muses Al mstgnsm neooewns MN ot imeswie MN ot osnssas mn Ot koney mn of btsonar MNot breast MN of carets Acaoenm *0< .03. r -- - "O oeserved beams. TABLE 2 Morwey Asaee *w Saecsa Cease or Aaea at Ses Oram or Ory Cttnw wontes WMiNmM 1animate Mea ............. .. .......... as 98 88 107 107 66 136 14S* 313 111 187 143 333 143 t_ t 273 400 -- 89 500* -- 186 -- --' 36* 75 110 ill 256 333 86 206 -- 538 Ory Claanwr Workers and PerchloroeOiyfenw/Brown & Kni! p r-- k-r W V- W ^ SL 038720 TABLE S sMWMtanCvarOMnMMibcmRteunM near or ns ov LWgot at (narnnmn n 1 ! 1 LsnySi gmsirimim <m MNdflMMr Qh99HWl/lMWil (Oil) i--i 5-9 10-14 *15 6/6.1 (98) 5/3.1 (161) 5/1.7 (294) 0/0.9 -- 1/1.4(711 4/0.7 (5711 2/0.4 (500) 1/0.2 (500) Total 16/11.1(136) 6/2.7 (296) * Aooraviaaou ussa sre: mn. mssgram nsecusm: SMfl. sanaanmm morwny rsno. these vara generally baaed on small numbers of observed mad expected deaths. Kidney eaaoar ni aiao elevated in three out of the four rsee/aex groups and two out of tba four union*. It la interesting that both kidney and bladder cancar war* found ia axoaaa. It can only ba apaculatad that thaaa two eaacar sitaa ara raintad to a enmaon etiology. Ia studiaa of cigarette laotm.1 aad of workers exposed to benzidine*. both sitaa were ele vated. Whan mortality from bladder ameer was axamiaad by latency aad exposure (baaed on employment ia PCS facilities) a pattern consistent with an occupational etiology was found. The increased risk for bladder can* car mortality occurred after 20 yean of latency vhich is similar to other studies of known bladdar carcino gens.10 Ia this study the calculation of both exposure aad latency associated with PCS are only estimates. V*'im it was assumed that petroieiua solvent* were used dur ing time periods of unlmown solvent use aad these time periods were not used in calculating exposure or la tency. For somo plants this assumption may bo isoorrset and PCS rather than petroleum solvents eould have been used. This would tend to iasreaee latency and exposure for tho cohort. However, the bias ** this creates depends on whether the bladder eanoer are affected more or loos than others in the cohort. Ia the analysis of workers employed only ia shops where PCS was the primary solvent, there was no ines risk ia mortality from bladder or kidney eanoer. There fore. the excess nsk occurred ia workers with a potential for mixed exposures to PCS and petroleum solvents. This finding doss not preclude Pd as tha exposure assoristsd with the exesas in urinary tract oncer but certainly weakens the possibility of an association. In addition, there ia experimental evidence that kidney cancer may bo related to exposure from petroleum solvent*. Recently, ia a study by Kitchen.11 vaporized, unleaded gasoliae induced kidney cancer ia exposed Fisher 344/1? rats. The chemical so-ucture and toxico logic properties of Stoddard solvent, the petroleum sol vent used ia dry cleaning, is mil** to those of gaso line.1* Cigarette smoking has also boon associated with an cxcaaa nsk of dsvoloping bladder cancer. Most studies have shown a twofold to fourfold excess risk of bladder cancer ia male smokers compared to that ia uoasmokers.1* Tho role of cigarette --"y in the excess risks observed ia this study cannot be determined quan titatively because so data were available oa the habits of the study cohort. However, according to Ar. eison.1* smoking is a weak confounder unless it j strongly associated with the divesee aad unless smoking habits between exposed aad nonexpoeed worker* differ drastically. The poeaible effects from smoking oa the risk for bloddor cancer in this population were calcu lated based on tbs method described by Axoisoo.1* Based oa these ~it can be concluded that smoking cannot account tor the threefold excess seen in t-*TM* cohort, la (set. if 100% of the population were heavy smokers, this would account for only a 56% increase in the risk. The most striking finding ia tho study was tba over whelming deficit ia mortality due to accidents. This may be due to tho demographics of the study population, which ia primarily lower socioeconomic aad inaar city (Chicago. New York City, Detroit, aad Oakland. Califor nia) workers. Therefore, it is passible that these workers did not own automobiles aad relied heavily oa inner city transportation systems which would account for the deficit in aaaspawation accidents. Tho two daaths from renal oalcuil, both of which were bund la the suboobort of workers employed only ia PCS shops was an unexpected finding that may have been a dianos oecsirsaoa. However, it is possible that exposure to PCS could bo related to this finding, although we know of no reports describing nephrotoxicity in humans xpoaed to PCS. renal effects have been observed m experimental --*-- studies.1** In addition, there have been reports in the literature describing the occurrence of urinary calculi due to chemical exposure. A person developed renal tubular aririoais aad urinary calculi after persistent toluene sniffing.1* aad ethylene glycol has boon associated with tbo production of calcium oxalate in renal tubules leading to formation of calculi In rats.1* The rate PCS might play ia these types of mechanisms whidh lead to unaary calculi is unknown. Several other epidemiologic studios of dry cleaner and laundry workers have been conducted. In a study by Blair at al`* a proportionate mortality ratio (PMR) analysis was performed on 330 death certificaxee ob tained from two local laundry and dry.cleaning unions. These deaths only represented a sample of the total number of deaths that occurred between 1957 and 1977 among tho union members. The sample of <--was act necessarily a probabilistic sample but included deaths that bad been identified by the researchers. Workers were included regardless of tho solvent used in the dry cleaning shop which employed the worker therefore, it was sot specifically a study of PCS expo- Joumal of Occupational Matficino/Vohjw 29 No. 6/Juno 1987 539 /-- ^ SL 038721 sure. Tha nil of mortality from all cancan combined waa found to bo higher than expected (S7 observed v S7.9 expected: P < .09). Among the eancor deaths. lung, ctrrut uton. and skin cancan ware elevated at a statis tically significant leveL Other malignant aooplaams found is excess worn uxtoanao. Uvar and Ischemia. Risxs for bladder, Iddney and paacraauc canoar vara not elevated, and breaat cancer risk was lover than ex pected. S*tx;? studied the mortality of female laundry and dry cleaner worken in Wisconsin by identifying 871 deceased workers from the occupational statement listed on death certificates. He tested for associations between occupation and cause of death by calculating specific PMBa for 25 causes of death. As in Blair's study the workers did not necessarily werk at shops using PCEIn contrast to Blair's study, the risk of death for all cancer, liver cancer, colon cancer, lung cancer and leukemia was not elevated. Statistically significant ex cess risks were found for cervix uteri, genital (unspec ified), and kidney cancer (7 observed r 2.7 expected). Bladder cancer risk was also elevated (5 observed v2.6 expected). Breast cancer nsk was less than expected. A study using similar methods was conducted by Duh and Asal1( in Oklahoma, where deaths from 440 laundry and dry cleaner workers were analysed. Again, no iden tification of solvent use was attempted: however, the use of petroleum solvents is more common among the dry cleaning shops in Oklahoma where more than 90% of the shape use this solvent, whereas the remaining use PCS. Duh and Asal found a statistically significant exeeaa nsk for lung and kidney cancer. Other elevated risks were found for cervix uteri, other female genital organa, and skin cancer. The nsks for liver, pancreas, and bladder cancer were lower than expected. The mortality rinks for those previous studies of dry cleaner workers along with those of the present study have been summarized in Table 8. There were several causes that exhibited consistent excess risks, including cancer of the cervix uten end kidney. A deficit waa observed in all studies for breast cancer. As discussed earlier, the results for cennx uten and breast are TUU Cawasnsrsior Gnaa^eeocc Hama ammw Morrsty Sanaa x Ory Cssnsr (ana Lanoyi wsmws* CauaeerOeaet as cancer KttMWi Uvar Pancreas Lung Slun Breast Ceres uten Sisoosr Kkmey LsuKenva area* (MM) tat 192 235 129 170T 429t 89 2097 93 200 227 KM* (PMR) 96 103 99 117 98 207 72 199t 199 297 97 0--1(SonT" 90 90 90 90 170 190 10 130 40 390 -- (SMS) 118 138 --t 172 114 -- 97 198 2987 200 --* IOR. tk < .09. * not< probably related to the socioeconomic status of' these occupational groups. Conclusion The excess risk for urinary tract cancer m tma study was somewhat unexpected. Because multiple causes of death were examined and this causa of death was not part of an a prion hypothesis, the finding may be due to chance. However, it was the only cause found to have a statistically significant excess in mortality and the excess nsk waa found in three out of the four race/sex groups end in three out of the four unions. The magni tude of the SUH and the pattern by latency and exposure to PCS waa eonaiatant with an occupational carcinogen. However, because of the limitation in the records used to identify the cohort, it la not possible to analyze the data by specific job or estimate of exposure to PCS. Therefore, a does response analysis for urinary tract cancer, ocher than by length of employment, is not possible. Sven the analysis by length of employment is limited as a surrogate of exposure because of missing data on solvent use. Inasmuch as many of the workers could have had confounding exposures to petroleum solvents in dry meaning shops, those with employment in PCS shops only were analyzed separately. In this "PCE only" suboohort there was no excess nsk for urinary tract canacr. Therefore, the confounding expo sure to petroleum solvents complicates any conclusions regarding the aaaaciatisa between PCS exposure ana ossoer of the urinary tract. Acknoedadgi lasatess (OU) latarat Iks Sale uaad is lasamss (or Oanuas FllUSr Jll ip*. Wa <tia iSsnt Or nag --asS tka Refarwncm L. M lusit Sitraay. iW X- Surra? Aarndjau I Smi at Haeitk. EdaaaOaa aaS Walter# I Ss/M7 saS Haeitk. poMlMOas Ha 7U.ua. 1ST7 (ata Sals kaas w aa at Sag 4. lMO>. A ftoaaasr * Tt Mftama tar PaaatMa <~trnmannin *ap rlawaa. Me. IX Daw* at Haeitk. E4u Sanaa aaS Waiters. Sanaaal Csaasr loaututa. wiMleim Ha- (tfUl TT-SM. IITT. A .Varnaa/ TunaaUfj Pratram TaahmnaJ JUparv as tea Taiaeai asr see CmeywasSnSs afTatracAlanmt*Tfma (Par^ndoroata Jtama) ia TMV5IMaai MO/Mdaa. Maoeaal laaittutaa of Heaitl . Uacaa* 1SSS (kaer# 4rsA). 540 Dry Claanwr Worker* and PwrcMorowtftyiana/Brown AvWpUi O r-- e-- W C #> iaa'lJl SL 038722 inilM I Oeaad JM 1! fell*- 134. 3. li Mil* NX. t.Mi IK. luffiu li MaiUeUaaero oaMM aoO MIM aoaiyaa. I Am 4tat I mi itecnu-ic 7. TToorir L Kim TJ. Siatof fW, n 41: (1--fripali p--w W mir MttilUr 14 tte IttM MM. ia fruaMi IF (M): Aimm 44 JtteO SMS of Cwwr 44 4IWI M rt)if7 --0 CmPM. Now Tore 14<IM rt4 l*n. n 343-MO. I. --4 EC: 1mwc 14 nMMt M tte ImU niH it aam- --Il`-- mam saO eowaa. ii BmoP W (ad): OMmmmim 4e prmaaaaa m tan Study at Cnnr M Oitr dm* Oia--am. OS Dm of H04114. fJ--" oo4 WtUuo MM Boutt Soroioo. .Iinml Clour luamw UMOOfiooli It, 1VTS. n 137-304. 9. Zsoo MOL Bum g. IHortta Ei 4i3iS4 iiqoiiwmiowo at blo44or iuwi. Arm inn> Xooltt 1973:27:1-7. 11. KlleOoa ON: Neoeiaeae nool oOmi at iioioortoO pMM to Ptaotor 34* nu. it SMIau MA (od): SonM CTaca 4 Abmm X) m Mi MMW ''-TTitfU Ian. 19*4. fMT.py U- 71. IX Soodaorw z& inoMo tyommrtoaa ta Cmoo ao (Mi- May'* ImduMMnt Mygtaoa mad Tamaataty. 4 X Now Tor*. Jo4a WU07 444 SoM. 1401. fM 34. tm mtc.34iM Irici3*o7 t'.onor). Scoot / Wort nvo Suita 1974:4.-40-103. 14. Xrafor UC Moor* RJ. Lottaao TK. at sb Roovrooi inaary Modi lOHOMl witt loiooo* --<w-T I M lUOilKt *1. 13. WUUaaa EC Saitt l* Oiaorttn M ! I Had 1900:44:718-733- -- am 1C Sloir 4. Oaooono P. Omutma 0: CooaaoarOoott aaaa louaArr oad try ---------T worsen. Am J FddUa Mmttd 1963:40:904 In. 17. Katt CM. Jawoot 0: Fraud* loaadrr aai try claaaiaf wortaro la Wlaoaaau: A orwlHy seoiywm. Am J FtdUa Xloaitt lMl:71JOt- X77. 1C Ditt *W, teoi OTL Mortality aaooc laootry aad dry ctmataa worttri ta 0kl*4eaa. do / FaaUm Mmlta 1444:74:1374-1300. Tho Artist's Orsam By irtiit I au ... reryone who hai tried to eroota wstthlsf which wao not here before him. with ao other teela aad material than the "--moiom. m af tha boau mhfe who haa and to am, oo manor bow eradely. oa tha wall of that tiaai oblivion. m tha tenet** of tha humaa spirit. `Elroy wao bota.** That ia primaniy ... ail... wo rror really triad to do. Aad I believe wo wlU all asraa that wo failed- That what ao mado oever quite m**,*hvd aad aoaar will "**. tha thape, the draam of perfection which wo inherited aad which drove u cod will eoatiBuo to drive u, atoo after aob failure. util anguish frees as tho fails still at lose Maybo it'* just ao wail that wo aro doomed to foil. aiaoe. aa loaf ao we do f*n aad the haad oonunuoe to held bleed, ao will try aain: whora. if wo ever did tbo dreaau match the ihape. tcaie that ultimate peak of perfection. nothing would remain bat to jump off tho other ado of it into suicide.... --Commoata by William Faulkner apoa receipt of National Book Award for fiction ia 1988. From "`Three Choara for Good Mark*': Writers oa Their Prisae" la Tho New Tort Tlmaa Book Ranaw. Nor 16. 1986* Journal of Occupational Modtemo/Volunio 29 No. 6/Jtmo 1987 SL 038723 Cr\ *"h r-i -- v> V- w A.. , > 541 f ( /V* /- UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON DC. 204S0 March 9, 1988 Hon. Lee M. Thaws Administrator U.S. Environmental Protection Agency 401 M Street. SW Washington. D.C. 20480 SAB-EHC-38-011 r- Dear Mr. Thanas: Thank you for your thoughtful response of August 3 to the Science Advisory Board's review of scientific evidence associated with exposure to oerchlotoethvlene. In your letter you asked the Board to provide further scientific advice on three issues that will subsequently bear on your risk management decision for this compound. The Board appreciates this opportunity for further scientific dialogue on these issues and hopes that its views in this letter can better promote consensus on the scientific issues under review. As noted in your letter, the assessment of the scientific evidence fran experimental animal studies centers on the relative significance for hunans of the production of rat kidney and mouse liver tunors. This question is applicable to a broad range of chlorinated hydrocarbon compounds--including dichlorcmethane, para-dichlorobenzene. trichloronethane, and trichloroethylen which produce tutors of the rat kidney and mouse liver under sane experimental conditions, Wiile recognizing the implications of such issues to these and other compounds, this letter is directed specifically to an assessnent of perchloroethylene. In responding to your letter, the Board's Environmental Health Cornuttee and its Halogenated Organics Subcommittee organized a scientific ^rkshop on August 12. 1987 to explore these and other issues with leading researchers in the field. EPA staff and msnbers of the public. An agenda of the workshop is attached. The Board has utilized the information obtained in this workshop, and the discussions anong Cornu.ttee and Subcommittee menders, to respond to your August 3 letter and also to advise the Agency on health effects evaluated in its ttaft Health Assessnent Document Addenda for DLchloratethane and Tri chloroethylene. The Board's findings and recottnerelations on these latter t>o compounds will be transnitted to you in separate letters. Our response to your specific questions follcws. Ouestion _1: Assuning that not all animal tumors are of equal significance to evVlua'tTrg hunan hazard, what is the Science Advisory Board's current consensus position, based on scientific evidence or professional judijnent, of the relative significance of male rat kidney or mouse hepatocellular tuners for hunan risk assessnent? SAB Response: In general, the Board's consensus conclusion on the significance of"male rat kidney tunors stsns tran recent reseerch (not yet published, but iru| ;ress) that indicates that for many halogenated organics, prooacly inclucirc perchloroethylene. the mechanism producing these type of tumors is procaolv -ct operative in honans and. therefore, may not be relevant for human risk assessment. This mechanism involves the metabolisn of the canpound in the liver and th bind ing of a protein (alpha-2u-globulin) with the metabolite as a conjugate molecule. This molecjle is filtered and accumulates in the kidney. Cne hypothesis is that the conjugate is more difficult to metabolize than the alpha-2u-glctulin alone. This protein then acconulates and is injurious to the cell. Repair :s followed by a _ cancerous formation at the site in a lew percentage of cases. Fran available scien tific evidence, this mechanism appears to be unique to male rats. Thus far. thirteen substances have been demonstrated to produce renal Conors in male rats through this mechanism including perehloroemylene, paradichlorobenzene and unleaded gasoline. Trichloroethylene, on the other hand, appears to produce renal Conors in male rats through a different (unknown) mechanism, thus creating important implications for honan health risk assessment. The Board's consensus on the significance of mouse liver tuners is that mechanistic explanations are not sufficiently well developed anc validated at this time to change EPA's present approach expressed in its risk assessment guidelines for carcinogenicity. It concludes that the generation of incus liver Conors by chemicals is an important predictor of potential risks tc hunans. Of the several mechanistic models under consideration (including regenerative hyperplasia, oncogene activation and tri-halcmethyl radical formation), the one most promising for iimnediate application to risk assess ment is characterized by proliferation of peroxisanes, an intracellular organ elle, in the liver. Peroxisane proliferation may be important for cgnpounds such as perchlcroethylene, but liver Conors observed after exposure to chlorinated solvents may involve different mechanisns. The importance of understanding the biologi cal mechanisms is that they may provide a basis other than the bioassay statistical analysis for low-dose risk estimation. A plausible mechanist (peroxisane proliferation or something else) may imply low-dose nonlinearity for sane substances that induce mouse liver Conors. However, different (pre sumably linear) mechanisms may operate for other substances, and these mechan ists may be consistent with linearity at low doses or a linear relationship to dose. These distinctions in low dose risk estimation should be explicitly included in the quantitative estimate of honan risk. Several substances that induce peroxisane proliferation in rodent livers, such as hypolipidenic drugs and the plasticizer di-ethylhexylphtalate (EEHP), also produce liver tonors in rodents. In suimary, however, a causal relation ship for this mechanisn is plausible but unproven. Sane scientists have reported the detection of oncogenes after administra tion of presumably non-genotoxic agents.