Document 4MpN3bgbBoXz8eBXYkKND2pQ

'E P A United States ' Environmental Protection Agency Research and Development EXTERNAL REVIEW DRAFT ECA0-C1N-405 April, 1908 pBO-215294 PB90-215294 -J DRINKING WATER CRITERIA DOCUMENT FOR 2.3.7,8-TETRACHLORODIBENZO-E-DIOXIN Prepared for OFFICE OF DRINKING WATER Prepared by Environmental Criteria and Assessment Office Office of Health and Environmental Assessment U.S. Environmental Protection Agency Cincinnati, OH 4'5 2 6 8______ REPRODUCEDBY | U.S. DEPARTMENTOFCOMM ERCE I NATIONAL TECHNICAL INFORMATION SERVICE i_______ . cpdiugfiPIO.VA 22161____ ' DRAFT: 00 NOT CITE OR QUOTE NOTICE This document Is a preliminary draft. It has not been Formally released by the U.S. Environmental Protection Agency arid should notv at this stage be construed to represent Agency policy. It Is being circulated for comments on its technical accuracy and policy implications. DISCLAIMER Mention of trade names or commercial products does not constitute endorsement or recomme dation for use. 11 I I FOREWORD Section 1412 (b)(3)(A) of the Safe Drinking Water Act, as amended in 1986, requires the Administrator of the Environmental Protection Agency to publish maximum contaminant level goals (MCLGs) and promulgate National Primary Drinking Water Regulations for each contaminant, which, in the judgment of the Administrator, may have an adverse effect on public health and which is known or anticipated to occur in public water systems. The MCLG Is nonenforceable and Is set at a level at which no known or antici pated adverse health effects 1n humans occur and which allows for an adequate margin of safety. Factors considered in setting the MCLG Include health effects data and sources of exposure other than drinking water. This document provides the health effects basis to be considered in establishing the MCLG. To achieve this objective, data on pharmacokinetics, human exposure, acute and chronic toxicity to animals and humans, epidemi ology and mechanisms of toxicity are evaluated. Specific emphasis 1s placed on literature data providing dose-response Information. Thus, while the literature search and evaluation performed In support of this document has been comprehensive, only the reports considered most pertinent In the deri vation of the MCLG are cited In the document. The comprehensive literature data base in support of this document Includes information published up to 1987; however, more recent data may have been added during the review process. When adequate health effects data exist, Health Advisory values for less than lifetime exposures (1-day, 10-day and longer-term, -10% of an Individual's lifetime) are Included 1n this document. These values are not used In setting the MCLG, but serve as Informal guidance to municipalities and other organizations when emergency spills or contamination situations occur. Michael B. Cook Director Office of Drinking Water 111 DOCUMENT DEVELOPMENT Debdas Mukerjee, Ph.D., Document Manager Environmental Criteria and Assessment Office, Cincinnati U,S. Environmental Protection Agency Helen H. Ball, M.S., Project Officer Environmental Criteria and Assessment Office, Cincinnati U.S. Environmental Protection Agency Authors Dipak K. Basu, Ph.D. Syracuse Research Corporation Syracuse, New York Denzil L. Tullis, Ph.D. Syracuse Research Corporation Syracuse, New York Scientific Reviewers Larry D. Anderson, Ph.D. Office of Drinking Water U.S. Environmental Protection Agency Washington, DC Editorial Reviewers Erma Durden, B.S. Environmental Criteria and Assessment Office, Cincinnati U.S. Environmental Protection Agency Judith Olsen, B.S. Environmental Criteria and Assessment Office, Cincinnati U.S. Environmental Protection Agency Document Preparation Technical Support Services Staff: C. Cooper, P. Daunt, C. Fessier, K. Mann, B. Zwayer, K. Davidson, J. Moore, Environmental Criteria and Assessment Office, Cincinnati Special Note: Since this document was developed from the comprehensive Information found 1n Ambient Water Quality Criteria for 2,3,7,8-Tetrachlorodlbenzo-jD-dloxln {EPA 440/5-84-007) and Health Assessment Document for Polychlorinated Dlbenzo-^dioxlns (EPA 600/8-84-014A), portions of this document were extracted from these two documents. 