^ ^ Steven H." RVynoldsT"ShaFi""J.""std^rs,' Rachel M. Patterson, Rooerx"R.'"KaVonpoT, Stuart A. Aaronson. Marshall w. Anderson. "Activated Oncogenes in B6C3F1 Mouse Liver Tonors: implications for Risk Assessment," Science Vol. 237 (September 11. 1987), pp. 1309-1316. SL 038725 /-i n /'N * ) Also, as you are aware, our increasing knowledge of the role of mechan isms of pranotion (later events in the carcinogenic process) may well clarify our understanding of cancer induction; certainly this is the case with dioxin and may relate to the halogenated hydrocarsons. Cuestipn_ 2: Wnat is the Board's view of the approach taken by EPA in using its" guidelines to infer human carcinogenic potential fran the total body of sci ntific evidence on perchloroethylene? SAB Responsei The issues regarding the application of the risk assessnent guidelines appear not to represent disagreement among scientists about scien tific evidence but, rather, the consequence of attenpting to fit the weights of vidence into necessarily arbitrary categories of risk. Since the weights of evidence, and uncertainties associated with such evidence, for perchloro ethylene and other compounds fall within a range of scientifically defensible choices, it may not be possible, in sane instances, to fit them neatly into only one risk category. Moreover, the more incanplete the data, the less precision one can expect in classifying a compound within EPA's cancer guide lines. In addition, the type of evidence that places a compound in a particular category may vary consideraoly frcm substance to substance within that category. For perchloroethylene, as with trichloroethylene, the Science Advisory Board concludes that the overall weight of evidence lies on the continuum between th categories B2 and C of EFA's risk assessnent guidelines for cancer. As perchloroethylene illustrates, the distinction between the B2 and C categories can be an arbitrary distinction on a continuum of weight of evidence. The "black-white interpretation" that you referred to in your letter is indeed troubling. Fran a scientific point of view, it sesns inappropriate for EPA and other agencies to regulate substances that are classified B2 and not to consider regulation of canpounds classified as C, regardless of the level of human exposure. In the case of B2. Bj_ or even A categorized canpounds where exposure levels are lew, ffiA may, with scientific justification, decline t reg ulate because the potential health effects appear to be trivial in magnitude. A substance classified as C (limited evidence in animals) for which human exposure is high may represent a much greater potential threat to human h alth. EPA and other agencies (including those in state goverrments) may, th refore, wish to take steps to reduce high exposures to substances in the C cate gory v*ienever there appears to be a potentially significant threat to human health (in the sense that the plausible upper bound estimate of potency times lifetime exposure is above the threshold where regulation may be judged appropriate). Indoor exposure to perchloroethylene, such as might be found in dry cleaning establishments not using the equivalent of good industrial hygiene practices, could merit action under this criterion. So might high levels of exposure to other solvents, pesticides or industrial chenieals that have been considered by the public as "safe" in the absence of sufficient evidence of carcinogenicity in animals. In many instances, this appearance of safety results fran not yet having the results fran well-designed bioassays such as those conducted by the National Toxicology Program. /"* ^ /A SL 038726 Finally, you noted the evaluation of perchloroethylene by the Internation al Agency for Research on Cancer (IARC) as an exar^le of evolving terminology for classifying potential carcinogens. In general# the Board believes that public understanding of caiplex scientific issues is enhanced when scientists and regulators can speak with a cannon voice, m view of its own experience of using the cancer risk assessnent guidelines and# in particular, having to address the issue of the scientific uncertainty that exists anong and within guideline categories# EPA should re-evaluate Its labeling systeo and methods for characterizing uncertainty. It should also review Wiether to be more con sistent with XARC*s teminology. Question 3i Is there research underway or anticipated that will clarify these rodent tutor responses and their relationship to human health risk assessment? ftoat additional research should be undertaken? SAB Response: Current researdi undertaken in various laboratories, including the National Institutes of Health# can reduce sane of the uncertainties assoc iated with rodent tunor responses. Research results and hypotheses presented at the Boards August 12 workshop has served to clarify our understanding of male rat kidney tunors and their significance for human risk assessnent. In addition# the Reynolds et. al. paper supplements our knowledge of activated oncogenes in mouse livers tumors. Several research efforts should be initiated to further narrow scientific uncertainty for perchloroethylene and structurally related camounds. Hues include: o Validation of mechanistic models for the rat kidney and mouse liv r tumors through experimentation with selected known carcinogens and non-carcinogena. o Development of isproved methods for assessing low dose response to envirormental pollutants that indujce peroxisane proliferation. Ouoe again# w are pleased to have this opportunity to present th views of the Science Advisory Board on these important scientific issues. Vie hope that the consensus stated above assists you In making the difficult risk manage ment decisions on perchloroethylene and other ccngomds. Executive Ccnmittee SL 038727 U.S. ENVIRONMENTAL PROTECTION AGENCY SCIENCE AOVISORY BOARD environmental health committee/halogenated organics SUBCOMMITTEE (COMBINED ROSTER) CHAIRMAN * *m * Or. Richard A. Griesemer, Director, Biology Division, Oak Ridge National Laboratory, Martin Marietta Energy Systems, Inc., P.0. Box Y, Oak Ridge, Tennessee 37831 CHAIRMAN OF THE HALOGENATED ORGANICS SUBCOMMITTEE Or. John Doull, Professor of Pharmacology and Toxicology, University of Kansas Medical Center, Kansas City, Kansas 66103 MEMBERS Dr. Seymour Abrahamson, Professor of Zoology and Genetics, Department of Zoology, University of Wisconsin, Madison, Wisconsin 53706 Dr. Linda Birnoaum, National Institute of Environmental Health Sciences, P.0. Box 12233, Research Triangle Park, North Carolina 27709 Or. George T. Bryan, Department of Human Oncology, University of Wisconsin, K-4, Room 528, 608 Clinical Science Center, 600 Highland Ave., Madison, Wisconsin 53792 Dr. James Bus, Pathology and Toxicology Research, Upjohn Company, Kalamazoo, Michigan 49001 Dr. Gary Carlson, Department of Pharmacology and Toxicology, School of Pharmacy, Purdue University, West Lafayette, Indiana 47907 Dr. Robert Dedrick, Chief, Chemical Engineering Section, National Institutes of Health, Bldg. 13, Room 3W13, Bethesda, Maryland 20892 Or. Philip Enterline, Department of Biostatistics, Graduate School of Public Health. University of Pittsburgh, 130 Desoto Street, Pittsburgh, Pennsylvania 15261 Dr. David Gaylor, National Center for Toxicological Research, Jefferson, Arkansas 72079 Dr. Ronald 0. Hood, Professor and Coordinator, Cell and Developmental Biology Section, Department of Biology, The University of Alabama and Principal Associate, R.D. Hood and Associates, Consulting Toxicologists, P.0. Box 1927, University, Alabama 35486 Dr. K. Roger Hornbrook, Department of Pharmacology, P.0. Box 26901, University of Oklahoma, Oklahoma City, Oklahoma 73190 p /'"A ' * 1 SL 038728 2 Or. Curtis Klaassen, Professor of Pharmacology, Mail *638, University of Kansas Medical Center, 39th and Rainbow Blvd., Kansas City, Kansas 66103 Dr. 0. Warner North, Principal, Decision Focus Inc., Los Altos Office Center, Suite 200, 4984 El Camino Real, Los Altos, California 94022 Dr. Karl K. Rozman, Department of Pharmacology, Toxicology and Therapeutics, University of Kansas, 39th and Rainbow Blvd., Kansas City, Kansas 66103 Dr. Stephen Safe, Department of Veterinary, Physiology & Pharmacology Texas A4M University, College of Veterinary Medicine, College Station, Texas 77843-4466 Dr. Robert Squire, 1515 Labelle Avenue, Ruxton, Maryland 21204 Dr. Thomas Starr, CIIT, P.0. Box 12137, Research Triangle Park, North Carolina 27709 Dr. Robert Tardiff, Principal, Environ Corporation, The Flour Mill, 1000 Potomac St. N.W., Washington, D.C. 20007 Or. Bernard Weiss, Professor, Division of Toxicology, P.0. Box RBB, University of Rochester, School of Medicine, Rochester, New York 14642 Dr. Ronald Wyzga, Electric Power Research Institute, 3412 Hlllview Avenue, P.0. Box 1041, Palo Alto, California 94303 EXECUTIVE SECRETARY Dr. C. Richard Cothern, Executive Secretary, Science Advisory Board [A-101F] U.S. Environmental Protection Agency, Washington, D.C. 20460 NOTE: This combined roster only include those individuals attending the meeting. si 38729 z/"* AM. TaoooL. SoppL L S-17 (19*3) Qhr$*ne*utVta19tt Young Scientists Award Lecture 1984: Spades Differences in Carctnogcuidty and Peroxisome Proliferation doe to Trichioroethyiene: A Biochemical Human Hazard Assessment C R. Bcombc Ai6m*r rut. Miirtirfill CiMm. SK10 *TJ. U.X. PLC AkoxL Trichloroethylene (TRT) administered to mice by gavage for 10 cooMcaxne days at doses of 50-2000 mg/kg body weight elicited dose-de- (up to 70055 of control values) ofhepatic cyanide insensitive peiimtoyi CoA oxidanoo (a marker of peroxisomal tf-oxidaaoa). No etlea was ob aais^* wh rats demonstrated no effect ofTRI on other cyanide insensitive paimitoyi CoA oxxdaooB or catalase A major metabolite of TIU. trichloroacetic add (TCA) when 10-200mg/kg body weight. stimulated hepatic cyanide insensitive paimitoyi CoA oxidation in both mice (up to 500* of control) and tats (up to 650* of control). Again, no effect upon activity was apparent. The of bimr ofTRI to TCA in isolated hepatocytes was markedly species depenrienr. The `intrinsic clearance' values (Vmax/iCm) for TRI in mouse, rat md human hepazocytes were 3.2 x 10"*, 1.2x 10"' and 5.25 x 10"* L-mm/lCT cells respectively. TCA induced peroxisoinai ^-oxidation in mouse and rat bepaiocytes. but had no effect upon this enzyme senmy in cultured human hepatocytes. It is postulated that the speries difference in bepatocaronogciiidty ofTRI (mouse positive: rat negative) is due to species differences in peroxisome proiifcraoon which in turn is a result of differences in the rate of forraaa n of TCA from TRI On this basis it is proposed that TRI presents no significant htnnan bepatocardnogemc hazard since. (1) human hepatocytes produced TCA at a rase even low than that of the rat. and (2) TCA was not a peroxisome proiifcraxor in human hepatocytes. I** 'erds: Trichloroethylene - Peroxisome Proliferation - Species Differences - Hcpetocdhi-ar Carcinoma I irarion ssmenr II j for 10 iose-dc* ensmve Xt was nmats Umiioyi -Tic adS osex of ifflit vi 50 % of cues of spedes use. rat un/10* md rat human tenicity xisome uon of uficam dTCA xisome irences fI l SL 038731 TactaioroctAytrat and 7 Trichloroethylene (TRJD, administered orally at high doses for 18 months has been shown to increase the incidence of hepatocellular carcinoma in B6C3Ft mice but not Osborne-Mendei rats (NCL1974V The interpretation of these studies has been confounded due to the presence of epoxide stabilizers in the TRL However more recent studies have demonstrated that pure TRI also causes hepatocellular carcinoma in B6C3F, mice (NT?. 1983) and Aldcriey Park (Swiss) mice (Bcombe and Pratt, unpublished data). Furthermore, no inraease in the incidmre f hepatocellular carcinoma was obsavad in Fisher 3*4 rats administered pore TRI (NT?. 1983). TRI has been extensively rammed for mutagenic potential, but many studies were bedeviled by the prance of mutagenic epoxide stabilizers. However, in general. TRI has been found to be only `marginally' mutagenic or non^nutagenic (Grain et al_ 1975; Simmon a ai_ 1977: Brotttetu et al- 1978: WaskeiL 1978; Bartsch et aL 1979; Slack-Erbra et aV. 1980). Covalent binding of trichtoractbyiem or its metabolites to protein. RNA and DNA has been illustrated in vino (Van Duuren and Bancrree. 1976: Bolt et .... 1977; Bolt and Fiber. 1977; Uehleke and Poplawsfci-Tabaretli. 1977; Banojee and Van Oauren. 1978). Howew. m vivo, only etuemeiv low (indisdngucfaable from protein binding) or zero binding of TRi metabolites to DNA has beat reported (Pitchman and Magee. 1982: Stea a ai. 1982V Hence, TRI docs not appear to be a genotoxic gr'opa. and probably exerts in caidnopnie potential via an epigenetic meek, cl Indeed. Schumann et aL (1980) and Stott et aV (1982) have speculated that TRI manifests it hrpafocareaogenicity due to cytotoxicity. Other epigenetic nurhanimw of chemical carmnogenidiy have been discussed (Thorpe et aL. 1982V one sudi nwhanom being the phenomenon of peroxisome proliferation. Several nonmntagmK g*1*1"*1' which are hepaioeaidnocentc to radats have been shown to chat hepatic peroxisome proiiferauoo (Hess et aV. 1965: Svoboda et ai_ 1967; Moody and Reddy. 1975: Reddy et aL. 1980V It has been proposed that a causal triarionship exists between peroxisome proliferation and the development of hepatocellular oremoma (Reddy et aL. 1980V The mcchanismls) involved in ncopiasie transformation after the administration of peroxisome proiiferaton is undear, however Ready et aV (1980: 1982) have postulated the involvement of reactive Q, species. Preliminary studies d this laboratory have shown TRI to eiibt hepatic peroxisome proliferation in mice but not rats. Hence, the present study was initiated to (1) investigate the possible involvement of peroxisome proliferation in tri chloroethylene carcmogetuaiy, (2) to explain species differences m such carcinoge- maty and (3) attempt to obtain a human hnynrH assessment using isolated and cultured human nepatocytes. pr* ^ ' I Method* ULElcmt Reagents Trichloroethylene (> 99.9 % by gas liquid chromatography, containing 0.02 % wrw triethyiaorine stabilnzr) was obtained from Imperil Chcmioi Industries PLC. Mood Drvmon. Cheshire. UK. Afl biochemical* were obtained from Sigma London Qiemieai Company (Dona. UK). Trichloroacetic add (TCA) and other rtirrmcals were purchased from BDH Qieracais Ltd (Liverpool. UK). Ceil culture materials were obtained from Flow Laboratories (Irvine. Scotland. UK). Anatiaii Male Aldericy Part rats (Wistar derived) and male Aideriey Part mice (Swiss) weghed 180-220 g and 25-50 g respeedveiy at the start of the studies. The animals were housed in suspended ""** steel wire mesh cages and fed throughout the studies with PCD diet (Special Dim Services Lid. WUham. Essex. UK) and allowed cap water ad libitum. Tbs "> were exposed to a iight/dark cycle of 12 hr (0600-1800 light). Ik mo studies Animals were administered trichloroethylene (50-2000 mg/kg body weight) or tnchlorsacenc add (10-200 mg/kg) dissolved is com oiL by gavage daily for 10 days. Control *""** received an appropriate volume of com oil vehicle alone (10 ml/kg body weight). The annnili were hilled by cervical dislocation 24 hr following the final ^<~>v of TKL TCA or corn oil. and the livers rapidly erased and weghed. The livers were homogenized in 4 volumes 20 mM THIS HO (pH 7.4) containing 5.4 cnM EDTA and 250 mM sucrose (SET butler). The homogenate was centrifuged ar 600 g (average) for 5 mm at 4 *C to remove connective tissue, intact cells and nuclei. The resultant supernatant was centrifuged at 15.000g (average) for 15 min at 4*C to sediment a heavy pellet consisting of peroxisomes, mitochondria and lysosomes. This pellet was suspended in SET buffer to a final protein concentration of 20- 30 mgimL Protein contest was estimated by the method of Lowry et al. (1951) using bovine serum albumin standards. Caralave activity and cyanide insensieve paimitoyi CoA oxidation (a per oxisomal fl-oririrtion murker) were dosmined spearopnotometnsslly in the resuspended I5.t00g pellets by the mahods of Seers .--a ,1952) and Bronfman ct aL (1979) respectively. Statistic* All values are expressed as Mean -- SEM. Smrisnca.' .igsificance was determined using Students t-test (two-uiied). a level of p <0.03 bang considered as significant. fcS SL 038732 C R. Econo* Jig 0.02% w/w dustries PLC. i from Sigma CA) and other C). Ceil culture i. UK). : mice (Swiss) ilex. cages and fed fltham. Essex, o a iighi/dark ly weight) or e daily for 10 te of com oil s (Inal dose of lie lives were .4 mM EDTA sged at 600 g id nuclei. The am at 4 *C to id lysosomes. ration of 20-- >- (1951) using ition (a per* ricallv m the (1952) and determined s significant. SL 038733 t t TdettancWytauv uc P,, jluuim Profcicruiea 9 Ja witro studjer CeO cuiturt. Hepatocytes were isolated from ms and mice by a two step in situ perfusion technique as described previously (Mitchell et aL. 1984). The isolated cells woe suspended in Liebowttz L15 medium containing foetal bovine serum (8.3 %), oyptose phosphate broth (8_3 ~), penicillin G (41J lU/ml). streptomycin sdlphate (8^2|ig/ml). glutamine (241 pg/ml). mmiin (io~* M) and hydrocortisone (10"* M). The Viability of the hepatocytes (> 95 was determined by trypan blue exclusion. 25 air Falcon assue culture flasks were seeded with 2 x 10* vubie hepatocytes contained in 4 ml ofculture medium. The flaskswere incubated at 37 *C in air. 4,24. 48 and 72 hr after seeding, the spent medium and any detached ceils were aspirated and fresh mrriitim applied. Human liver was obtained from brain-dad renal uansnlam don rs after compliance with ethical and legal requirements. The liver was sliced (0.5 mm slices) by band and digested in Hank's buffer containing 0J Si (w/v> 0.1 % (w/v) 120u boding doth and centrifugation for 2min as 100 g. Ceils were then treated as for tat and mouse hepatocytes except ascorbic add (50mgfl) was added to the Trichloroacetic add. dissolved in N.N-dimexhyiformamide was added to the monolayer cultures at each 24 t\pur medium The amount of distethyifor- munirir new exceeded 10 pi per flask (4 mi) and this concentration produced a obvious cytotoxicity and had no effect upon the parameters measured. 96 bouts after verting, the hepatocytes were harvested. The medium was diaemded and the ceils washed in 2 mi SET buffer. The ceils were removed from the flask by napmg with a rubber policeman into 1 mi of SET bulTcr. The ceils were disrupted by sotucauon and the resultant homoenate used for the determination of protein contest and cyanide insensitive paimitoyi CoA oxidation. Metohoiivn of TRJ by freshly isolated hepatocytes Rat. mouse and human hepatocytes were isolated as described earlier, and suspended in the complete Liebowitz Ll5 culture medium. TRI (0.02-2 mM) was added and the cells incubated at 37 *C in <*!"< 25 ml Pierce * Reach-flasks* for periods up to 1 hr. Formation of product was tioear over the time periods utilized. Initial rates for TCA formation were ea)gtii*twt and kinetic parameters estimated graphically using Woolf plots (S/V versus S). Trichloroacetic acid was extracted from acidified incubation mixtures using dicthykther and derivttized with diazomethanc. Quanutauon was by gas-liquid chromatography (Carbopak C SP1000 0.1 K 80-100 mean. 3 Teet long, 145 *C) with electron mpturc detection. A /N ,, , ,, ^ i V- - 10 C R. Sou Ft*. L piiono^ CoA (30-2000 wkrdaT) by <br 10 Vatom an MaaaxSEM z Rat. 7.70 *a (3); Mm J4<U3 (3) p<<LQ3. In moo effects of mddoroetitficne om impaste peroxisomal enzyme activity The admmisxraxoa ofTRI to mice for 10 consecutive days resulted in 2 dose-related increase in hepebe perexaomai S-ondsnon (cyanide insensitive paimitoyi CoA ondaoon). a 7-Toid anon being observed ax a dose ofTRI of2000 mg/kg/day. No agniiiant aiteraoon in peroxisomal tf-omdshon was observed in rets after a similar treatment regnacn (Fig.l). C.ifaiase. also a peroxisomal marker enzyme, was effectively in both specs of animal (Table 1). Electron microscopy indicated an increased peroxisome volume density (expressed as * of cytoplasmic volume) in max but not rats (Table ZV In otoa offsets of trichloroacetic acid on hepatic peroxt:umai enzyme actnnty Trichloroacetic acid (TCA) is a major metabolite of TRL The administration of TCA to rets and once led to dose-related inocass in cyanide insensitive paimitoyi CoA oxidation in both sposea. At doses of200 mg/kg/day for 10 days. 6J-fold (rat) and 4.8^'->.d (mouse) increase in peroxisomal 0-oxidation were observed (Fig. 2). TCA. in common with TTU. had little, if any. effect on hepauc catalase activity (Table 3V Once again peroxisome volume densities were increased concomittantly with 0-oxidauon aenvity (Table 4). n 67 SL 038734 C R. Senate I heUnaot 11 7oA muilnnw a? tetm rua of sO_S3 (S) ante i <0.03. ctamy a dose-related ainmoyi CoA ag/leg/day. No after a similar enzyme, was n microscopy n cytoplasmic aetmtty (lustration of jve paimitovi . 6.i-foid(rati rved (fig. 2). aiase activity comutantly Si 3S735 RU ite ptewfl CoA o: (10-300 ng/kg/day) b Vote* am MousSEM (a--^5V donate raw* of < - ***. J-Il ~2 (3); Mow. 10J0U7 (4) ote NAO* tram rapteDte control p<O01 R*. A. Edna of S-oxidanoQ actrrtue of rat. hmm lad hi Cdto te minute u loniM ia Mnteut and m innate of and for 1 date Altar Uaa umb p . n piate teUmayi CoA oateatete Vaiuaui Moaa-SEM ( --a icofeaicat (nm typical rat ate t hfpatocvw puBUiMiu. For te teteitepwi teMa in MoaxSEM <n J I for 2 laOmdoal lite : pi o ^ f'1 ' ' " L ^ V * J Tafcfa L Eflin at tncMofoakrim oa OapaiK nmaat anwcy a nn am nun Dom at CiaiantKV'-an*1) (VkvOar) 0 50 100 200 500 1000 2000 Maaat oeoos (toot 0.11 0.03 ( 9C) L0904Z (132) 1.00041 (132) 014*019 (103) 0.750.03 ( 91) 0.71 0.0 ( 95) Hu 090*013 (100) 090 0.09 (102) U1 0.09 (115) 141 0.11 (133) 042 0.09 ( 15) 041 0.03 ( 91) 042 0.01 ( 15) <uy%. Coocroa TmM* 1 0 500 1000 1500 tea 1404 NO 14 04 11 0.6 Vataaaan MaatSHM (a*4l ND. MMaaaa Min 14 04 4.7 145 !9.13 2L4 3.6 TaMalElteM Don at CaiaiantCt'* **') m na im nuet Moan te 0 0.09 0.02 (100) 0.70 0.03 (100) 10 171 0.04(102) 010*0.04(113) 30 100 *0.04 ( 93) 1.03 0.14 M3I) 50 041 0.10 ( 14) 0.M 0.12 (113) 100 0.71 0.04 (I0Z) 073 4.00 < 90) 30 1.05 0.43 (152) 051 0.04 ( 76) Aw 10-200 aykvOa? at I far 10, i 4av*. Gintrou remvaO 5EM (a--*-0. Va^aa a SL 38736 /' to C. R. I CMyt. Castrate inu i SL 038737 TaU4. 13 tn- (acytepc Rais Mice 0 1J*<L3 ta<u 30 *2t03 10.7 too 3^*03 IOil2J 200 7.9*U ju : cod is coni oil for 10 ooc : Mm:SEM (n J-5X ND. not < : dayv TaM*3.K^MTa mi Mm Rat Hm Km (sM> 3t 131 51 Iff (190. 163) [pucUmmnir cafe) 107 If tf 7 3.6 <L2:2J) Woolf 1 :$EM (t4 Tor no i , o*2 for I vniuKa (Lewiir rafe) 3.1a 10"* 1.2*10*' 3.23 10-* (4.110-*: t.7*i0*) Its Me vein ofperoxtsomai 3-oxsdatiom The exposure of cultured rat and mouse hepatoeytes to TCA resulted in doserelated ineirasei in cyanide insensitive paJmitoyi CoA oxidation m botn spedes. However, woes human hepatoeytes were """* no stimulation of peroxisomal 4-oddanoo was observed (Fig. 3). This lade of response is believed n t to be indicative of lack of viability of the human cells, because cells from the same liver responded to pheaobarbitone and 0-naphthoflavone as inducers of mixed function osidase actrvuy (unpublished data). MciaboiLan of trtchioroethyiene by isolated hepatoeytes The kinetics of biotransformauon of TR1 to TCA were examined using freshly isolated hepatoeytes. Km values for TCA fonnanon woe similar in rat and human cells. However a considerably lower Km for the n-tcuon was manifest in m use bepatocytes (Table 3). Vmax values were Highest in mouseceils, followed by rat cells 70 14 c*. and human ceils. Damson of `inainste deanace' or 'capaaty' factors (Vmax/Km) illustrated 30-fold and 3-fold differences in efficacy ofTCA formation between mouse and tax hepatocytes and rat and human hepatocytes respectively. This work has shown crkhioroethyieae to stimulate hepatic peroxisomal 0-oxidation (an Ht0. yarning oxidase) in mice but not rats. This increase in axzyme activity was accompanied by a paralld increase in the number of and the volume density of peroxisomes (expressed as fS of cytoplasmic volume). No etTect upon hepatic raralasc was observed. Although these experiments were performed in Alderley Park rats and mice, unrilar data has been obtained utilizing B6C3F, mice and Osbome-Mendd raa: the strains used in the NCI canca bioassay (unpublished data). Reddy et aL (1980) have postulated a causal relationship between peroxisome proliferation and the hepatooumnogenieity of several non-mutagcnic <-**----< to rodents. These amhois suggest that inoeased peroxisomal 0-oxidaaon of fatty adds leads to the fonnsnon of increased steady state coocentzadons of HtOt (Mannaera a aL. 1979), whki in torn could lend to increased pexoxidanve damage of cellular compoucsus indqdmg DNA. This in turn could lead to a mutational event and evenmalty canca according to the jotnaocmutation hypothesis. The potenoai for `reaemeOj' cytotoxicity and genotoxidry may also be increased due to the lack ofa pualki increase in ataiase. a major H:0;-detoxifying enzyme, rnrirrd. eviaence for paoxidanve damage to the liver of rodents following the administration of peoxuome prolifcratora has been presented (Laiwani et aL. 1981: Reddy et aL. 19S2L Also, in this laboratory, the administration of mchloroethykse (1000mgykg/day) for two months and longa to B6CZF, buck baa resulted in the depoeinon of lipofuxcra -- an indicator of oxidative damage and ageing (Miqud et aL. 1977). If this hypothesis is correct the spades difference in susceptibility to TUI- induced hcpaiocaxcmogeaesu may be due to the speaes difference in peroxisome proliferation. The present study has identified TCA as a peroxisome proliferaur in both mice and cats. TCA is a major metabolite of TRJ (Butler. 1949); so why is TRI a peroxisome prohfexator in mice but not in rats? Previous studies (Bcombe a aL. 1982: Green, personal communication), involving the oral adminiuradon of TRL have demonstrated linear lcinrnrs for TCA formation in miceand saturation kinetira in rats. That is. as the dose ofTRI is increased proportionally snn, TCA is produced in mice. However, in similar experiments with rats, one rr-.ca a point where no more TCA is formed and the TR! Is siusirrarsd from the rat unchanged. Detaiies lunette studio have shown that the T.aximur. ` raurvai" dux; '.'TCA able to he produced in rats, irrespective of the dose of 77.1. about * * 'n^.kg. This value ts noteworthy, since in the present study no peroxisomal eeiyrae -aiuenon was observed below doses of iOragrkg TCA. r't Si 038738 C R. Bcasi neity' factors CA formation i respectively. peroxisomal tis increase m *r of and the ie>. No ciTect performed in 36Cjr, mice (uopubiished i peroxisome chemtcais to on of Tarry | S Of HjO; fiove damage a mutaaonai n hypothesis, be increased ring enzyme, allowing the Iwani et aL. utrauon of 3F, mice has lamage and icy to TRIperoxisome n both mice iy is TRI a uiiucauon), kineucs for se ofTRI is . in similar ted and the shown that rspecuve of he present 50 mg; kg SL 038739 Twchlarowayk-- add Pm 15 This suggests this rats produce ngaflidem TCA from TRI to eiiat peroxisome proiiferanon, thereby arpiamnig the lack of peroxisome prolifennon following TRI admimstzanon to ms. Furthermore, if peroxisome proliferation is causally related to tumour development, these observations would offer a mechanistic explanation of the spumes diflemee in eatcinogenioty of TTU. This hypothesis demands that TCA should dibx hepatocellular carcmoma m both mo and mice: accordingly a two year ttndy ofTCA admmntraaon to rats and mice has started. How do them observanaos help with a possible human hazard assessment? Firstly, peroxisoms proiiferanon and the concomitant incrase in peroxisomal 0* oxidation have beat evoked asmdicstors ofhepatocaranogemary and. secondly, it is postulated that the difTerenca m peroxisome proiiferanon and caidnogemcsty of TRI are due to differences in the ability of rats and mice to produce TCA. These presentstudies on the in vitro metabolism ofTRI to TCA have shown that mouse hepaioeytes have a 30-Toid greater propensity for TCA prooucuon than rat hepatocyies. which in turn are 3-ames more active than human cells. Therefore, it would appear that, m human liver TCA would be produced in smaller quantities than in rat liver and hence m insafTicirni amounts to elicit peroxisome proliferation after exposure to TRL Furthermore, the addition of TCA. the proximate peroxisome protiferaior. to human cells did not result in increased peroxisomal l-oxidanoo. Hence an mthnacspcacs difference in biological response appears to exist between rodent and human hepaioeytes. Intummary, it is poemlated that the spmea difference in the hepaiocardnogw* nidty ofTRI is due to species dtenua in peroxisome proliferation, which, in turn, is a result of difTerenca in the rue of formation of TCA from TRL On this basis it is suggested that TRI presents no significant hepatocardnogeoie hazard to man since <1) human hepaioeytes produced TCA at a rate less than rat hepatocytes and (2) TCA was not a poomoms proliferator in human hepatoeyto. Indeed this assertion is substantiated by amoral epidemiological studies which have de monstrated no increased of liver rumours in humans exposed to trichloroethylene (Asdson et si. 1778; Maick et aL. 1979: Novotna et aL. 1979; Paddle. 1983). Airtiiw O. AnQguoeX.HvivMeiCHaWWxa.MoiimaMP.dg Vrrmnr a(1V7S) Acoaotmndyon Itx--rjM S and Vo Pn--o 1L (1971) C--Max Mnd--f of thn anmaopta meftloiooiByitM m anyone hbdhmoI pnnaiand tonopoooiONA ie mt. rRn J>: T7S-T10. Baraea H. Malawi)* C Bum A aae Fhnao G (1977) Muufeue and aikvtaoaf flmiDOliiB of iiwiow rtlaoxiwa<iww MO nnawww prertneod *T n u or huteaa ihvr bbml Ana Tosaoot 41: J<W7I. ftTiaoSaor JWUVJDAi I u eraskdewn ofhjOwnw 19S: 133--(40. Bolt HM and Fiber JG <1977) IrimmO Mediae dOiamuM cUrytmo to oaowoMta Eonroo HaJtb Pinyon 21: 107-uZ Bed HM. BnaOtw A. WetowMi C G3 DL uO BoM W (1X77) Inrannucm <m "*Cjicftli11milifmm apoor mil rmt l/w amMao: ^m*S of ndwonwiy and mwok pnxoa Mndidj of iiMiaholiiM lex Aim Oeea* tema Hold 39: IO>*l 11. BroniMos M. Indoa NCand La Own f <t979) Fany aod Wincaw Bioodva in Comm a: I0XM03*. f -- ^ /e /'v O O \J * -W 1 CJU > C. 2mm K aad Fm D (1*71) < PUM TmmI t: 4IMIL OLFtma IS aadGm T(190TWr ; 24: < ai m' cneaioronaytaaa ia 1 Emm IE*adTB**a7:S*J2. I rrCA) fomoaa iwoan mm ia mddowataytaaa (TM. TB* 0(1973) Mimnatyai 124: 200917. Hm L Sudtfi W aad Kit v (INS) Nnw of I by ky* Uiwmat NO. Baddy MK. whSA wri BaMyJK(19Bl) I t at two* ------- prta . &Bf ait BoaMaoaa m tan M [ rhlnra 4 <? ) tylidlaoH* I ite !! pir/kl tin. Car....ipami 2: 643-430. m ----'*--L"***fT*TT)"--------------------------------- -- r--r---. J BM Qm 1*3: 20S-I73. emumttotiun. fawraUttamj"'124-124? W,W*"""*** ^ ,mn* M--wCP.Pia--U.TB--JaadOtfr9idpn(l979)l m J Bio* Ohb 234:4304391 ot," M(UUKym [rota eoauoi Mqm J. Otn J. im0. SO mm Jotmom JE (1977) Ufawai: Rad amtim.la-.emm tidaabn Biatop.VH.pp m-492 (Ed WA Pryor). Irm--fnaa (7 Yatkk MidbUl AM. Bndyn JW HmN CX (1904) ! Moady OC aad Baddy JK (l*l) BpamM (nnabady) pratiteaaM no M < . Tm4 Dnal *5: 497-304. SHEW 9bM No<NIH) 74-MX ' '~ M * CAS N TM1-4- i <190 NnM Tiiiaidnu hva draft Raponar 4: 747-741. Miwm Dand A. Matak B (1979) Aor pay ' :1m l nwaot'i 31: 121-122. PmrnrnGunnTt--------- nn niiimin KAyto*uoiaowa:Jmamdy by ladmvy USaa MmmPNONS KMOtadrCIadk rtaaato i*CO aad iau i ond (fear ONA ia as mb mob. 1 Tmnl Eawrcm Hcaitn 9: 797-413. o/a Baddy JK. AanMTDLaiifipMadMI Hfpgtmdoie mua ptronMwfmil'nunfwa m aavat dan at' rtumnl araaooa. Mama 33: 397-391. Baddy JlLUlwaat NO. Baddy M K aid Qom BA (190 Emww amnialaooa at raiwli lipot'aaoa m tda inw datnc da AM. Quan 39 ot fhanracnt 53: 237-21*. ToaaeDt Apgk Sanaa V9. KaMoaBn K aad Tatwf BC (1977) Ma dtadtadnalng BA Bridpa aad FH Sotafcl. pp 249-2*. 2w"tf.Snn '(atlaad. Annndan. Ska- >an A. Rc..l R_ Fnafet C aad Udkka R (1990) TriedoranlirlMt vapovn do imyradat dominant Itta. -muaxtoaa n malm on And Towcot *t: 37--M. Stair WT.Quan." -- --------------->------------ ---.... . at ineMoraat.-- *r ia Mot aad ran Tiaal Aprs '`--irnncoi 2; IJ7-15I. Svoboda DJ. Gran :( aad AzatooifDL(19d7) Mkaododw ia cxocr-mrataiN 33: 127-132. -nU. J Cell Biot r 038^ S^ C R. Scoaoc 1 yo*L 1 Environ Thcrso7: S4-i 4ctmUD0 iy*o 'TRTL The ncra tod po*nm rodueod by avt^WHaiBBoau MUnlidiMt>2* css 2; 64S-450. Ui t&c folia pMool so* ary pu-uuaomxl fitly daUbno mod i (New US fed plain-....... AS Ho 79-01-d. tmdy bynMtutry ..r--' siitruon form s r siuasluorexxnt r. Tam* Appi i/ica io anolun* asl ?p 2S9-US- do oat prooucs 'jiar usicracuons arils. J Ceil Bi 1 SL 038741 Tti PratiCnoo* Thosp* , Bofe HM. aOn-f.hiiiilwpSs^ KooF 0901 is ms*. Earroc V<aoofrapp Ho 4. Euiopsss < > sod Toncotofy Cents*. Idpa. Uchkta H sad PoptawiSs-Tsboswtfi S U9T7) tsmoroMs lii*ita> of *C.l*Mlod ao In* ooaamami a w* sad in woo. AM Tasm 37: 239-294. Va Dwe BL sad lump S (19W1 Comm ipirtiis of MMOotaa of tbs WsLMHiLn97I>AM*yofsfaoaWMSwyofosM>*MsoOtOswttOo*iM Mrax Rm57 content ad kidney 138. S_ and OkahaRa. mctaUotluonctn m .238-266. D.(l983).Concene ortans of ns after Fundam. Appi. Tax- dmuifn-tluoncm. In nd Biology of Cad431. Elicvicr/NonA neiiu. to The Chem> of Cadmium (M. icr/Nonh Holland. ein and Uie nephro* tm Symposium (M. pp. 101-107. Freid- TOXICOLOGY wvOAftnJIDMLUMACCLOCY 92. *0^- i 1211983V The Role of Trichloroacetic Acid and Peroxisome Proliferation in th Differences in Carcinogenicity of Perchloroethytene in the Mouse and Rat J. Odum. T. Green,1 J. R. Foster, and P. M. Hext Imperial Chemical !ndustnts PLC. Central Toxicology Laboratory. Aldtriev Park. Sr Macclesfield. Cheshire SK1Q 47V. Inued Kingdom Received JuneS. 1997; accepted September 21.19S7 Thc Role ofTriehioroacene And and Pcroxuomc Proliferation in the Differences in Circmo- getuary of Perehtoraeihylene to the Mouse and Rat. Odum. i. CaZ>. T_ FoileR. J. R.. and HSXT, P.M. 11988). Toxicol. Appl. Pharmacol. 92.103-112. Father 344 no and 86C3FI mice ofboth ictus were exposed to 400 ppm perchforaethyiene (PER) by mhalanoo. 6 hr/day for 14, 21. or 28 days or to 200 ppm for 28 days, inemsed numbers of peraaisoaMs were seen under the elemun and increased peroxisomal cyuudc-tnanunt peiautoyt CoA oxida- non was iiuenunl (3.6-fold..... .. in in males and 2.1-fold increree m frnialm u the liven of me expoeed to PER. Hepeac caiaiase was not loarered. Pmxaome proiiforanon was not oteervad io rat liver or in the kidneys of either speem. Thchloreaeenc sod (TCA), a known ntnnnis n and hepaae perextsome proiifcreaag stent, was found to be a metor metabolite of PER. Blood levels of this metabolite ronamed in man and mm durum and for 48 hr after a antic 6-hr exposure to 400 ppm PER showed that peak Mood IcvMi in mm were 13 times hithm than those seen in tarn Comparison ofareas undm the curvn om the ume course ofthe experiment showed that ram were exposed to 6.7 times mom TCA than nux. The difference in metabolism ofPER to TCA in mm and tats leads to the spamesdifference in hepatic peroxisome pradfoanoo which is believed to be the bass of the speomdifesare in bepaiocaictnofematy. Pnoxisome proiiferauoo docs not appear m play a role in the apparent caroaopemcity of PER m the ret kidney, e ins mi> r>w. lac. Percfaloroethylene (PER) (1.1.2.2-tetrachloroethylene) is a volatile liquid which is used extensively io the dry cleaning industry and as a general degraasam in manufacturing in dustry. A significant increase in hepatocellular car cinoma has been observed in male and fe male mice but not rats in two carcinogenicity bioassays of PER. In the first study (NCI. 1977) Osborne-Mendel rats and B6C3F1 mice received PER by gavage in corn oil at doses of approximately 500 or 1000 mg/kg. Both dose groups showed about a 50% inci dence of hepatocellular carcinoma in mice. In the second study (Mennear eta/.. l986)Fi- 1 To whom eonmoondenee should be addressed. seber 344 rats and B6C3F1 mice were ex posed to PER by inhalation (mice 100 or 200 ppm and tats 200 or 400 ppm 6 hr/day). In creased hepatic tumor incidence was again observed in mice. 50% in low and high dose males and 26 and 72% in low and high dose females. In the latter study a low incidence of kidney tubular adenocarcinoma was bserved in male rats at the highest dose. The species difference in hepatocarcinogenicity is "<! to that seen with trichloro ethylene (TRI) (NCI. 1976; NTP, 1983). TRI has been shown to induce peroxisome prolif eration in mouse liver but not rat liver, after oral administration (Elcombe el aL. 1985). A causal relationship has been suggested be tween hepatic peroxisome prolifeiati n and 103 A/"**- O W- th* *- 004l-00X/g S3J CMMWimuwa SL 0387A2 Z't+ZmT ^ 'T- -i ' . 104 ODUM ET AL. hepatocellular carcinoma in rodents (Reddy et ai. 1980) although no such relationship has yet been shown between renal peroxi some proliferation and renal tubular adeno carcinoma (Reddy et ai.. 1975. 1982). The species difference in hepatic peroxisome pro liferation elicited by TRI is believed to be the basis of the species difference in carcinogenic ity (Elcombe era/.. 1985). Trichloroacetic acid (TCA), a major me tabolite of TRI (Green and Prouc 1985) has recently been shown to be carcinogenic in the B6C3F1 mouse (Herren-Freund etai. 1986). TCA has also been shown to be responsible for peroxisome proliferation in TRI-dosed mice (Elcombe. 