1v TABLE OF CONTENTS Page I. S U M M A R Y ......................................................... 1-1 II. PHYSICAL AND CHEMICAL PROPERTIES............................... II-1 CHEMICAL STRUCTURE AND SYNONYMS........................... PHYSICAL PROPERTIES........................................ STABILITY................................................ SUMMARY.................................................. II-l II-l 11 -4 11-7 III. TOXICOKINETICS................................................ III-1 ABSORPTION.............................................. III-1 Absorption From the Gastrointestinal Tract........ III-1 D I S T R I B U T I O N ............................................ METABOLISM ............... EXCRETION................................................ SUMMARY.................................................. 111-6 III-13 III -17 III-23 IV. HUMAN EXPOSURE.................................................. IV-1 EXPOSURE ESTIMATION ............................................ IV-1 Drinking W a t e r ........................................ . D i e t .................................................... Air...................................................... IV-1 IV-2 IV-4 S U M M A R Y ...................................................... IV-5 V. HEALTH EFFECTS IN ANIMALS ...................................... V-l EXPERIMENTAL ANIMALS ...................................... V-l Acute Toxicity............................... Subchronic Toxicity ............................... Chronic Toxicity................................... V-46 V -53 TARGET ORGAN TOXICITY................................... V-58 Hepatic Effects ................................... Immunological Effects ............................. Other Organ S y s t e m s ............................... V-58 V -62 V -64 OTHER EFFECTS................... ......................... V-66 Carcinogenicity ................................... Mutagenicity............................. .. Teratogenicity and Reproductive Toxicity.......... V-66 V -95 V-108 SUMMARY.................................................. V-l 21 v V-l TABLE OF CONTENTS (cont.) VI. HEALTH EFFECTS IN HUMANS. ..................................... VI-1 CLINICAL CASE STUDIES..................................... EPIDEMIOLOGICAL STUDIES. ............................... HIGH RISK SUBPOPULATIONS.............................. SUMMARY................................................. VI-1 VI-5 VI-61 VI-62 VII. MECHANISM OF TOXICITY..... ..... ............................ VII-1 RECEPTOR-MEDIATED TOXICITY . ........................... VII-1 2.3.7.8- TCDD: Segregation of Activity with the Ah Locus....................................... 