1985). Quantitative differ ences in the metabolism of TRI in rats and mice and hence in circulating levels of TCA (Prout et ai.. 1985) may lead to the species difference in peroxisome proliferation and consequent carcinogenicity. TCA is reported to be a major metabolite of PER in mice and rats (YUner, 1961; Daniel 1963; Deleant et ai.. 1985) and may therefore elicit the same response when animals are exposed to PER. In view of the lack of mutagenicity of PER (Greim et ai.. 1975; Baruch et ai.. 1979) this may be the basis for the species difference in carcinogenicity. In this paper the pathological and bio chemical changes in rat and mouse liver and kidney, with particular regard to peroxisome proliferation, were determined after inhala tion exposure to PER. Blood levels of TCA in rats and mice exposed to PER were also determined. The animal strains and dose lev els used in the 1985 PER bioassay (Mennear et ai.. 1986) were adopted in order to assess the relevance of our results to the develop ment of tumors in these animals. Materials METHODS I.I.L2-Tctiachlaraeihyfcne (Atuiv pads. 99.9*> and tnddoreemic acid fAnelar padc, 99* pun) wn obtained from BDH Chanucah PLC (Poo*. Dona. UK.). BiachcsmJf were obtained from Sigma Chemical Co. (Poole. Donee UK). Animals Male and female Fucher 344 tan <160-130 j) and male and female B6C3FI rake (23-23 |) were supplied by Charles River i Margate. Kent. UK). Animals were multiply boused in suspended uainlcis steel who man cages in tong-tom inhilstion exposure chambers, equipped with a (2-hr light cycle, prior to. during. and after exposure. They received food (PCD diet. Special Diets Services Ltd.. Withxm. Esses. l'K) and water libitum before and after, but not dunng esposure. Exposun to PERfor up to IS Days Exposure. Male and female nts and mice (5 per group) were exposed to conctnoanew at 200 or 400 ppm of PER for* hr/day for 14.21. or21 conmcuiive days. Con trol animals were eapomd matronly, but otherwise were treated in a manner nmdmto that ofthe ten animals. Exposures were whole body m stainless smcl chambers (Doe and Tiirtwm, 1911) having an internal volume of approximately 3.4 mJ. The dwnthsn were air coodtnoned to have a oommai npmmre of 22*C and rela tive humidity of40-60%. Theaw*ow through the chamban eras 300 Iften/mia. Atmombcm were generated by pasnng repotand PER into the input air of the caambars. Atmospheres in the ten chambers were analyzed for PER by gaseftromamgrapby (CO on a Hewlett-Packard IMOA GC (Same tomanoe dctactori fitted with a Porapak PS column (IJnxi ora). The column tempera ture was I93"C helium earner ps 30 ml/min. Eighteen hours after the taampoture period, were tailed by overexposure w hahwhanc (Fluothanc. Imperial Chemkal Indmuim PIC Phaimaeeuoals DivtBoe) and exsanguinated. The Even and kidneys were rapidly teraored. wmghwL and then divided to provide nous far light mkramopy. demon microscopy, and biochenucal analyse. Light microscopy. Shorn ofEvcr and kidney were fixed in 10* ocuoxl budded fotmol Miine. dehydrated through an amending ethanol wnes. and embedded in paraffin wax. Sections (3 ml vert cut and stained with hematoxylin and eosia. Electron microscopy. Tioum were fixed in 3* glutaraJdchyde in 0.1 m sodium phosphate buffer, dehydrated, and embedded in epoxy rehn, Secuows (t >o) woe cut and sained with I* tntaidiar blue m 1* borax for light microscopy. Areas were mleeiad from the centnlobular repoas of dm liven and the S3 lepoos of the proximal tubules ofthe kidney far ctnuu mmomopy. Ultrathin SL 038743 f+ ** r\f\ ^ u W W .i perchloroethylene and peroxisomes 105 TABLE 1 PEROXISOMAL CYAMIDt-lNSE-NSrnVH pALMtTOYl. COCNZYME A OXIDATION IN RaT ANO MOUSE UV1E and Kidney*m ExiqmitoPER CN-insensave {wimitoM CdA oxidation (nmol/mui/mt protein) Concentnoon (ppmi Duration (days) Mouse Liror Rat Kidncv Mouse Rat Male 0 zoo 400 400 400 Female 0 ZOO 400 400 400 14-28 ZS 14 21 Z3 14-23 28 14 zi 23 3.16 n 1.06* IU9 = 4,4" 11.98 = 2.86" 13.90 = 3.27" 18.64 = 3.61" 9.01 = 1.62 16.68 = 3.32" 14 40 = 127" 18.74= 1.68" 17.99 = 2.33" 10.26=0.31 12.95 =0.93* 13.68 = 1.68" 12.94=0.81" 13.61 =0.89" 12.62 =0.77 13.76= 1.06" 14.90 = 1.91* 13Jl = 2J1* 14.14 = 1.90 3.37* 6.92 7.69 8.70 8.18 2.48 2.83 2-39 128 2.49 137 = 0--9 2.98=0.21" 197=0.65 144 = 0.49 176=0.33 1.98=0.21 3.11 =0.43" 136 * 0.23" 168 = 0-26" 141=0.19" Salt. Control animals were exposed to air only for |4, 21.or 28 day*. * Values are x = SD. n 5 except (or contrail. where n - 13. * Mouse kidney* were pooled acetxdinf to poup; valuesaic Statistically ppitew. p < 0.03. " Statistically spuBouit. p < 0.01. and text atrite and viewed and photofrapbed in a JOEL JEM I0OCX electron micreacopa. Morpoometnc analy se of peroxisomo waa performed according to the pea* eni principles of Weibel ft oL (1964) oo electron micro |i min ofiitii nfrytnpliim n i migniftrwinn nf?1 flftft Bioditmtcai anaiytts. Secoona ofEver and Iddaey ra mining after Baton had been taken for light aad electron mkweeapy were placed in ice-cold sucroee (230 bm) EDTa (3.4 mMl Tria-Hd (20 an) bufler. pH 7.4. Mouse kidney* were pooled according to group. Hotnog- enaias (23% w/v approximately) vrora prepared using a Teflon glass homoycmxer at 4*C Homogenates were centnfriged at 3000ff for 3 min at *C The supernatant* from the kidney homogenates were stored at-7(rCunal wed. Supernatants (tom Use iiver homogenates wcr fur ther centnfriged at 13.000f for tS min at 4*C. aa de scribed by Elcombe <r aL (1983). Tim supernatants were disardd and the pellets (eootaininc pooairomes) were mupended to the above buffer and stored at -70"C The protein content of the liver and kidney fractions wea determined by the method of Lowry at oL (1931V The acBvtnea of the peroxisomal enzymes tatalew and ryanxie inmiamve peinutoyt coenzyme Aoaidaaewere determined by thn methods ofBemiand Sizer< (932) and Brontean <r oL (1979V resoecuvety. -- 7*Ct Concrmrations in Blood arirr Expamrr to PER Raa and auce were exposed to 400 ppm PER for up to 6 hr. A impall killed at tune points of lees than 6 hr rot capemri in (lass denccatoo at a flow rate of 3-10 liSBi/tmn. Item expoaed for the frill 6 hr rot homed in the tone ana chambers described afow. Atmospheres were gemsmied by vaponmng PER into the m stream aad warn momtoted by pm dramupe phy. Croups of three tea or three mice were killed by apoHn m COi and Wed by midias puncmie at inter* vet* from the start of exposure until 48 hr ptmexpoeure (see Fig. 2V TCA was extracted from btaod asdmaibad by Preut m mL (1913) aad the methylated mmrlm west nalifl oe a Hewiert-Packnrd 3890a pa chromato graph 6fdaithn skill un mpoite detector. A glasscoiumn (2 m x 2 mmV pecked with Pempak PS and oper and at IKTC with a nitrogen earner gas flow of 23 mi/ min, waa k1 for the analysis. Under them coodidoea TCA had a tetenoon amt of 3.7 min. The limit ofdetecnon far TCA in Mood waa 02. me/mV 106 ODUM ET AL. RESULTS Effects ofExposure to PER The mean analyzed concentrations of PER for the 28 days of exposure were 193 and 389 ppm for the rats and 196 and 395 ppm for the mice. These were close to the target levels of 200 and 400 ppm. No significant clinical ab normalities were seen in rats or mice exposed to either concentration of PER. Liver TABLE2 Peroxisomal Catalase activity in Rat and Mouse Liver after Exposureto PER Cooccntntion (ppm> Duration (davit l"sla*a (tac*1 m protein'1) Most Rat Male 0 400 400 400 Female 0 400 400 400 14-28 14 21 28 14-28 14 21 23 1.05 s 1.17* 1.12S0.19 1J0s0.IT* 1.44 sOJ4* 1.79 s0.12 1_56 =0.09 1.15 s 0.0J 1.76 sOJS 1.62 s 0.26 US = 0.57 1.62 s 0.21 1.90 = 0.15 1.56 = 0.25 1.44 = 0.15 1.77=0.50 1.59 = 0.24 Exposure of B6C3F1 mice to 400 ppm PER for either 14. 21. or 28 days resulted in small but statistically significant increases in liver/body weight ratios up to 1.2- and 1.3fold in males and females, respectively. F344 rats exposed to PER showed no changes in liver/body weight ratios. Cyanide (CNVinsenstttve paimitovl CoA oxidase, a marker for peroxisomal ^-oxida tion was significantly increased in mouse liver after exposure to PER (Table I). This enzyme increased to a similar level in males and females but the control rate ofCN-insensidve paimitoyl CoA oxidation was higher m females. Therefore the increase over control rates was tower in females than males and maximum response (seen after 28 days expo sure to 400 ppm) was a 3.6-fold increase in males and a 2.1 -fold increase in females. In contrast, only small increases in CN-insensitive paimitoyl CoA oxidadon were ob served following treatment of F344 rats with PER (Table 1) although these were some times statistically significant. The maximum increase (l ,3-fold) was in males exposed to 400 ppm for 28 days. The basal activity of CN-insensitive paimitoyl CoA oxidadon was noted to be approximately 2-fold greater in F344 rats than in B6C3F1 mice (Table 1). Catalase, another peroxisomal enzyme, was largely unaffected in mice and rats ex- Vofe. Control animals ww npnarrl mar only 14.21. or 2S days. * VaiuM wi: SD. it - 5 raeept for coomb, when n m IS. * Statistically signiftaaLp < OUOS. posed to PER (Table 2X The only increases (up to 1.4-fold) were observed in male mice exposed to 400 ppm. By light microscopy the liven f mice ex posed to 400 ppm PER showed centhl buiar eosinophilia and centhiobuiar fatty vacuoialion. Both effects were seen to a similar extent in males and females and the numbers ofani mals affected increased from 14 to 28 days. Similar effects on lipid were seen at the elec tron microscope level in mice exposed to 200 ppm for 28 days or 400 ppm for 14,21. or 28 days. Extensive lipid accumulad n was ob served in centhiobuiar hepaxocytes. The lipid was present in the form of large droplets. 2to 5-um diameter, lying free in the cytoplasm of the cells (macrovesides). and small drop lets. 0.1- to 0.5-ttm diameter, contained within the cisterna ofthe endoplasmic reticu lum (microvesicles). Figure l sta ws the ultrastructural appearance of a centhiobuiar hepatocyte from (a) an untreated male mouse and (b) a male mouse exposed to 400 ppm PER for 28 days. Electron microscopy showed proliferation of peroxisomes in the Ftc. l. (a) (jUrastructural appearance of a centnlobular hepacocytc from an untreated male B6C3F1 mouse showing nucleus IN)and peroxisomes! H ).x6300,(b)UUrastniaBfalappearneeofaeeninlobular hepetocytc from a male mouse exposed to 400 ppm PER 6 hr/day for 28 days. The cell shows an accumula tion of lipid in the form of tarfe droplets (L) and small vesicles and a proWerioon of peroxisomes (R ). The nudeua is seen at(N). *6300. Insert: higher magnification ofperoxisome showing electron dense cote, x 19.800. 107 '''i *- lUo ODUM ET Al_ TABLE 3 Morphometric analysis of Hef vtic Peroxisomes in Mice and Rats Exposed to PER Exposure Coacesmnon (ppfflt Dureuon (davit Perousome volume cytoplasm) Moure Rat Male Female Male Female 0 :s IS = 0.6* 15=0.3 3.1 =0.1 3.3=0.7 n II hn. ZOO :s 3.2= 1.5* 44*0.6* 4.7 = 1.9 * II *ca 14 4.9= 1.5* 13=0.6 17=0.5 400 :i 5.4= t.3* 4.1 *0.9* 13=0.7 12= l.l 400 :s 6.0= !.4" 4.3 = 1.2* 3.4- 1.4 3.4= 1.0 * Vaiua (x = SD. a-5) ire calculated from three microinpta per vumil. with 37 J pomu applied to each micro- inph. * Suusically jifninnhL p < 0.01. cemriiobuUr region of the mouse liver (Fig. lb. Table 3). The proliferated peroxisomes were small (<0.5 and the majority re* tained the central nucleoid (Fig. lb. insert). Exposure to 400 or 200 ppm resulted in sia~ osncaily significant increases in the volume of cytoplasm occupied by peroxisomes (Ta ble 3). Exposure of male mice to 200 or 400 ppm PER also resulted in a decrease in mitochon dria after 14 days but this was followed by mi tochondrial proliferation in those animals subsequently exposed to 400 ppm. The effect was not seen in females. Concomitant with these changes, exposure at either level for any of the time periods investigated resulted in a decrease in the amount of normal rough en doplasmic rebcuium in the cells. Light microscopic examination of livers from rats exposed to PER showed centrilobu- lar hypertrophy in both sexes with a concom itant loss of glycogen. The effects in males were ofsimilar intensity in both the 200- and 400-ppm dose groups and there was little evi dence of progression of the lesion from 14 to 28 days in the 400-ppm group. Results sug gest that the males were more sensitive to the liver hypertrophic effects of PER since no effect was seen in females exposed to 200 ppm for 28 days. Electron microscopy showed a time-de pendent proliferation of smooth endoplas mic reticulum in the liver in both sexes which correlated well with the centrilobular hyper trophy. The males were more susceptible than the females. There was n dose-or timedependant increase in peroxisomes in the liv en ofeither sex (Table 3). Kidney No increases in kidnev/body weight ratios were seen in rats and mice exposed to PER. The effect ofPER on peroxisomal cyanideinsensitive palmitoyi CoA oxidation in rats and mice is shown in Table 1. Insufficient mouse kidney tissue precluded the measure ment of this marker in individual animals Consequently values could not be tested for statistical significance. Slight increases were seen in 4-oxidation in male mouse kidney, the maximum being a 1.6-fold increase after 21 days exposure to 400 ppm. Small increases in this marker were also observed in female rat kidneys after exposure to PER (Table l) up to a maximum of 1.6-fold. There was no effect of PER on renal cata lase activity in rats or mice of either sex (data not shown). PERCHIOROETHYLENE AND PEROXISOMES 109 to. and the metabolites which have been identi fied support this assumption (Yllner. 1961: Daniel. 1963: Bonse et ai. 1973: Sakamoto. 1976). Alkylation of nucleotides by reactive epoxides has been described for other chlori nated aikenes such as vinyl chloride (Laib and Bolt. 1977) and vinylidene chloride (Re itz et ai.. 1980). DNA binding has not how ever been demonstrated after treatment of rats and mice with PER (Schumann et ai.. 1980) nor does PER induce gene mutations FlO. Blood leveii of TCA in mice tnd ran exposed to PER (400 ppm) for 6 hr and recovery. Value are means "ith three animals to each ume point in bacteria (Baruch et ai.. 1979: Greim et ai.. 1975; Bronzetti et ai.. 1983). These observa tions led to proposals (Schumann etai.. 1980) that PER-induced liver tumors in B6C3F1 mice are a result of recurrent cytotoxicity and No compound-related changes were ob enhancement of the high spontaneous inci served in the kidneys of either species at the dence of liver tumors found in this strain of light or electron microscope level. mouse. The results of the present study suggest an Blood Leveis of TCA after Exposure to PER alternative hypothesis for the mechanism of PER-induced carcinogenicity, that of peroxi Blood levels of TCA in rats and mice dur ing and after a 6-hr exposure to PER (400 ppm) are shown in Fig. 2. Peak blood levels of TCA (approximately 130 isft/mi) in mice were reached 3-4 hr after the end ofthe expo sure period and thereafter declined with a haif-tife of 7-8 hr. Forty hours afterexposure, levels persisted at 3-- 10 Mg/ml. In contrast blood levels in the rat reached a plateau of approximately 7 pg/ml after 3 hr ofexposure declining to 4 Mg/ml 48 hr after the end of exposure. Comparison of the concentrations of TCA to which the two species were ex posed. by caiculauon of the area under the curves, shows the mouse to have been ex posed to 6.7-fold more TCA than the rat. some proliferation leading to cancer forma tion via an epigenetic mechanism (Reddy et oL. 1980). Exposure of male and female mice to PER for up to 28 days resulted in a signifi cant proliferation of peroxisomes in the liver as measured by peroxisomal 0-oxidation (Ta ble l) and morphometric analysis of electron micrographs (Table 3). The induction of per oxisomal d-oxidadon in this study was not accompanied by increases in catalase (Table 2). This phenomenon has been observed after administration of other peroxisome prolif eraters (Cohen and Grasso. 1981; Reddy and Lalwani. 1983. for review) and is believed to lead to increased levels of hydrogen peroxide in the cell, causing oxidative damage, cyto toxicity, and possibly DNA damage. How. ever, a definite link between such changes DISCUSSION and the eventual development of cancer re mains to be established. The bepatocarcinogeniciry of PER in the Although increases in the activity of per mouse has been known for some yean but no oxisomal enzymes were observed in the livers satisfactory mechanism, either genotoxic or of rats after treatment with PER (Table 1). epigenetic, has so far been proposed. The first these were slight compared to the changes step in the metabolism of PER is believed to seen in the mouse and could not be corrobo be xidatzon to an epoxide (Reichert, 1983) rated by electron microscopy (Table 3). Sim- 110 ODL'M ET AL. iiar results after treatment of rats and mice with PER have recently been reported by Goldsworthy and Popp (1987). A sixfold in crease in hepatic CN-insensitive palmitoyl CoA oxidase was observed in B6C3FI mice dosed PER by gavage at 1000 mg/kg for 10 days. No such increase was seen in F3-U rats. This species difference in peroxisome prolif eration is identical to that found by Elcombe et ai. (1983) after TRI was dosed by gavage to mice and rats. There were however differ ences in the pathology of treated liven be tween the present study and that reported by Elcombe et ai. (1985) for TRI. PER-induced peroxisome proliferation was observed in the centrilobular region of mouse liver. The in duced peroxisomes were small and retained the nucleoid core, whereas after TRI treat ment they generally lacked the nucleoid core. Exposure to PER also resulted in a concomi tant accumulation of lipid in centrilobular cells with periportal ceils unaffected. The rea sons for these differences are unknown but may be due to the effects of other metabolites or the parent chemical. TCA. the major metabolite of PER (Dekant et al.. 1985). is a known hepatic peroxi some proliferator in both rats and mice (El combe. 1985) and is the metabolite responsi ble for increased peroxisomes in mice exposed to TRI in previous studies (Elcombe et ai.. 1985). In the present study, TCA aris ing from metabolism of PER only induced hepatic peroxisome proliferation in mice be cause of the much higher concentrations of this metabolite in mouse blood. The lack of a response in rats indicates that a threshold concentration of TCA has to be reached in order to induce peroxisome proliferation in rodent liver. The low blood levels ofTCA ob served in rats compared with mice correlates with the lower rate of oxidative metabolism of PER in rats than mice (Schumann et al., 1980: Ikeda and Ohtsuji, 1972). Recent studies have confirmed that TCA is in fact a carcinogen in B6C3FI mice (HerrenFreund et ai.. 1986). TCA dosed to male mice in drinking water at 5 g/liter for 61 weeks pro duced a 50o tumor incidence compared to a 5% incidence in the control group. Peroxi some proliferation was observed in the livers of treated animals. Thus the species differ ence in the carciaagenicity of PER between rats and mice may be explained by the marked difference in blood levels ofTCA and a mechanism which induces peroxisome pro liferation. The effect of PER on the kidney m mice and rats was minimal (Table 1). No com pound-related changes were seen at the light or electron microscope level in regions of the nephron where peroxisomes are known to be most prevalent (Beard and Novikoff. 