2.3.7.8- TCDD and Related Toxic Halogenated Aryl Hydrocarbons: Structure-Activity Correlations . . . VII-2 VII-4 M E T A B O L I S M ............................................. VITAMIN A DEPLETION...................................... LIPID PEROXIDATION ...................................... ENDOCRINE IMBALANCE..................................... SUMMARY................................................. VII-B VII-10 VII-10 VII-11 VII-15 VIII. QUANTIFICATION OF TOXICOLOGICAL EFFECTS .................... V1II-1 INTRODUCTION V1II-1 NONCARCINOGENIC EFFECTS.................................VIII-6 1-Day HA......................................... .. V1II-7 10-Day HA ......................................... VIII-10 Longer-Term Exposure. . . . . .................... VIII-10 QUANTIFICATION OF NONCARCINOGENIC EFFECTS.................VIII-H Derivation of 1-Day HA............................... V1II-14 Derivation of 10-Day H A .......................... V111-15 Derivation of Longer-Term HA......................... VIII-16 Assessment of Lifetime Exposure and Derivation of a D W E L .............. VIII-17 CARCINOGENIC EFFECTS ................................... QUANTIFICATION OF CARCINOGENIC EFFECTS ................ EXISTING GUIDELINES, RECOMMENDATIONS AND STANDARDS . . . SPECIAL CONSIDERATIONS ................................. VI11-19 VIII-23 VI11-26 VII1-27 f Synergistic Effects ............................... VI11-27 High Risk Subpopulatlons.......................... VIII-28 SUMMARY............................................... .. VI11-28 IX. REFERENCES..................................................... IX-1 v1 LIST OF TABLES No, T1tie Page II-1 Solubility of 2,3,7,8-TCDD. .............................. II-2 11-2 Physical Parameters of 2,3,7,8-TCDD...................... 11-3 111-1 Percentage of 2,3,7,8-TCDD 1n the Liver of Rats 24 Hours After Oral Administration of 0.5 mi of Various Formulations Containing TCDD............ .................. 111-3 II1-2 Tissue Distribution of 2,3,7,8-TCDD....................... 111-7 111-3 Elimination of 2,3,7,8-TCDD............................... 111-18 V-l Lethal Doses of 2,3,7,8-TCDD Following Acute Exposure . . . V-2 V-2 Toxic Responses Following Exposure to 2,3,7,8-TCDD: Species Differences.......... ............................ V -11 V-3 Estimated Single Oral LD50 - 30 Values for PCDDs........... V-12 V-4 Immunological Effects of 2,3,7,8-TCDD 1n Animals.............. V-29 V-5 Effects of Chronic Exposure to 2,3,7,8-TCDD In Laboratory Rodents......................................... V -54 V-6 Carcinogenicity Bioassays of 2,3,7,8-TCDD Administration by the Oral Route............................. V-68 V-7 Carcinogenicity Bioassays of 2,3,7,8-TCDD Administered by the Dermal Route.......................... V-72 V-8 2,3,7,8-TCDD Intake and Mortality In Male Sprague-Dawley R a t s .......................................... V-74 V-9 Benign and Malignant Tumors 1n Rats Ingesting 2,3,7,8-TCDD........................................... .. . V-75 V-l0 Liver Tumors In Rats Ingesting 2,3,7,8-TCDD.................. V-76 V-ll Tumors That Were Significantly Decreased In Rats Following Exposure to 2,3,7,8-TCDD........................... V-79 V-12 Tumor Incidence 1n Mice Treated with 2,4,5-TCPE Contaminated with 2,3,7,0-TCDD............................... V-81 V-l3 Assessment of the Initiation and Promotion Activity of 2,3,7,8-TCDD 1n Laboratory Animals . . .......... .. . V-87 V-l4 The Results of Mutagenicity Assays for 2,3,7,8-TCDD 1n Salmonella typh1mur1um ................................... V-96 LIST OF TABLES (cont.) No. Title Page V-15 Studies on the Potential Teratogenic Effects of 2.3.7.8- TCDO Contaminated 2,4,5-T , ................. , . V-109 V-16 Studies on the Potential Teratogenic and Reproductive Effects of 2,3,7,8-TCDD ................................... V-113 VI-1 Distribution of Tumor Types in Two Case-Control Studies of Soft-Tissue Sarcoma.................. .......... VI-23 VI-2 Exposure Frequencies In Two Case-Control Studies of Soft-Tissue Sarcoma..................................... VI-24 VI-3 Relative Risks of Soft-Tissue Sarcoma 1n Relation to Exposure to Phenoxyacetlc Acids and Chlorophenols In Two Case-Control Studies ......................... VI-26 VI-4 Distribution of Histological Types of Soft-Tissue S a r c omas.......................................... VI-31 VI-5 Midland County Soft and Connective Tissue Cancer Deaths 1960-1981 .......... VI-40 VI-6 Other Occupations (M1nus/Forestry/Agr1culture). . . . . . . VI-47 VI-7 Other Occupations (M1nus/Forestry/Agr1culture/ Woodworkers)....................................... V I -48 VI-8 Analysis of Stomach Cancer Mortality 1n a Group of West German Factory Workers Exposed to 2,3,7,8-TCDD . . . . VI-52 VI-9 Reanalysis of Stomach Cancer Mortality 1n a Group of West German Factory Workers Exposed to 2,3,7,8 -TCDD . . . . VI-55 VI-10 Stomach Cancer Mortality In Three Studies of Workers Exposed to Phenoxyacetlc Acid Herbicides and/or 2.3.7.8- TCDD...................................... VI-57 VIII-1 Acute Toxicity of 2,3,7,8-TCDO. . ..................... .. . VI11-8 VI11-2 Effects of 4-13 Weeks Exposure to 2,3,7,8 -TCDD.......VI11-11 VIII-3 Effects of Long-Term Oral Exposure to 2,3,7,8 -TCDD. . . . . VIII-13 VIII-4 Carcinogenicity Bioassays of 2,3,7,8 -TCDD by Oral and Dermal Exposure ................... . ................. VIII-21 VIII-5 Summary of Human Potency Estimates for 2,3,7,8-TCDD . . . . VIII-24 vl 11 No. VIII-6 VI11-7 LIST OF TABLES (cont.) Title Page Responses and Parameters of the Koclba Feeding Study. . . . VIII-25 Summary of Noncarclnogenlc and Carcinogenic Effects for 2,3,7,8-TCDD........................................... VI11-24 lx ADI AHH bw BCF BromoPeCDD DCDD DHBA DMSO DNA EC/GC E50 FEL GC/HS GC/SIM/MS GI HPLC HRGC HRMS HxCDDs 1 .p. LC50 ld50 LOAEL LRMS 3-MC LIST OF ABBREVIATIONS Acceptable dally Intake Aryl hydroxycarbon hydroxylase Body weight Bioconcentration factor Br omopen tac hi 0rod 1ben zo-j>-dioxin D1chlorodlbenzo-p-d1ox1n Dimethyl benzanthracene Dimethylsulfoxide Deoxyribonucleic acid Electron capture/gas chromatography Median effective dose Frank-effect level Gas chromatography/mass spectrometry Gas chromatography/specl f1c Ion mon1toring/mass spec trometry Gastrointestinal High performance liquid chromatography High resolution gas chromatography High resolution mass spectrometry Hexachloro derivatives of d1benzo--d1ox1ns Intraperitoneal Concentration lethal to 50% of recipients Dose lethal to 50% of recipients Lowest-observed-adverse-effect level Low resolution mass spectrometry 3-Methylcholanthrene X MFD NICI NOAEL NOEL OCDD PCDDs PCP PeCDDs PPb ppm ppt RBC RNA SA 5 ,C . TCDDs TrlCDD 2,4',5-T TWA UV WCOT LIST OF ABBREVIATIONS (cont.) Mixed function oxidase Negative 1on chemical Ionization No-observed-adverse-effect level No-observed-effect level Octachlorlnated d1benzo--dlox1ns All polychlorinated d1benzo-2 -dlox1ns Pentachlorophenol Pentachloro derivatives of d1benzo--d1ox1ns Parts per billion Parts per million Parts per trillion Red blood cells Ribonucleic acid Satellite association Subcutaneous Tetrachloro derivatives of d1benzo--d1ox1ns Tr1chlorod1benzo--d1ox1n 2,4,5-Trlchlorophenoxy acetic acid Time-weighted average U1travlolet Wall-coated open tubular xl EPA . United Ste'es Environmental Protection Agency Research and Development DRINKING WATER CRITERIA DOCUMENT FOR 2,3.7,8-TETRACHLORODIBENZO--DIOXIN HNAt-ORAft-. ECA0-C1N-405 em be f , 14& 1 h~PY t\ ' Prepared for OFFICE OF DRINKING WATER Prepared by Environmental Criteria and Assessment Office Of f i ce of Heal th and Envi ronment al Assessment U.S. Environmental Protection Agency Cincinnati, OH 4 5 2 6 8 DRAFT: DO NOT CITE OR QUOTE NOTICE This document Is a preliminary draft. It has not been formally released by the U.S. Environmental Protection Agency and should not at this stage be construed to represent Agency policy. It Is being circulated for comments on U s technical accuracy and policy Implications. I. SUMMARY 2.3.7.8- Tetrachlorod1benzo-i)-d1ox1n (2,3,7,8-TCDD) 1s one of the most toxic and environmentally stable tricyclic aromatic compounds belonging to chl*orinated d1benzo--dlox1<ns. It 1s a contaminant formed In the production of 2,4,5-trlchlorophenol. It Is also a contaminant of a few chlorinated phenoxy acids (especially the herbicide 2,4,5-tr1chlorophenoxy acetic a d d and sllvex) and hexachlorophene. 