19691. Similarly the increases observed in peroxi somal enzymes were slight and not related to dose or exposure: Peroxisome proliferation is therefore unlikely to play a role in the carci nogenicity of PER in the rat kidney and fur ther lnvesnganom are needed to establish an alternative mechanism. Metabolism ofPER in man is known to oc cur at a very stow rate (Fernandez aaL. 1976: Monster et aL 1979). It is also a saturable process, saturation occurring at the low inhalahooal exposure level of 100 ppm (Ikeda et ai.. 1972:Ohtsukia/.. 1983). Consequently man is exposed to lower concentrations of TCA than mice or tats. Furthermore. TCA does not induce peroxisome protiferanon in vtiro m human hepaiocytes (Elcombe. 1985); indeed the response of primates to the induc tion of peroxisome proliferation by other agents is generally much lower than that of rodent species (Cohen and Grasso. 1981: Reddy and Lahoni. 1983). In conclusion this study demonstrates that quantitative didactics in the metabolism of PERtoTCAiamiceandratsteadt prolifer ation of peroxisomes in the livers of mice but not rats. The known carcinogenicity of TCA in B6C3F1 mice and the correlation between hepatic peroxisome proliferad n and cancer in rodents strongly suggests that TCA-induced peroxisomeproliferation is the basis of the species difference in hepatocarcinogenicity of PER. The limited capacity of humans PERCHLORQETHYLENE AND PEROXISOMES 111 to metabolize PER coupled with an intrinsic deficiency in response to TCA as a per oxisome proiiferator indicates that PER is unlikely to cause hepatocellular carcinoma DOR. 3. E_ ano Tinston. D. J. (19811. Novel chambers for long term inhalation studies. In Prooaaings of'the Inhalation Toxicology and Technology Symposium (8 K. 1. Leong. Ed.1. pp. 77-88. Ann Arbor Science Pub. Ann Arbor. Ml. in man. ElCOMBL C. R. 11985). Species differences in caromogeoscity and peroxisome proliferation due to trichloro ethylene A biochemical human hoard asamament. ACKNOWLEDGMENTS Arch. Toxicol. (Suppi.) t. 6-17. Elcombc. G R_ Rost. M. S_ and Pratt. L S. < 19151. The authors thank Mr. S. Millward and Mr. 1. Basnets for carrying out the inhalation exposures and Mr. N. Cowans and Mr. W. M. Provan for thetr help with the TCA blood lev el study. Biochemical, histological, and uluastructurai changes in rat sad mouse liver following the admimuraaon of mchloroethylene: Posable relevance to roeam differ ences in hepatocarcinogemcity. Toxicol. Appl. Phar macol. 79. 365-376. Fernandez. J.. Guberman. E.. and Caperos. J. REFERENCES (19761. Experimental human exposures to tctnchloroethylene vapour and elimination in breath after inha lation. Amor. Ind. Hyg. .Assoc. J. 37,143-150. BaHTSCH. H.. Mauvveille. c.. Barun. A, and PIanche. G. (19791. Mutagenic and alkylating me tabolites of hxlo-tthvlenes. chlorobutadiene and dkhlorebutene* produced by rodent or human liver mam Evidence for oatrane formauon by P430 linked mscrosomal mono-otygenasea Arch. ToxtcoL 41.249-277. Goldsworthy. T. t_ and Popp. J. a. (19ET). Chlon* sated hydrocarbon induced peroxisomeenzyme activ ity in relation io species and organ carcinogenicity ToxtcoL Appl. Pharmacol. SB. 225-233. Green. T- and Pxout. M. S. (19831. Spaom differ ences in response to trichloroethylene. 1L Biotransfor- Beard. M. E-. and Novikopf. A. B. (19691. Dwnbu- mason in rats and mice. ToxtcoL Appt. Pharmacol. 79. hon of peroxisomes (microbodie m the nephron of 40i-m. the rat. A cytochcmioi study. J. Cell Bid. 42. SOI-- Genu, M- Bonse. G. Raowan. T~ Reichert. D.. SIS. and HinsCHLER. D. (19731. Munoewmiy in ruto Burs. R- F.. and Sizer. 1. w. < 19521. a spcctropboto and potential carcinogenicity ofchlorinated cihytcnes metric method of measuring the breakdown ofhydro gen peroxide by catalase. J. Burl. Chem. 195.13J-I40, BONSE. G-. L'REaN. Th.. REICHERT. D.. AND H0OCH. Ler. D. (19751. Chemical reactivity, metabolic oxrane formauon and biological raacuvtty of chlonnaied etfiyienes in the isolated penused rat liver preparation. Btocnem. Pharmacol. 24,1129-1834. Bronfman. M.. Inestoosa. N. C. and Leighton. F. (19791. Fatty acid oxidation by human liver peroxi somes. Biochem. Biophys. Res. Common. EE. 1030- 1036. Bronzettt. G- Bauer. C.. Corsl C- Dei Carra. TORE. R-. GaLU. A.. NlERL R-. AND PAOUNL M. (19831. Geneuc and biochemical studies on perdilorocthyiene "in *aro" and "in vivo." Muiat. Rat 116. 323-331. Cohen, a. j.. and GRaSSO. P. (I9S1V. Review of the hep*uc response to hypoiipidaemic drugs ta rodents and assessment ofin toxicoiogml tramocanee to man. Food Cosmrt. Toxicol. 19.585-605. Daniel. J. w, (19631. The metabolism of "Cl-labeled mchloroethylene and tetnchlotoethylenc in the rat. Biocntm. Pharmacol. 12.795-802. DtRANT. W., Hang. R-. and Henecmur. D. (19831. Absorption, elimwsuon and metabolism of teoachltN methylene. Saunyn-Schmtedeberts Arch. Pharmacol. (Suppi.) 329, R24. as a function of metabolic oairane formation. Biod*m Pharmacol. 24.2013-2017. Herren-Freund. S. I_ Pereira, m. a_ Olsen. G.. and De angelo. A. B. (19861. The cuonogcaeity of mchloroethylene and its metabolites, tnchloroacznc sad and dichloroaeeuc acid in mouse liver. Proc. Amtr .Assoc. Cancer Res. 27.91. IKETM. M_ and OKTSUJL H. (19721. A comperauve sudy of the excretion of Fujiwara tncuoo-pomuvc m unne of humans and rodents given akhloro or tctnchloro-dcnvauves of ethane and eth ylene. Br J. Ind. Mad. 29,99-104. Ikeba. M- Ohtsuii. H- Imamura. T.. and Komoike. Y. (1972). Urinary excretion of total wchloro-compounds. tnchloroethanoi and tnchloraeeeiic acid as a measure of exposure to trichloroethylene and teuaehloroethyteee. Br. J. Ind. Mad. 29.328-333. Lair. R. i_ andBolt. H. M. (1977>. Aikyianon ofRNA by vinyl chloride metabolites in vitro and in vivo: For mauon of I AV-ethenO-adenostne. Toxicology t, 183195. Lowry. O. H.. Rosebrough. N. S,, Farr. a. l_ and Randall. R. J. (1951). Protein measurement with the Foiin phenol reagcnL J. Biol. Cham. 193.265-Z75. MEnnear. J. H.. Claru. W.. Ragan, H_ and MILLER. R. (1986k. Toxicology and carciDogmucsiy rt A*" L ,. w . ->Md 112 ODUM ET AL. studies of inhaled tenehioronhylene i PERO in male and female F344 rata and 36C3FI mice. Toxicologist 4,135. Monster, a. C_ Boersma. C_ and stunweg. H. (1979). Kinctn of tetrachioraetnylene in volunteers: Influence of exposure concentration and work load. In. Aren. Occur. fmmi Health 42.303-309. National Cancer institute (1976), Carcinogenesis Bloat lav of TnchloroethUene. CAS No. 79-01-6. DHEW pub. No. (NIH) 76-601 National Cancer Institute (1977). Bioasserv ot'TrtreehloroethUene for Possible Carcinogenicity. DHEW pub. No. 77-413. National Toxicology Program (1983). National Toxicol ogy Program draft report absmeta on nine chemical armnoccncss animal tnoaauva. Chem. Regal. Rtp. 6. 767-76*. Ohtsukl T., Sato. JC. Koizumi, a, Kumai. m.. and IKEDa. M. (1913). Limned opacity of humans to metabolisc tetnchloroethylcnc. In. Arch. Environ. Health SI. 381-390. Prout. M. s,, Provan, w. m_ and Green. T. (19*3). Species differences in response to trichloroethylene. I. Pbarmacokincus in rats and mice. Toxicol. Appi. Pharmacol. 79.3*9-400. Reddy, J. K- azarnoff. D. L. and Hignite. C. E. (1980). Hypoiipidacmsc bepane peroxisome prolifcraton form a novel class ofcbesuol carcmopcns. Harare (London 2*3.397-39*. Ridoy. J.1C. and Ulwanu N. D. (1983). Carcinogenmi* by hepatic peroxisome praiifcraion: Evaluation of the nsk of hypobomettnc drags and industrial plasooxen to humans. CRC Crit. Re*. Toxicol 12. 1-58. Reddy, j. 1C. keomkakamtha. T. p.. and Tao. m. s. (1975). Mtanbody (ptroxHomei protiferanon in mouse kidney induced by methyl dofenapate. VirchmnArdi. B Ceil Pathol 17.295-306. Reddy. J. K.. Warren. J. R_ Ridoy. M. K.. and Lai.. wanl N. D. (1982V Hepanc and renal eiiect of peroxtsomc pralilcmsasK Biotapol impliatrana. Ann. .V. Y Acad. Sci. 3S6v*l-l 10. Reichert. D. (1913V Biotogkal xenonsand interactions oftetiachlorocfkytene. Muter. Res. 123.411 -t29. Rettz. R. H_ WaTanabr. P. G.. McKenna. M. J.. QUatt. J. F- and GtNRJNC. P. J. (1980). Effects of vinyiidene chloride on DNA synthesis and DNA re pair in the tat sad mouse: A comparative study with dimethytnmwimina. Toxicol Appi. Pharmacol 52. 357-J70. Sakamoto. N. (1976). Metabolism of tctrachloroethylenc in gurnet pi^yapdn./ Health IS. U-|6. Schumann a. m_ Quasi-. J. F,, ano Watanase. P. G. (1980). The pharmacokinetics and maeromolecutar interactions of perchioroethyicnc tn mice and mo as related to onengensaxy. Toxicol. Appi. Pharmacol 35. 207-219. Weiam. E. R_ Staubu. w,, Gnage. H. R_ and Hess. F. (1964). Conetnod morphometnc aad biocnemm msdieson the kwoSL LMorpbomctnc model stereo. logialmeUsomand normal morphometnc data for tat liver, y. CeUBioL 21.68-91. Yllneh. S. (1961). Urinary membotita of l4C-tenachloronhylene in imrr Harare(London 191.820. 038751 Ip. /\ t . '. j ' ta Ctula* utlk t MM t(t UkUtU M) HI* (HIM III (TAM. TJUOO) iM Mnuu et tl* UtMl (MIM ay a humuI >1 a# trail* mm MUMM it IMM at : nau < lunt* y cat aetata >* tl(K .illMta. Maanauta .ay eta aa rana ra vara na MtiaaM iliuaua Itaataa at tea aaa eaa aataa aaaeia Mat alls aMtiHaialaaaaiae .tieiaiiMiituiaa .IataaaaiaaaaIsauUiamam* a* '1 laiaaat. uaanaitia S taOCl Mila :aa trine ^ .aaeiral Itrtner treat* * let* metTlvetian. i anttaeeateattt trey* live matt''rettea 4 -.aaa tt tiaitiu :;n aae* la eaclr S *aa a alQI f lnaa I r Mpaa uaMaaa a( ____________ (MX) aaa tu rata oaaaaa la aarlr (>S> 3a -- el ! iiauaau attar R aaa MUraa *? S has |a Uieti ti------- mm ttt*at*tat iota la ***** C(. U tt* turn Ut*n, aottaata eate rattiat* * 5OX a OM e( ttu oaa at ON* attaaaia. T>eee tiaUai* ***** Mlu toot Itaeataaaaa aaaaaaaitl 11 ay ta ^tiaaai at lamaianini at SVDX La toll ctalM *-**aaaaa*. vui ;iaa*aat>aaa la earl* 1 tttay rare eeaai- tttaa HeaeeatTtea la earl* ra* Sj taeea aetata ~**a tot tala* la W Maitiiu iallaatay IVW 1tear*eat ** lay feaaaaa oa nil t treat*la* Miuiaeal claa *r at naan *1 ttnayaa m. Weretree ** IX (raata -Mia aaa uan:. e* me aaa rat till) Manat eat mu 1*1/. a Lallan rlataa (Mt)` ti SL 038752 '*flOCZS!MCS 0i* AJ.cn VC_ 7 \ *< >1 1988 a* - / '> .1 ^ Xc i 225 19th Jtreei, N W , Suite iOO. Wasningron. D C 29036-2411 '2021 223-5S9C March 13, 1990 0 6 i V E ti MAH : : Mr. Donald J. Ames Assistant Chief stationary Source Division Air Resources Board Attn: Ferchloroethylene P. 0. Box 2815 Sacramento, CA 95812 Division Air Resource* Board Dear Mr. Ames: On February 1, 1990, HSIA submitted comments on the draft technical support document for the proposed identification of perchloroethylene as a toxic air contaminant. On page 26 of these comments there is a typographical error. The first sentence on page 26 currently reads "[t]his calculation shows, in other words, a potential risk of 98,000 in 1,000,000.'* The correct sentence should read "[t]this calculation shows, in other words, a potential risk of 980.000 in 1,000,000." Enclosed is a corrected page 26. Please insert this corrected page into the HSIA comments. Sincerely, a Q=r-- Enclosure Thomas A. Cortina Director of Administration SL 038753 p /*> "" ^' V/ . * * ^ 26 CORRECTED PAGE This calculation shows, in other words, a potential risk of 980,000 in 1,000,000. Needless to say, this is a very large and very detectable potential risk. The example of a 30-year exposure at the TLV may be unrealistic, however. Thus, the table presented at the end of our comments shows similar calculations for different fractions of the TLV and for the lower and upper bound of the risk estimates in the draft report. It can be seen from this table that even the lower bound on the risk estimate range shows extreme (e.g., 7,000 in 1,000,000) potential lifetime risks. Assuming that the exposure scenarios are realistic, either the calculated potential risk is real, does exist, and has gone undetected, or the calculated unit risk estimates are wrong. Past manufacturing plant experience would indicate that particularily the low end of the ppm-year scenarios are very consistent with past practices and may even be under estimates. For example, drycleaning workers, who have a geometric mean exposure of 22 ppm with a skewed distribution, may hav an average exposure generally exceeding 25 ppm. Vet the drycleaning industry has been the subject of numerous epidemiology studies, none of which have detected a risk of the magnitude shown in the table. This is true as well for other industries that use perchlorethylene. Thus, we are left with the conclusion that the unit risks estimates in the draft report vastly overestimate the potential risk to humans. SL 038754 International Fabricare Institute 12251 TECH ROAD SILVER SPRING. MD 20904 (3011 622-1900 January 31, 1990 Mr. Donald J. Ames, Assistant Chief Stationary Source Division Air Resources Board P.0. Box 2815 Sacramento, CA 95812 StatioDnJevri/iion .WRosot" Dear Mr. Ames: RE: Comments of the International Fabricare Institute on the Preliminary Draft of tne lecnmcai support. Document for "Proposed identification of Perchloroethylene as a Toxic Air contaminant" The International Fabricare Institute and the California Fabricare Institute-- resDectively the national/international association and the state association tor retail drycleaning and laundry businesses--wish to submit the following comments on the Air Resource Board's proposal to identify perchloroethylene as a toxic air contaminant. At the onset, we wish to express the need for possible additional comments on the future "final draft" report beyond the limits of only "...comments on the Executive Summary and revisions made to preliminary draft report" as expressed in the ARB cover letter, with an official release date of December 28, 1989-- and actual receipt at our offices on January 8, 1990--a scant three weeks has been available for review and preparation of comments on a complex and comprehensive document. Given the likely impact of designating perchloroethylene a carcinogenic air pollutant, versus the extremely limited time constraints given for initial comments, we request the opportunity to provide further substantive comment as needed. I. Impact on the Drycleaning Industry of Designating PCE a Carcinogenic Air Pollutant --cording to a national survey of equipment and plant operations conducted in *988 by IFI, 79.OS of all retail drycleaning plants in the United States use ,3uE exclusively, while an additional 9% of plants use PCE in addition to another solvent. While no break-out figures are available specifically for California, we would expect that PCE usage would be the same or slightly htaher than national figures. SL 038755 THE ASSOCIATION OF PROFESSIONAL DRYCLEANERS AND LAUNDERERS Stationary Source Division Air Resources Board ? There are no known or anticipated substitutes for PCE in the drycleaning industry. Of the other solvents currently used, mineral spirits (eg, Stoddard solvent) have been in use since the 1920's, but because of combustibility are prohibited from new installations in virtually all urban and suburban areas of the country. The limited used of fluorocarbon 113 in drycleaning (approximately 6* of plants) is expected to phase out in the near future because of decreasing availability and rising costs connected with implementation of the Montreal Protocol provisions. Obviously, F-113 is not a viable substitute for PCE. Similarly, methyl chloroform (1,1,1--trichloroethane)--which after its introduction as a specialized drycleaning solvent two years ago is now used by only approximately 50 plants in the nation--is not a viable substitute because of ozone depletion concerns and its likely inclusion under the Montreal Protocol. Finally, "emerging" solvents such as KCFC 131a or 141b have chemical and/or physical properties which make them either unusable or potentially unsafe (eg, explosivebility) for the drycleaning of textile goods. Additionally, recent statements indicate that HCFCs are being viewed by producers as an interim solution and may be phased out within approximately ten years. With no viable substitutes available or anticipated for PCE in drycleaning, the central issue then becomes that of. potential emission limitations which would be likely to be imposed following designation of PCE as a carcinogenic air pollutant. One example of possible emission limitations is the South Coast Air Quality Management Districts's "Screening Risk Calculation" in prooosed Rule 223 of March 1989. Using the ARB Technical Support Document (TSD) upper-bound risk estimates for PCE, the SCAOMD's Screening Risk Calculation can be rearranged to project maximum "allowable" daily emissions; these would range from 0.35--1.5 Ib/day. For a typical small drycleaning plant cleaning 1,500 lbs. of garments a week (300 lbs/day), this would eguate to an emission factor range of 0.1--0.5 lb PCE/100 lbs. cleaned, or the equivalent of 140,000--700,000 lbs cleaning/drum of PCE. Emission limits In this range--and their equivalent solvent mileages--far exceed that possible under RACT, BACT, and MACT--in fact, outstrip that of the best operating plant in the nation by an order of magnitude or better. Our industry has consistently taken the position that if PCE is shown to be a probable human carcinogenic, then aooropriate measures should be adopted to protect Industry employees and the public. However, we also believe strongly that any such determination must be based on sound scientific data and consistent and rigorous interpretation of any such results. r> <'> *''*T, "* SL 038756 Stationary Source Division Air Resources Board 3 Significant evidence nas arisen indicating that PCE, while carcinogenic in mice, is unlikely to be a human carcinogen. In concert with this, EPA's Science Advisory Board has suggested that PCE is a specific example of a compound that does not cleanly fit into EPA's current carcinogen ranking system. That ranking system is, of course, now undergoing review with respect to the possible inclusion of additional categories. The significant questions about the weight of evidence for possible human carcinogenicity of PCE clearly indicate that designation as a carcinogenic air pollutant is unwarranted. Moreover, the incalculable damage to the drycleaning industry which would occur from such a designation could never be recovered. As an industry, we believe that significant steps can be taken in reducing emissions of PCE in all media...but that designation of DCE as a carcinogenic air DOllutant is not only not necessary or warranted, but disastrous. The impact on drycleaning of a carcinogen designation for PCE is not overstated--nor is our commitment to achieving significant reductions in PCE emissions by working with the Air Resources Board in the development of standards. II. Comments on ARB Technical Support Document Part A Rather than the 4,200 drycleaning facilities in California estimated in the TSD, the just-released 1987 U.S. Census of Service Industries reports 2,450 retail drycleaning stores, 916 coin-oo laundry /cleaning stores, and 157 industrial laundry facilities in California. Based on IFI national industry statistics, PCE is used in apDroximately 88* of retail stores, Jl5-20* of coin-ops and 32-5% of industrial plants. Applyina these distributions gives adjusted figures for PCE drycleaning stores in California of approximately 2,200 retail, 140-200 coin-op, and 3-8 industrial -- or approximately 2,300--2,400 facilities overall, a significant reduction from the estimated 4,200 facilities. The 1987 Census of Service Industries statistics also provide a method for calculating the approximate total PCE consumption by the drycleaning industry. As reported, total retail drycleaning receipts in California for 1987 were 'jproximately S518 million. Jsing an IFI receipt factor for 1987 of S2.52/lb cleaned--as developed in the annual IFI operating cost survey--gives approximately 205.5 million pounds cleaned per year. This annual poundage figure, multiplied by the 88* of the stores which use PCE, gives a value of approximately 181 million pounds cleaned in retail PCE stores in California. 