2,3,7,8-TCOD 1s considered relatively stable toward heat, a d d s and alkalies. It begins to decompose at 500C and virtually complete decomposition occurs within 21 seconds at a temperature of 8Q0C. It 1s very slightly soluble In water (0.2 pg/i). In aquatic media, the compound Is expected to persist for a long time since It is likely to remain sorbed to sediments and biota. 2.3.7.8-TCDD 1s readily absorbed by mammals following either oral or dermal exposure. Because of Its relatively high 11p1d solubility, 2,3,7,8TCDD 1s rapidly distributed to tissues with a high lipid content. The Uver also represents a major site of accumulation In many species. Metabolism occurs slowly, and the polar metabolites are excreted 1n the urine and feces. 81l1ary excretion of 2,3,7,8-TCDD metabolites also occurs. Unmetabollzed 2,3,7,8-TCOD Is also excreted In the feces and 1n the milk of 1actat1ng animals. There are great differences' 1n species sensitivity to 2,3,7,8-TCDD, with LDggS ranging from 0.6 vg/kg bw 1n the guinea pig to 5 mg/kg bw 1n the hamster. In all species tested, thymic atrophy and severe weight loss are characteristic of 2,3,7,8-TCDD poisoning, with death occurring several 01290 1-1 09/17/84 days to weeks following exposure. In rats, rabbits and mice, 2,3,7,B-TCDD produces acute liver Injury which 1s not observed 1n either monkeys, hamsters or guinea pigs. Suppression of the immune system has been observed 1n mice, rats and guinea pigs. Clinical case studies and epidemiological * ... studies have Implicated 2,3,7,8-TCOD as the causative agent 1n the develop ment of chloracne, hyperpigmentation, altered liver function, porphyria cutanea tarda and hirsutism In humans. In many respects, the type and extent of effects observed are similar to those observed 1n nonhuman primates, though there are differences In the dermal symptomology elicited. Chloracne Is the major dermal finding 1n humans. In monkeys, common dermal effects include loss of hair, toenails and fingernails. ^ 2,3,7,8-TCDD has been demonstrated to be teratogenic 1n rats, mice and rabbits, and fetlddal 1n monkeys. The major toxic signs and terata observed were cleft palate In mice, edema, hemorrhage, and kidney anomalies 1n rats and extra ribs 1n rabbits. Some epidemiological studies have indi cated a possible teratogenic effect 1n humans, but the evidence 1s not suf ficient to reach a firm conclusion. - In vivo and Jm vltro mutagenicity tests have produced Inconclusive evi dence as to the mutagenicity of 2,3,7,8-TCDD; however, a number of bioassays have demonstrated this compound to be a potent animal carcinogen. Adenomas or carcinomas of the thyroid, hepatocellular carcinomas, carcinomas of the tongue and hard palate, and adenomas of the adrenal gland have all been produced 1n rats and mice. Some evidence from human epidemiologic studies associate exposure to herbicides contaminated with 2,3,7,8-TCDD with soft tissue sarcomas and nonHodgklns lymphomas; however, the exposures to 2,3,7,8-TCDD were always compounded with exposures to herbicide chemicals. 01290 1-2 08/ 10/84 These epidemiologic studies are consistent with the position that 2 ,3,7,8TCDD is probably carcinogenic for humans. Because 2,3,7,8-TCDD Is usually found in association with other materials (e.g., chlorophenols, phenoxyacetic adds, combustion products, etc.), it Is not presently possible to evaluate the carcinogenicity of 2,3,7,8-TCDD by itself In humans. A few possible mechanisms of toxicity have been proposed for 2,3,7,8TCDD. These Include receptor mediated toxicity, metabol1sm/dispos1tIon, vitamin A depletion, Increased lipid peroxidation and endocrine Imbalance. All of the proposed mechanisms may account for some of the toxic effects of 2.3.7.8-TCDD and/or the species differences in sensitivity to the toxin. However, further research 1s needed on the mechanlsm(s) for toxicity of 2.3.7.8- TCDD. Six metabolites have been Identified In dogs exposed to 2.3.7.8- TCDD. The major metabolite Is 1,3,7,8-tetrachloro-2-hydroxydlbenzo>-d1ox1n. A 1-day HA of l.OxlO-3 pg/i, for a 10 kg child was calculated from a single-dose oral LOAEL In guinea pigs, the species most sensitive to the toxicity of this compound. A 10-day HA was calculated by dividing the 1-day HA by 10. The resulting 10-day HAwas l.OxlO-4 pg/ft for a 10 kg child. A longer-term HA of 1xl0~5 pg/S, for a 10 kg child and 3.5xl0~5 pg/a for 70 kg adult wereestimated from a LOAEL derived In a 3-generatlon reproductive study In rats (Murray et al., 1979) along with the rationale developed by the EPA. A DWEL of 3.5X10" 5 pg/i. has been derived for lifetime exposure for an adult. However, the carcinogenicity risk assessment Indicates lower HAs for lifetime exposure to 2,3,7,8-TCDD. Based on a linearized multistage model, and dose-response data for the Incidence of tumors of the liver, lung, hard palate or nasal turbinates In 01290 1-3 09/23/87 female rats, the c o nc e n t r a t i o n s of 2,3, 7 , 8- TCDD in drinking water that would result 1n Increased lifetime cancer risks of 1 0 ' " , 1 0 " 5 , and 1 C T 6 w e re e s ti m a te d to be 2 . 2 x l C T s , 2 . 2 x 1 0 " * and 2 . 2 x l C P 7 vg/i., respectively. 01290 1-4 09/23/87 II. PHYSICAL ANO CHEMICAL PROPERTIES Chemical Structure and Synonyms 2,3,7,8-Tetrachlorod1benzo-p-d1ox1n (2,3,7,8-TCOD) CAS Registry Number: 1746-01-6 Chem. Abst. Name: 2,3,7,8-tetrachlorod1benzo[b,e]{1,4)-d1ox1n RTECS Number: HP35000 Synonyms: D1ox1n; TCDBD; TCDD; 2,3,7,8-tetrachlorodlbenzodloxin, 2,3,7,8tetrachlorodlbenzo-1,4-d1ox1n (IARC, 1977). Physical Properties 2,3,7,8-TCOD has a molecular formula of C.l,zH,4C14,0,c and a molecu- lar weight of 321.9. In the pure form, 1t exists as colorless needles with a melting point of 303-305C (Cruimett and Stehl, 1973). In a chloroform solution, the maximum absorption of 2,3,7,8-TCDD occurs at 310 nm, with a molar absorption coefficient of 5562 (NRCC, 1981). The available solubility data (Table II-l) Indicates that 2,3,7,8-TCDD 1s a highly lipophilic substance. Values for other physical properties of 2,3,7.B-TC0D that are available 1n the literature searched are given 1n Table II-2. 01300 II-l 09/18/84  ! ... TABLE II-l Solubility of 2,3,7,8-TCDD* Solvent Solubility (ppm) Water Lard oil Benzene o-D1chlorobenzene Chloroform Acetone n-Octanol Methanol 2 x 10'* 44 570 1400 370 no so 10 Source: Adapted front Crumrtet and Stehl, 1973 ' 01300 11-2 08/10/84 TABLE II-2 Physical Parameters of 2,3,7,8-TCDD Parameter Vapor pressure (mm of Hg) Octanol/water partition coefficient Sorption partition coefficient (Koc) Value 1.7 X 10' 1 .4 X 106 6.9 X 10` 1.9 X 10 9.9 X 10s 3.3 X 10* Reference NRCC, 1981 NRCC, 1981 Mabey et al., 1981 U.S. EPA, 1984a NRCC, 1981 Habey et al., 1981 01300 '11-3 09/1B/84 Stability 2,3,7,8-TCDD Is considered relatively stable toward heat, acids and alkalies (Albro, 1979). It begins to decompose at a temperature of 500C with virtually complete degradation at 80DC within 21 seconds (Stehl et 1r al., 1973). Ga m a radiation degrades 2,3,7,8-TCDD (FanelH et al., 1978). The four transformation