038757 p wv Stationary Source Division Air Resources Board 4 Even assuming a relatively low average "solvent mileage" of 8,000 lbs clothes cleaned/700 lb drum of PCE (as opposed to current best estimates of J10,000 lbs/drum), calculated PCE consumption by retail stores would be approximately 7,900 tons per year. In the absence of current data for PCE consumption factors for coin-op and industrial facilities, using the same ratios between industry sectors as reported by EPA (TSD Part A, Appendix A-2) gives a total for all sectors of drycleaning of approximately 11,000 tons PCE/year, rather than the 18,100 tons/year estimated. It should be noted that the results of this calculation are consistent by ratio with the reduced number of plants reported in the Census. Similarly, using this method of calculation with a higher number of plants~eg, those estimated in the TSD--would give a higher PCE consumption which would be very close to that originally estimated. One other likely cause for the higher ARB number for drycleaning PCE use is the data cited by producers of 14,930 tons of PCE "shipped in 1985 from U.S. producers to California for drycleaning. *' In general, we have found that drycleaning consumption of PCE has frequently been overestimated in the past by PCE producers via the use of numbers such as this. Specifically, it should be noted that PCE is not shipped directly to drycleaning stores. Instead, it is purchased from "solvent distributors from whom the same drycleaning grade PCE is frequently purchased by other users of PCE--ie, businesses outside the drycleaning industry--as the purity level is greater than that of industrial-grade PCE. Unfortunately, the solvent producers will often equate sales of drycleaning-grade PCE to distributors directly with sales to drycleaning plants, when such is not the case. We would be happy to refine these calculations in concert with the ARB as needed. The estimate in t.he TSD of air emissions as a faction of total PCE consumption in drycleaning (ie, 0.88 lb PCE emitted/lb PCE used) is significantly high, most likely because of an under estimation of the quantity contained in hazardous wastes. Absent a small quantity generator waste exemption in California, these PCEcontaining drycleaning wastes are drummed and sent to solvent recycling facilities, such as those of Safety Kleen. As most of the retail drycleaning sector relies on standard-sized cartridge filters as part of their drycleaning system, and distills solvent at fairly constant rates relative to poundage cleaned, the total residual PCE in cartridge and still wastes is relatively constant at approximately 3.2 lbs PCE/100 lbs cleaned. Solvent mileages of 8,000--10,000--15,000--20,000 lbs cleaned/700 lb drum of PCE are equivalent, respectively, to consumption rates of 8.8--7.0--4.7--3.5 lbs PCE/100 lbs cleaned. s'* SL 038758 Stationary Source Division Air Resources Board - After subtracting the relatively constant 3.2 lb PCE/100 lbs cleaned waste loss from the above numbers, air emissions equal, respectively, 64*--54%--32%--9^ of total PCE use, not the 88* estimated in the document. Again, we would be happy to provide a more in-depth analysis of these factors with ARB staff. Part A also touches upon indoor air concentrations of PCE, some portion of which may be attributable to residuals in drycleaned articles. As part of this discussion, it is noted that data is available on indoor air levels of PCE from several European studies, but that this data may not be representative of California. We believe that the ARB is correct in feeling that this data may be nonrepresentative. The common European practice of installing so-called "unit drycleaning shoos" in blocks of residential apartment rows has presented problems. Typically, a unit shop is often found with apartments on either side of the plant, above it, and behind it...with no means of ventilation other than through the front door. Fortunately, this is not a common practice in the U.S. The drycleaning industry is concerned about the possibility of PCE in drycleaned garments contributing to indoor air levels. To this end, we are currently finalizing a draft test program with the cooperation of U.S. EPA. Testing under this program will evaluate possible contributions of drycleaned garments to indoor air levels of PCE and the possible operational changes which might be used to minimize any PCE residuals. III. Comments on ARB Technical Support Document Part B The evidence suggestive of PCE's carcinogenicity, when scrutinized carefully, consists primarily of two findings of liver tumors in the B6C3F1 mouse. The scientific community has repeatedly cautioned against reliance on liver tumors in this strain of mice as an indicator of possible carcinogenicity in humans. As one example, researchers have identified a cellular oncogene in this strain, and this has been postulated as the likely cause for its extreme susceptibility to tumors. Given the absence of any valid finding of carcinogenicity in other animal species--as discussed in the following--let alone in humans, PCE should not be classified as a "potential" fie, probable) human carcinogen by the Air Resources Board. Classification of PCE as a probable human carcinogen reverses at least three contrary determinations by EPA and/or its scientific advisors. The U.S. EPA's 'uly 1985 Health Assessment Document (HAD) on PCE concluded: According to the Agency's Proposed Guidelines for Carcinogen Risk Assessment (published November 1984), the cancer evidence of PCE In animal test systems is limited and the cancer evidence in epidemiologic studies is inconclusive. The overall weight-of-evidence classification for PCE would be Group C, ie., a possible human carcinogen. -- Si 38?59 n '"'PGV Stationary Source Division Air Resources Board P HAD at 1-4, 9-73 (emphasis added). The Agency's Science Advisory Board also concluded in 1984 that "Insufficient evidence exists to classify the chemical as a probable carcinogen for humans." Letter from Herschel E. Griffin, m.o. and Norton Nelson, Ph.D. to EPA Administrator Ruckelshaus, January 4, 1985. In addition, EPA's Risk Assessment Forum Classified PCE in Group C. See 50 Federal Register 46887. All of these determinations were made "aTter publication of1tPA's 1984 proposed guidelines for classifying carcinogens ana reflected the criteria specified in the guidelines. The critical issue presented by EPA's 1986 HAD Addendum and by the AR8 Technical Support Document is whether subsequent experimental findings warrant a reversal of the earlier classification. The Addendum cites two studies in support of its conclusion: (1) the 1977 NCI gavage study, which is described as having provided "positive evidence of hepatocellular carcinomas In mice," and (2) the 1985 NTP inhalation study, which the Addendum describes as "demonstrating that PCE can induce carcinogenic effects in both rats and mice through inhalation exposure." Both the 1977 and 1985 studies have been severely criticized by the scientific community. Shortly after the 1977 gavage study was completed, NCI's own advisory committed, the Clearinghouse on Environmental Carcinogens, expressed serious reservations about the study as to both mice and rats. Clearinghouse members characterized the study as "poorly-designed," primarily because the animals received toxic doses of PCE. In its official statement, the Clearinghouse observed: "the significance of the bioassay ... is significantly blunted by the evidence of toxicity in both the low and high dose groups," and noted that the effects observed with both rats and mice were "prima facie evidence for dose schedules in excess of the maximum tolerated dose." see transcript of proceedings of National Institutes of Health Clearinghouse on Environmental Carcinogens, Data Evaluation/Risk Assessment Subgroups, September 26, 1977 at 144. Thus, NCI's own advisory committee (the Clearinghouse), as is abundantly clear from the transcripts of the relevant Clearinghouse meetings, long ago repudiated the 1977 bioassay as reliable evidence of carcinogenicity in either species. NTP's conclusion that the 1985 inhalation study demonstrated "clear evidence" of carcinogenicity in both rats and mice has been similarly Questioned. Dr. Robert A. Squire, previous Director of the animal bioassay cancer testing program at the National Cancer Institute, has reviewed NTP's report, including an on-site review of tissue slides from the test animal. Dr. Sauire has noted several factors which cast doubt on NTP's finding of "clear evidence" of carcinogenicity in male rats and "some evidence" in female rats: o There currently are no generally accepted criteria for staging leukemias in rat. The ad hoc criteria used in the NTP study neither were reviewed in advance by experts in the field nor were similar to criteria used for staging leukemias in other species. n r\ >J* SL 038760 Stationary Source Division Air Resources Board o There was an abnormally high incidence of leukemia Tn control animals, indicating some other environmental factor which affected the observed leukemia rate in test animals. o The leukemia finding is of doubtful relevance to ootential effects of PCE on humans. The observed incidence of leukemia in rats is "a questionable endpoint for predicting human risk. There is no comparable human disease." o The finding of kidney tumors in male rats was not statistically significant and, in any case, occurred only in conjunction with nephrotoxic doses of PCE. Dr. Squire also questioned the relevance of the NTP's findings of increased mouse liver cancers to humans. He noted the widely-recognized susceptibility of mice to develop cancer when exposed to substances which are also toxic to the mouse liver. For this reason. Dr. Squire concluded: The mouse liver is generally not an appropriate surrogate for carcinogenesis risk in humans particularly where Hike PCE^ the compound tested is non-genotoxic and is administered at toxic levels. Additionally, Clement Associates, Inc., in an audit of the 1985 NTP study, also identified a number of deficiencies. These included deficiencies in animal handling and identification, as well as general problems with the testing laboratory serious enough to require corrective action by NTP. Clement also expressed concerns similar to Dr. Squire's about the relevance of observed findings of rat leukemias, particularly in view of the arbitrary staging criteria used by NTP, and about the significance of the rat kidney tumors. Indeed, Clement concluded that the observed mononuclear cell leukemia in rats was unrelated to PCE exposure. Significantly, the Science Advisory Board's Environmental Health Committee, Halogenated Organics Subcommittee, at its May 1986 review of the 1985 NTP findings concluded that all of the 1985 rat data, including the finding of leukemia in females, must be considered at best equivocal and is of doubtful relevance to humans. The SAB Subcommittee in fact rejected all the evidence cited in the Addendum in support of GrouD B2 classification for PCE, except the findings of mouse liver tumors. While the Subcommittee accepted the 1985 positive findings in mice, it hod reservations about whether those findings reflected a true carcinogenic mechanism. Regardless of the Subcommittee's reservations, these findings '..early do not represent "sufficient evidence" of carcinogenicity in animals under EPA's guidelines. Evidence is considered sufficient only if an increased incidence of tumors is found SL 038761 ^ , w- 1 * t Stationary Source Division Air Resources Board 8 (a) in multiple soecies or strains; or fb) in multiple experiments (preferably with different routes of administration or using different dose levels): or (c) to an unusual degree with regard to incidence, site or type of tumor, or age at onset. *9 Federal Register 46294, 46300 (November 23, 1984). Therefore, for the 1985 mouse liver tumor data to be considered sufficient evidence, it would have to be corroborated by at least one other test utilizing the same species or strain of mouse or utilizing another species. As noted, the 1985 study yielded no valid findings with regard to rats. The 1977 NCI study was repudiated by NCI's own advisory committee for use of doses exceeding the maximum tolerated dose in both species. Even assuming NCI's 1977 findings of increased mouse liver tumors were reliable, this would still not be "sufficient" evidence. Positive findings of liver tumors in the B6C3F1 mouse in two for more) experiments cannot be viewed as more than "limited" evidence of carcinogenicity. The clear weight of scientific authority is against the use of the mouse liver tumor as a virtual sole indicator of effects in humans. Moreover, the species- specific effect of PCE on mice is well-documented. When NTP repeated the 1977 NCI gavage bioassay in 1983, it attempted to prove that liver damage from PCE is not a necessary precursor of liver tumors in the B6C3F1 mouse. In fact, the data demonstrated a contrary proposition--that hepatotoxicity is a prerequisite for hepatocarcinogenicity in this strain. In mice, tetrachloroethylene was carcinogenic to the liver, but only at levels which were hepatotoxic, indicating that there are critical levels of cellular damage which may be necessary before there is an increase In cancer risk. This clearly has implications for human risk assessment and one may conclude that no-effect or threshold levels would probably also exist in man where there is no hepatotoxicity. These findings are consistent with the absence of a genetic mechanism for PCEinduced mouse liver tumors. Dr. Marvin Kuschner, Dean of the School of Medicine at the State University of New York at Stoneybrook, and a former member of the SAB Subcommittee on Airborne Carcinogens, as early as 1981 hypothesized that PCE is a liver toxin which, in the B6C3F1 mouse, damages liver cells. Again, under this theory, exposure to PCE at levels below that which destroys liver cells would not result in tumors in mice or other species. More recently, researchers have discovered a cellular oncogene in spontaneous liver tumors in B6C3F1 mice. This finding also "supports the concept that the B6C3F1 mouse is hypersusceptible to liver development" and indicates that this animal "is dissimilar to the genetically diverse human population In its ability to activate, with a very high frequency, a specific tumor-associated oncogene." T. Fox P. Watanabe, Detection of a Cellular Oncogene in Spontaneous Liver Tumors of B6C3F1 Mice, 228 Science at 596-97 (1985). This ver tumors in :E or, indeed, SL 038762 Stationary Source Division Air Resources Board 0 any other substance. In recognition of these severe limitations, EPA's guidelines specifically provide that mouse liver-only tumor responses, even if replicated, should not be accorded the same weight as positive findings in multiple experiments with some different species. Thus, the guidelines state: Under specific circumstances, such as the production of neoplasms that occur with high spontaneous background incidence, the evidence may be decreased to "limited1' if warranted (eg., there are widely diverging scientific views regarding the validity of the mouse liver tumor as an indicator of potential human carcinogenicity when this is the only response observed, even in replicated experiments in the absence of short term or other evidence). 