processes (photoreaction, biotransformation, hydrolysis and radical oxidation) that control the fate of a chemical In aquatic media do not appreciably transform 2,3,7,8-TCDD (Matsumura et al., 1983). In organic solvents, 2,3,7,8-TCDD undergoes reductive photodechlorlnatlon at wavelengths <320 nm (Crosby et al., 1971; L1bert1 et al., 1978). In aqueous solution, hydroxylatlve dechlorination has not been seen. PUmmer et al. (1973) reported that a 2,3,7,8-TCDD suspension 1n distilled water remained unchanged when Irradiated with a sunlamp for an unspecified time. In contrast, 2,3,7,8-TCDD 1n methanol solution, or a benzene solution of 2,3,7,8-TCDD in water 1n the presence of a surfactant, underwent substan tial photodegradation under sunlamp or sunlight Irradiation (PUmmer et al., 1973; Crosby et al., 1971). The surfactant, 1-hexyldecylpyrldlnlum chlo ride, sensitized the photodecomposlt1on of 2,3,7,8-TCDD 1n aqueous solution (Botre et al., 1978). In order to explain the longer ha 1f-11fe of 2,3,7,8TCDD (1.6 year vs. 1 year) 1n a model laboratory ecosystem than 1n an out door pond, Matsumura et al. (1983) and Tsushlmoto et al, (1982) speculated that photolysis 1s the most likely cause. In the outdoor environment, where the Intensity of sunlight 1s higher compared with the laboratory experiments (100 w fluorescent lamp over 40 cm of water surface), algae-medlated photosensltlzatlon of 2,3,7,8-TCDD was speculated to have caused some photodecom position of this compound. From the available Information, It 1s difficult 01300 1 1-4 04/05/84 to predict the fate of 2,3,7,8-TCDD In aquatic media under environmental photolytlc conditions. In the presence of hydrogen atom donating substrate(s) 1n surface waters, photolysis may be a significant fate process. I 2,3,7,8-TCDD exhibits relatively strong resistance to microbial biodegradation. Only 5 of -100 microbial strains that have the ability to degrade persistent pesticides show slight ability to degrade 2,3,7,8-TCDD (U.S. EPA, 1980). Ward and Matsumura (1977) reported that the half-life of 2.3.7.8- TCDD In sediment-containing Wisconsin lake waters was 550-590 days. In lake water alone, -70% of the 2,3,7,8-TCDD remained after 589 days. Using an outdoor pond as a model aquatic ecosystem, Tsushlmoto et al. (1982) and Matsumura et al. (1983) estimated the apparent half-life of 2,3,7,8TCDD to be -1 year. Although biodegradation may have been responsible for part of the degradation, other Investigators (Huetter and Philippi, 1982) have reported the virtually complete lack of blodegradablHty of 2.3.7.8- TCDO. The biodegradation half-life of 2,3,7,8-TCDD can be estimated from the theoretical rate constant values based on relative rates of transformation reported 1n the literature or on structure-activity analogy values given by Habey et al. (1981). Assuming the biotransformation rate constant of lxl0"iO mi, cell" 1 hr-1 (Habey et al., 1981) and the concentration of microorganisms capable of degrading 2,3,7,8-TCDD as 5xl05 cell ml-1 (Burns et al., 1981), the half-11fe of biodegradation Is estimated to be >1 year. 01300 11-5 04/05/84 Although several Investigators Implicated volatilization as one of the major reasons for the observed disappearance of 2,3,7,8-TCDD from aqueous i solution during microbial studies, little quantitative Information regarding the volatilization of 2,3,7,8-TCDD from aquatic media Is available. 