49 Federal Register at 46300. Since there is no substantive short term or other evidence (eg., genotoxicity or mutagenicity) for PCE, the 1977 and 19B5 mouse data is, at most, a severely limited indication of possible human carcinogenicity. Telling evidence for the lack of human carcinogenicity comes from the available epidemiological evidence for PCE. With a unit risk factor (as calculated by ARBI of 31 -- 144 x 10-6/ppb, evidence of an increased risk should be strongly discernible in drycleaning worker cohorts, even those of small size, considering the typical levels of exposure in the range of 40-60 ppm which have existed for 40 years or more. A unit risk factor of 31-144 x 10-6/ppb is equivalent to 0.031--0.144/parts per million, and greater than unity for 50 ppm exposures, ie, in the range of 1.55- -".20/50 ppm for lifetime exposures. Even corrected for daily duration of exposure and worxing span factors, the risk factor is purportedly in the ranqe of 0.26-1 .20/50 ppm. Significant deficiencies exist with most epidemiological studies which have been reported, to the point where critically-disabling flaws disqualify these studies as a possible predictor of potential human carcinogenicity. Specifically, save for the retrospective cohort mortality study by NIOSH, no study has been done where exposure was known to be limited to PCE. In fact, each of these other studies has either specified or acknowledged exposure to multiple solvents, including carbon tetrachloride, petroleum solvents, and others. A further limitation of these studies has been an inability to identify work occupations, with the result that the cohorts have consisted cf n unknown mixture of laundry plant workers, drivers, and other unexposed employees. SL 038763 o rv /' r ' Stationary Source Division Air Resources Board 10 While the full NIOSH cohort of 1,500 workers could not be fully identified as to solvent exposure, a subcohort of approximately one-third of the full grouD was known to have had only PCE exposures in their working careers. In the suEcohort--the only PCE-specific exposure group ever studied--there was no overall increase or cancer, and no site-speci?ic increase in cancer. In conclusion, we believe that the equivocal bioassay data, coupled with the lack of any confirming human data where such would be expected, does not support a determination of potential/probable human carcinogenicity for PCE. At the same time, our industry believes that until final evidence is available to establish an even-stronger determination of non-carcinogenicity, a continued effort to reduce in-plant exposures and environmental emissions represents the most prudent of courses. To that end, we offer a commitment to the Air Resources Board to work jointly in developing viable, innovative approaches to significantly reduce environmental emissions from our industry. Sincerely, William E. Fisher Assistant General Manager/ Vice President copy to: Caffey Norman Earl Nichols, Paul Ceccarelli Charles Riggott, Jon Meijer Fax to G.laumann SL 38764 County of San Diego February 7,1990 R, J. Sommerviiie Air Pollution Control Officer Robert Rood Substance Evaluation Section Air Resources Board Attn: Perch!nro-,2th P.O.Box 2815 Sacramento, CA 95812 COMMENTS ON PROPOSED IDENTIFICATION OF FERCHLOROETHYLENE AS A TOXIC AIR CONTAMINANT, PART A REPORT Even though the perchloroethylene usages in Table III-l (Section III, page A-7) were directly referenced from Appendix A, these two sources do not agree and the difference is unclear. CARD predicts a proportional increase in dry cleaning emissions due to an increase in state population (Section III, page A-13). However, there is no mention whether tighter OSHA regulations in the work place and associated phasing-out of transfer systems were considered. For dry cleaning operations, CARB uses an EPA quoted reduction value of 70% (Section III, page A-S) for facilities equipped with refrigeration units, while the San Diego Air Pollution Control District (APCD) has been using 90% as the accepted reduction value. This factor reflects control standards in current district rules and this discrepancy could mean an underestimation of perchloroethylene emissions from dry cleaning operations in San Diego County. Little mention was made of emissions from filter or distillate waste which has been shown to contribute significantly to overall perchloroethylene emissions. The report gives the indication that throughout the state degreasers are significant contributors to perc emissions. San Diego County currently has approximately 20 or the estimated 350 perchloroethylene degreasers in California (Section III, page A-9) and the emissions are estimated from inventories to be 30 tons per year. This means that degreasers may not contribute to perc emissions in some distnets us significantly as in others. J ^ r\ ^N ' k ,w.) SL 038765 AIR POLLUTION CONTROL DISTRICT 9150 Chesapeake Drive. San Diego, California 92123*1095 Air Resources Board Robert Rood 2 February 7,1990 Emissions from state-wide maskant operation were not mentioned as significant contributors, while operations in San Diego County may be relatively large emitters of perchloroethylene. Thank you for the opportunity to comment. STANLEY J. ROMELCZYK Senior Air Pollution Control Engineer SJR:jl SL 038766 II. AIR RESOURCES BOARD STAFF RESPONSES TO COMMENTS ON PART A SL 038767 V k. W w **/ A. COMMENTS FROM CITIZENS FOR A BETTER ENVIRONMENT 1. Comment: The Part A report does not adequately assess total public perchloroethylene exposures for all indoor environments. Specifically, Part A neglects the exposure of workers, consumers and others in indoor microenvironments such as hospitals, schools, offices, and workplaces. Exposure to a consumer at a dry cleaning counter should be assessed. Response: The database for indoor exposure assessment is very limited. The report emphasizes indoor monitoring studies that were designed to provide representative data for Californians. So far, these types of studies have concentrated mainly on residential environments. The studies reviewed include ones in which 1.) only personal exposure data was collected, 2.) personal exposure data was collected and indoor and outdoor concentrations were measured, and 3.) only indoor concentrations were measured. Although studies discussed in the report do not specifically target consumers at dry cleaning counters, the personal exposure studies include workplace exposures and exposures in consumer-accessible environments. For example, in the Total Exposure Assessment Methodology (TEAM) studies conducted in 1987, several participants noted visits to dry cleaning shops among their personal activities. In addition to the personal exposure studies, an Environmental Protection Agency (EPA) study of the indoor environments of different public buildings is reviewed in Part A (Wallace et al., 1987; Sheldon et al., 1988). These public buildings include a hospital, a school, two homes for the elderly, and several offices. None of these public buildings are located in California. We note that the California Occupational Safety and Health Agency (Cal-OSHA) has the specific mandate to review and regulate workers1 exposures and workplace air levels. 2. Comment: Information should be added to assess the multiple routes of exposure from the different sources of perchloroethylene in indoor air. Response: Currently, we do not have specific data to breakdown the perchloroethylene sources of exposure in indoor air. However, in keeping with the California Health and Safety Code section 39660.5, we assessed exposure from indoor air and discussed the relative contribution of indoor air to total exposure based on the available data. SL 038768 -V. J. 3. Comment: The European data on perchloroethylene exposure by ingestion should be related to exposure in California by identifying the mechanisms by which perchloroethylene contaminates the food supply. Cancer risks associated with the ingestion of perchloroethylene in food should be presented in the report. Response: The European data do not furnish a sound basis for assessing perchloroethylene exposure by ingestion in California because: 1.) the data are incomplete and dated, and 2.) the difference in dietary habits between the European study group and Californians can not be adequately assessed. Although exposure through media other than air is included when information is available, the purpose of the ARB's Part A report is to identify mechanisms by which persons are exposed to airborne toxic substances. The Part B report prepared by the Department of Health Services assesses the cancer risk based on the ARB's exposure information. Some airborne toxicants, especially those of a particulate nature, contaminate food or water by deposition, adsorption, or dissolution. Due to its volatility and relative insolubility in water, the contribution of airborne perchloroethylene to concentrations of the substance in food or water is expected to be negligible. 4. Comment: The report should include exposure and cancer risk assessment for: 1.) average and maximum indoor air concentrations, and 2.) daily maximum, chronic, and worst-case exposures from the combination of air and other environmental media. Response: The revised Part A report Includes available data on maximum indoor air concentrations. Also, the daily dose via indoor air inhalation includes 24-hour average maximum concentrations. There are sufficient data on ambient concentrations and near source emissions to assess the cancer risk for exposed populations. The cancer risk assessment is summarized in the Staff Report/Executive Summary and more fully described in the Part B report. However, the database for perchloroethylene exposure via environmental media other than air is very limited and is not necessary to support our reconmendation that perchloroethylene be identified as a toxic air contaminant. 5. Comment: Superfund Amendments and Reauthorization Act (SARA) Title III data for toxic air contaminants should be included in reports. Also, the geographical distribution of the SARA Title III emissions should be presented. r SL 038769 Response: The ARB staff has not used information from the SARA Title III, section 313 database in toxic air contaminant identification reports for the following reasons: 1.) only those industries which manufacture, process, or use chemicals above specified thresholds are required to report, 2.) emissions from utilities, military installations, hazardous treatment plants, agriculture, and motor vehicles are not required to be reported, 3.) the database relies entirely on self-reporting, that is, industrial facilities estimate their own emissions, and 4.) the emissions reported by a facility are not audited. Using a variety of information sources has enabled the ARB staff to develop more complete emissions inventories than the SARA Title III reports. The information contained in the Federal SARA Title III section 313 database is available to the public through the following agencies: Office of Hazardous Materials Data Management Environmental Affairs Agency P.0. Box 2815 Sacramento, California 95812 Phone: (916) 327-1848 or 327-1849, Pollutant Characterization Section Noncriteria Pollutant Programs Branch (MD-15) U.S. Environmental Protection Agency Research Triangle Park, N.C. 27711 Phone (919) 541-5371 6. Comment: In section IV C, Exposure to Perchloroethylene Near Emission Sources, Part A should include the individual and population cancer risk for the population exposed to emissions from the eight South Coast facilities modeled by the ARB staff. Response: The individual and population cancer risk for the population surrounding the eight South Coast facilities is in the Staff Report/Executive Summary and the revised Part B report. 7. Comment: Part A should Include the emission levels for the eight South Coast facilities discussed in section IV C, Exposure to Perchloroethylene Near Emission Sources. SL 038770 C/-h r* rj o A. A .J Response: The revised report Includes the estimated total emissions, as well as the range of emissions, for five facilities centered on the City of Industry and three facilities centered on Burbank. COMMENTS FROM INTERNATIONAL FABRICARE INSTITUTE 1. Comment: The results of a 1988 national survey by the industry indicate that 79 percent of all retail dry cleaners use perchloroethylene exclusively and 9 percent use perchloroethylene plus another solvent. California perchloroethylene-usage Is probably the same or higher. Response: The information regarding the extent of perchloroethylene-usage by the dry cleaning industry is included in the revised Part A report. 2. Comment: There are no known reasonable substitutes for perchloroethylene in the dry cleaning industry. toaonso: The purpose of the identification phase of the toxic air contaminant program is to determine whether or not a compound should be listed as a toxic air contaminant. If perchloroethylene is identified as a toxic air contaminant, the possibility of substituting perchloroethylene with another cleaning solvent will be considered in the risk management phase of the process. 3. Comment: One example of emissions limitations Is the range (0.35 to 1.5 pounds per day) in the South Coast Air Quality Management District's (SCAQMD's) Proposed Rule 223. This limitations range far exceeds that possible under Reasonably Acceptable Control Technology (RACT), Best Available Control Technology (BACT), or Maximum Available Control Technology (MACT). In fact, it is more stringent than the emissions limits achievable at the best operating plant in the nation by an order of magnitude or better. Response: The comment above should be directed to the SCAQMD. The Part A report provides exposure information for the proposed identification of perchloroethylene as a toxic air contaminant and does not propose or endorse any district control measure. If perchloroethylene is identified as a toxic air contaminant, all possible control measures will be considered in the risk management phase of the process. 4. Comnent: The ARB staff has overestimated the number of dry cleaning operations in California. The number of dry cleaners using perchloroethylene in California is approximately 2400. v .. . / 038771 si* Response: The revised Part A report states that there are approximately 3,000 dry cleaning operations in the state using perchloroethylene. Based on permitting records, the estimated total number of dry cleaners using perchloroethylene in just the Bay Area and South Coast air quality management districts is approximately 2600. 5. Comment: In Appendix A page A-2, the ARB staff estimates that 18,100 tons/year of perchloroethylene are used by California dry cleaners based on sales by solvent producers. The perchloroethylene-usage by California dry cleaners is overestimated because solvent producers equate the sales of dry cleaning-grade perchloroethylene with sales to dry cleaners when, in reality, other businesses use this grade of the solvent. Response: The estimate in Appendix A used data from solvent producers for 1985 while the estimate in Chapter III of Part A (12,857 tons) was based on a survey of California halogenated solvent distributors for the 1987 inventory year. In order to avoid confusion, Appendix A (Methods for Estimating Usage and Emissions of Perchloroethylene in California) is not included in the revised Part A report. The methodology for estimating usage and emissions from each source is now included in the text of the report. The 1987 estimate in the revised Part A report relies on the distributor's records of the establishments to which they sold perchloroethylene and not on the grade of perchloroethylene sold. 6. Comment: The ARB staff use an overly high perchloroethylene emission factor of 0.88 pounds emitted for each pound used in dry cleaning. Response: The 0.88 emission factor for nation-wide dry cleaners developed by Wolf and Myers (1987) is based on subtracting the amount of solvent waste generated in 1984 from the total amount of perchloroethylene used that year. The difference is assumed to be perchloroethylene emissions. The ARB staff believes that the 0.88 factor is appropriate for estimating 1987 perchloroethylene dry cleaning emissions. Currently, the staff expects that overall perchloroethylene emissions from the California dry cleaning industry are decreasing due to: 1.) the adoption of dry cleaning control measures by several air pollution control districts, 2.) the institution of lower permissible exposure limits for workers by the California Occupational Safety and Health Agency (Cal-OSHA) in 1990, 3.) the adoption of DHS hazardous waste regulations requiring the storage of filter and still-bottom waste in air-tight containers, 4.) the trend to replace transfer units with SL 038772 dry-to-dry units, and 5.) the trend for large industrial cleaners to use soap and water rather than perchloroethylene. This information is included in the revised Part A report. COMMENTS FROM SAN DIEGO AIR POLLUTION CONTROL DISTRICT 1. Confront: The amount of perchloroethylene-usage shown in Table III-l, page A-7, does not agree with the amount shown in Appendix A, page A-l. Response: The perchloroethylene-usage shown in Table III-l Is based on a survey of halogenated solvent distributors for the 1987 Inventory year while the usage shown in Appendix A is based on the amount of perchloroethylene shipped to California in 1985. To avoid confusion in the revised Part A report, the methods for estimating usage and emissions of perchloroethylene In California are described by sourcecategory in the body of the text. 2. Comnent: On page A-13, the ARB staff predicts an increase in dry cleaning (and perchloroethylene emissions) due to a projected increase in California's population. Was the institution of tighter Cal-OSHA regulations in 1990 considered in this prediction? Response: The preliminary draft of Proposed Identification of Perchloroethylene as a Toxic Air Contaminant was Issued before the Cal-OSHA permissible exposure limits of 25 parts per million (ppm) became effective in April 1990. The revised Part A report discusses the likely trend toward decreasing perchloroethylene emissions from dry cleaners (please see the response to comment B 6 above). In the revised report, the projected increase in population is linked to a predicted increase in perchloroethylene-usage and emissions for industries other than dry cleaning. 3. Comment: On page A-8, the ARB staff uses an EPA estimate of 70 percent reduction in perchloroethylene emissions from dry cleaning facilities equipped with refrigerated condensers. Based on control standards in the San Diego Air Pollution Control District rule, the district estimates a 90 percent reduction in emissions. Is San Diego underestimating perchloroethylene emissions from dry cleaning in the county? BeSRonse: The San Diego rule specifies that "90 percent or more by weight of the halogenated organic compounds be removed by the device ..." but does not specify the type of air pollution control device to be used. As mentioned in Part A, carbon adsorbers (most often associated with transfer dry cleaning operations) are estimated to reduce p. o rn s* ^ * > ..................... J 038773 SL emissions by about 95 percent while refrigerated condensers (most often associated with dry-to-dry dry cleaning operations) reduce emissions by about 70 percent. In transfer operations, washed clothes are physically transferred to a drying unit, thus furnishing an additional opportunity for perchloroethylene emissions. In dry-to-dry operations, these emissions are avoided by washing and drying clothes in the same unit. The total reduction in perchloroethylene emissions using the dry-to-dry unit with a refrigerated condenser should be very close to that achieved using a transfer unit with a carbon adsorber. Both systems are capable of the 90X reduction specified by the San Diego District rule. 4. Comment: In Part A, little mention was made of emissions from filter or distillate waste which has been shown to contribute significantly to overall perchloroethylene emissions. Response: Given the use of air pollution control devices such as carbon adsorbers and refrigerated condensers, filter and distillate residues may be a significant source of perchloroethylene emissions. Once the residues are removed to air-tight containers in accordance with hazardous waste regulations, they cease to contribute to dry cleaning emissions. This information is reflected in the revised Part A report. 5. Confront: On page A-9, the Part A report indicates that degreasers are a significant source of perchloroethylene emissions throughout the state. Actually, degreasers may not significantly contribute to emissions in some districts. For example, San Diego has only 20 degreasers (out of California's estimated 350 degreasers) with estimated emissions of 30 tons per year. Response: Some districts are expected to have more degreasers and, therefore, more emissions from degreasing than other districts. Currently, there is insufficient data to estimate perchloroethylene emissions from degreasers in each district of California. Such information should become available as data from the AB 2588 Hot Spots Emissions Inventory are analyzed. 6. Comment: Emissions from state-wide maskant operations were not mentioned as significant sources of perchloroethylene emissions. Such operations in San Diego County may be relatively large perchloroethylene emitters. SL 038774 A "N Response: The halogenated solvent distributors surveyed by the ARB staff could not identify the purchasers of 642 tons of perchloroethylene sold In the 1987 inventory year. An unknown amount of the 642 tons was expected to be used by the semi-conductor industry in maskant operations. Thus, in the Part A report, the perchloroethylene emissions from maskant operations are included in the emissions attributed to the Miscellaneous source-category. * sj SV 03*715 III. DEPARTMENT OF HEALTH SERVICES STAFF RESPONSES TO COMMENTS ON PART B SL 038776 DEPARTMENT OF HEALTH SERVICES STAFF RESPONSES TO PUBLIC COMMENTS ON THE JANUARY 1989 REVISION OF PART B OF THE DRAFT REPORT TO THE AIR RESOURCES BOARD ON TETRACHLOROETHYLENE (PCE) General Comments 1- Comment: The statement in Chapter 1 of the draft document that PCE is used for coffee decaffeination is incorrect (Halogenated Solvents Industry Alliance, February 1, 1990 [HSIA], page 27). Response: The reference to use of perchloroethylene in decaffination of coffee vas eliminated. 2. Comment: Chapter 1 of the draft document states that the Occupati nal Safety and Health Administration permissible exposure limit (OSHA PEL) for PCE is 50 ppm for an 8-hour time-weighted-average (TWA). The OSHA PEL has recently been changed to 25 ppm for an 8-hour TWA (HSIA, p. 27). Response: The revised PEL has been added to the document. Topic: Hazard Identification 1. Comment: The weight of available scientific evidence does not support the conclusion that PCE is a probable human carcinogen (HSIA, p. 4). Significant questions about the weight of evidence for possible human carcinogenicity of PCE indicate that designation of PCE as a carcinogenic air pollutant or as "a 'potential' (i.e., probable)" human carcinogen is unwarranted (International Fabricare Institute, January 31, 1990 [IFI], pp. 3, 5-10). Response: Experimental studies have indicated that PCE is or its metabolites are genotoxic and can produce cancer in laboratory animals. PCE induced DNA strand breaks in liver and kidney cells of mice treated 111 vivo PCE induced transformation of rat embryo cells. It induced sex-linked recessive, lethal mutations in Drosophila. PCE induced gene conversion and mitotic recombination in yeast. PCE has been shown to be mutagenic to plants ia vitro. When mice were exposed to PCE by oral or inhalation administration, it produced hepatocellular carcinomas. Exposure of rats by inhalation to PCE produced an increased incidence of leukemias. Since PCE is genotoxic and can produce cancer in laboratory animals, it should be considered a probable human carcinogen. 2. Comment: Applying the criteria of IARC (the International Agency for Research on Cancer) does not justify hazard identification premised on a determination that it is more likely than not that PCE poses a human cancer risk (HSIA, p. 21). Response: As indicated by IARC's preamble to Supplement 7, several agents exhibited evidence of carcinogenicity in experimental animals prior to evidence being obtained from epidemi 1 gical studies r case 0 SL 038777 1 reports. There are 44 agents for which there is sufficient r limited evidence of carcinogenicity to humans and all 37 that have been tested adequately experimentally produce cancer in at least one animal species. And as stated in IARC (1987) "although this association cannot establish that all agents that cause cancer in experimental animals also cause cancer in humans, nevertheless, in the absence of adequate data on humans, it is biologically plausible and prudent to regard agents for which there is sufficient evidence of carcinogenicity in experimental animals as if they presented a carcinogenic risk to humans." Consequently, since there is sufficient evidence of carcinogenicity in experimental animals for PCE, it is considered a probable human carcinogen. This is indicated in the IARC monographs on the evaluation of carcinogenic risks to humans Supplement 7 (1987). 3. Comment: Chapter 1 of e draft report states that the EPA is likely to classify PCE in its ri assessment category B2, but EPA staff have indicated that the Age. / has not reached a final decision on classifying PCE. The EFA's Science Advisory has placed PCE "on the continuum between" categories B2 and C. This conclusion should be reflected throughout the document wherever EPA's classification of PCE is mentioned (HSIA, p. 20). Response: Perchloroethylene is currently classified as a B2 carcinogen by EPA. Discussions with EPA staff indicate that the agency still supports the B2 classification and has made regulatory decisions based on this determination. For example, in January 1991 EPA set PCE drinking water standards based on the agency's position that PCE is a probable (B2) human carcinogen. However, it is likely that this compound will undergo continual review at EPA. 4. Comment: California's carcinogen risk assessment guidelines ("Guidelines for chemical carcinogen risk assessments and their scientific rationale," State of California, Health and Welfare Agency, Department of Health Services, November 1985) suggest that PCE should not be identified as posing a cancer hazard to humans (HSIA, pp. 21-22). Response: As indicated in California's Carcinogen Risk Assessment Guidelines, the document on the Health Effects of Tetrachloroethylene does discuss the animal bioassays of cancer in detail. The document also discusses the pharmacokinetics and other factors pertaining to the potential genotoxicity and carcinogenicity of PCE. The guidelines also indicate that IARC follows all of the suggested procedures necessary t properly identify a chemical as a potential human carcinogen. The uncertainties of evaluating PCE as a carcinogen are discussed throughout the chapter on toxic effects in animals. The uncertainties in the pharmacokinetic and metabolism data are also discussed in the document. It is the conclusion of DHS staff that the overall weight of evidence regarding PCE indicates that it is a potential carcinogenic hazard to humans. 5. Comment: The mixed results of the in vitro genotoxicity studies of tetrachloroethylene oxide mentioned on page 3-22 of the draft document sh uld be Interpreted cautiously. The Halogenated Organics Subcommittee of the EPA Science Advisory Board has noted that PCE is not mutagenic, 2 038778 Sb and chat Cecrachl roethylene oxide is not a demonstrated metabolite of PCE and is apparently not carcinogenic (HS1A, pp. 18-20). Response: The determination that PCE as a genotoxic agent is not based solely on the genotoxicity of tetrachloroethylene oxide. It is simply indicated in the document that in addition to some positive genotoxic studies on PCE itself, mutagenic activity has been determined from PCE metabolites. 6. Comment: Overall, the draft document's evaluation of the epidemiological data is consistent with that of EPA's Science Advis ry Board. Since that Board's review, results have become available fr m the most complete epidemiological study of the industry conducted to date [Brown DP and SD Kaplan (1987) Retrospective cohort mortality study of dry cleaner workers using perchloroethylene. J Occup Med 29:535] (HSIA, p. 17). This study shows no increased risk of cancer in dry cleaning workers exposed only to PCE (HSIA, p. 18, IFI, pp. 9-10). This was the only group ever studied where exposure was known to be limited to PCE (IFI, pp. 9-10). Response: As indicated in the health effects document, there is inadequate evidence of carcinogenicity in humans. This conclusion is based on a number of epidemiologic studies including the study of Brown and Kaplan. Topic: Dose-Response Assessment 1. Comment: The draft report should be revised to reflect more clearly the qualitative differences in metabolism of PCE among rodent species and humans (HSIA, p. 4). Response: The differences in metabolism of PCE are discussed in Section 2 on pharmacokinetics and metabolism as well as the Sections on quantification of PCE's carcinogenic potency. In addition, numerous citations are given to other authors that have also reviewed the metabolic data on PCE. Furthermore, in the revised risk assessment of PCE, the differences in metabolism among rodents and humans are taken into account through use of a pharmacokinetic model. 2. Comment: If the final report includes an estimate of potential risk, it should incorporate all available scientific data. Pharmacokinetic information is not reflected in the draft document. The draft document is therefore inconsistent with California's carcinogen risk assessment guidelines. In light of this and the availability of extensive pharmacokinetic data, the rejection of a physiologically-based pharmacokinetic model in favor of an approach based on default assumptions appears to be an abuse of DHS' discretion (HSIA, pp. 4-5, 24). Response: The document has been revised along the lines of HSIA's comments. That is, pharmacokinetic data which were incorporated in the document in the previous draft had been revised with additional SL 038779 V/ 3 pharmacokinetic information. This has resulted in a IS-fold lower estimate of upperbound risk. A risk estimate based on pharmacokinetic information was used in the current document to establish the range of risks and the best value for the upper bound of risk. 3. pnmmenf Page 5-2 of the draft document notes that using the metabolized dose to calculate cancer potency would result in a higher potency value than would using the administered dose. This is mathematically correct but misleading and irrelevant. Metabolized d ses will lead to higher potency values, but equivalent doses will be smaller. Risk estimates themselves can be higher or lower depending upon the metabolism and distribution of the particular compound (HSIA, p. 27). Response: The statement regarding the higher potency for metabolized dose is given for information to the reader. Since there has been discussion in the scientific literature indicating that pharmacokinetic doses may imply a lower risk to humans, the reader might be confused seeing higher potency values in the document. Consequently, the statement regarding higher potency value will remain in the document in order to clearly state to the reader that the potency values for the metabolized dose throughout the document may be higher than those for the applied dose. As indicated in the comment, in terms of actual risk to humans, the use of a pharmacokinetic dose does not itself imply chat there will be higher or lower risk to humans. Instead, the risk estimate will depend upon the model chosen, the parameters used, and the assumptions made regarding the parameters and the model. 4. Comment The statement on page 1-9 of the draft document that range of potencies reported by EPA (2.9 to 9.5 x 10*') includes the DHS range is incorrect (HSIA, p. 27). Response: The range of unit risks reported in the Health Effects document on tetrachloroethylene is 3 to 106 x 10*' per jig/nr of PCE. This range includes the potencies reported by EPA. 5. Comment: There is no good scientific basis for risk estimates, based on default assumptions, that are 20-fold higher than those being considered by EPA (HSIA, p. 22). Response: Instead of using the default assumptions, the range of risk reported in the document now incorporates the pharmacokinetic information. Consequently, the previous risk estimate was lowered 15fold. 6. Comment: The gavage bioassay reported by the National Cancer Institute in 1977 does not appear to be a "properly conducted bioassay" as required by the California carcinogen risk assessment guidelines (HSIA, p. 6) . Response: The quality of the NCI 1977 study is discussed extensively in Section 3 and Section 5 of the document on the Health Effects of Tetrachloroethylene. Due to various shortcomings in the study, the risk r "J 4.0 4 03878 SL estimate from that study is not included in the range of risk or the best value for risk recommended by the Department. 7. Comment: Much relevant research is neither discussed nor referenced in the draft document (HS1A, p. 16). [The commentor submitted copies of the following articles: (1) Brown DP and SD Kaplan (1987) Retrospective cohort mortality study of dry cleaner workers using perchloroethylene. J Occup Med 29:535. (2) Elcombe CR (1985) Species differences in carcinogenicity and peroxisome proliferation due to trichloroethylene: a biochemical human hazard assessment. Arch Toxicol Suppl 8:6. (3) Odum J, T Green, JR Foster and PM Hext (1988) The role of trichloroacetic acid and peroxisome proliferation in the differences in carcinogenicity of perchloroethylene in the mouse and rat. Toxicol Appl Pharmac 1 93:103. (4) Herren-Freund SL, MA Pereira, G Olsen and AB DeAngelo (1986) The carcinogenicity of trichloroethylene (TCE) and its metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA), in mouse liver. Proc Amer Assoc Cancer Res 27:91, abstr. 356. The document should address several recently published works that apply physiologically-based pharmacokinetic (PB-PK) models. (HSIA, pp. 23-25). Response: The study by Brown and Kaplan is discussed in the health effects document in Section 4 under "Epidemiologic Evidence for Carcinogenicity in Humans." The study of Elcombe (1985) deals with TCE and is referred to in Section 3 under the subheading\ "Hepatic Toxicity." The work of Odum et al. (1988) was added and is discussed in Section 3 under the subheading "Hepatic Toxicity." The abstract of Herren-Freund et al. (1986) has not been added to the document, however, an article by the authors with the same title published in 1987 has been incorporated into Section 3 under the subheading "Tests for DNA or Chromosomal Damage". Studies applying PB-PK models for PCE have been discussed in the revised Health Effects document and pharmacokinetic information has been incorporated in estimating carcinogenic risks. 8. Comment: Although it true that PB-PK models do not account for inter individual variability [this is stated on page 5-51 of the draft document], the same statement could be made for other models, including the LMS [linearized multistage model], unless the surface area factor is viewed as an individual tissue sensitivity factor (HSIA, p. 28), Response: The standard approach used in risk assessment by EPA and DHS incorporate assumptions that would take into account inter-individual variability. That is, the use of an applied dose and the linearized multistage model are health protective assumptions which have a scientific basis. These health protective assumptions were made to incorporate potential inter-individual variability within the human population. Consequently, when a new procedure is suggested to replace an existing procedure, questions regarding incorporations of factors that protect certain populations need to be considered. The commentor compares the linearized multistage model with the pharmacokinetic model. This is an inappropriate comparison in that use of a phamacokinetic model does not preclude use of the linearized multistage model as well. In the DHS document both models are used together. Instead, the comparison should be made between use of the pharmacokinetic model and use of an applied dose model. The current perchloroethylene document SL 038781 5 uses a pharmacokinetic model and an estimate of the uncertainty to account for population differences in metabolism. In this way, inter individual differences can be taken into account. The surface area correction is used to consider the difference* in sensitivity of tissue response between rodents and humans. 9. Comment: Use of body-surface area correction factors is not appropriate in the case of PCE; body weight provides a better basis for dose adjustment (HSIA, p. 23). Response: The use of a surface area correction factor in the adjustment of rodent risk to human risk results in a difference of rodent to human risk of less than three fold. The use of the surface area correction factor is discussed extensively in Section 5 of the risk assessment document. In short, the surface area correction factor is used in order to take into account interspecies differences in tissue response to perchloroethylene. 10. Comment:: The q* correction on the bottom of page 5-8 should be clarified to differentiate between exposure period and observation period. If the dosing is for only one year but the animals are observed for an additional year, and the natural lifespan of mice and rats is assumed to be two years (104 weeks), the correction factor should be 104/Te raised to the unity power, not the third power (HSIA, p. 28). Response: The recorrection refers to a shortened lifespan resulting in a shortened exposure. This has been clarified in the document. The correction referred to in the comment is an adjustment for shortened exposure as described in Section 5 under the topic of "Dose Adjustments". For this correction the length of the exposure, Le, is simply divided by the lifespan of the animal. 11. Comment: The range of unit risk estimates presented in the draft document is inconsistent with human experience. Calculations with these unit risk estimates for persons occupationally exposed to PCE yield very large, very detectable, potential risks, and indicate that the unit risk estimates vastly overestimate the potential risks to humans (HSIA, pp. 25-26, 29; IFI, p. 9). Response: A number of epidemiologic studies have been conducted on workers most likely exposed to perchloroethylene (PCE), those working in the dry-cleaning industry. In these studies it is not known how often they were exposed to PCE or the concentrations to which they were exposed to. Furthermore, confounding factors such as low economic status, smoking, alcohol use, and exposures to other carcinogenic solvents, make it difficult to link human exposure to PCE with cancer. This is discussed in Section 4 under the heading "Epidemiologic Evidence for Carcinogenicity in Humans." One of the more complete studies was conducted by Brown and Kaplan (1987). In this study, exposures ranged from 3 to 22 ppm (i.e., 12.5 ppm average). There were 1,690 workers in this study with 619 workers primarily exposed to PCE. Assuming a length of employment of 5 years, the lifetime average exposure w uld be 0.21 ppm '' ' 6- 03872 si. ^ ((5/7)(8/24)(5/70)(12.5)-Q.21). Multiplying the risk times the exposure and the number of persons in the study ((1690) x (0.21 ppm) x (56 x 10" 3)(ppm)'1 - 20 indicates that an increase of 20 cancers would be expected. In the study there were 142 observed cancer deaths while only 123 were expected, i.e., an increase of 19. Thus, the estimate is close to the value expected. SL 038783