2,3,7,8-TCDD may undergo some water-mediated evaporation 1n aquatic media (Matsumura et al., 1983). A transport model to estimate 2,3,7,8-TCDD volatilization from a cooling pond on an Industrial site based on measured concentrations 1n the pond bottom sediment and pond surface area led to an estimated rate of 15-16 mg/year {Thibodeaux, 1983). Using the formulas of L1ss and Slater (1974), a vapor pressure value of -10"* torr (0.1 m Pa) and a solubility value of 6.2X10" 10 mole/t, NRCC (1981) calculated the volatilization half-Hfe for 2,3,7,8-TCDD to be 6 minutes from water of 1 cm depth and 10 hours from water of 1 m depth. Evaporation half-Hfe 1s directly proportional to water depth and Inversely proportional to mass transfer coefficient (Thibodeaux, 1979). The limitations of the L1ss-Slater theory to predict the rate of volatilization have been discussed 1n the NRCC (1981) document. The L1ss-Slater model does not consider terrestrial matrices (suspended solids, sediments, biota, etc.) normally encountered In natural surface water. Employing a computerized EXAMS model for two stan dardized aquatic ecosystems and the Input parameters for 2,3,6,8-TCDD, as discussed 1n NRCC (1981), volatilization has been estimated to account for 100% of the fraction lost and biodegradation has been calculated to be 0%. The volatilization half-Hfe for 2,3,7,8-TCDD has been estimated to be 5.5 and 12 years from pond and lake water, respectively. However, 1t should be remembered that these are estimated values and experimental confirmation of these values Is not available. 01300 11--6 04/05/84 Data from microcosm experiments Indicate that 2,3,7,8-TCDD 1s highly sorbed to sediments and biota (Isensee and Jones, 1975; Ward and Hatsumura, 1978). More than 90* of 2,3,7,8-TCDD 1n an aquatic medium may be present 1n thet adsorbed state (Hard and Hats..umura, 1978; Hatsumura et al., 1983). This 1s not surprising considering the low water solubility and the high octanol/ water partition coefficient. In fact, the equation of Karlckhoff et al. (1979) predicts a sorption partition coefficient value of 104 for 2,3,7,8TCDD 1n sediments containing 2* organic carbon (NRCC, 1981). Summary 2,3,7,8-TCDD has a molecular formula of C,1.2H.4C1.4Q_2 and a molecu- lar weight of 321.9. In pure form, 1t exists as colorless needles with a melting point of 3Q3-305*C (Cruimett and Stehl, 1973) and 1s relatively reslstent to degradation by heat, acids and alkalies. It 1s very slightly soluble 1n water (0 .2 ppb) and somewhat soluble 1n organic solvents (Cruimett and Stehl, 1973). The compound has a low vapor pressure (1.7x10** nrn of Hg) and a high octanol/water partition coefficient (NRCC, 1981; Habey et al., 1981). From the available Information, 1t 1s difficult to predict the photo lytic fate of 2,3,7,8-TCDD 1n aquatic media. In the absence of hydrogen donating substrates, photolysis does not appear to be a significant process (PIInner et al., 1973; Crosby et al., 1971). 2,3,7,8-TCDD exhibits rela tively strong resistance to microbial degradation (U.S. EPA, 1980; Huetter and Philippi, 1982). The eslmated biodegradation half-life of 2,3,7,8-TCDD 1s >1 year (Mabey et al., 1981; Burns et al., 1981). Hydrolysis and radical oxidation do not appear to be significant processes for 2,3,7,8-TCDD 1n aquatic media (NRCC, 1981). 01300 1 1-7 04/05/84 Little quantitative Information regarding the volatilization of 2,3,7,8TCDD from aquatic media 1s available. Theoretical modeling of 2,3,7,8-TCDD (EXAMS model) provides a volatilization ha 1f-11fe for 2,3,7,8-TCDD of 5.5 and 12 years from pond and lake water, respectively (NRCC, 1981). However, no* experimental confirmation of these values 1s available. Experimental data show that this compound 1s likely to remain sorbed to sediments and biota In aquatic media (Isensee and Jones, 1975; Ward and Matsumura, 1978). 01300 11-8 03/15/84 III. TOXICOKINETICS The toxicokinetics of 2,3,7,8-TCDD has been Investigated 1n a number of laboratory animals, and there are several recent reviews on this subject (Neal et al., 1982; Gaslewlcz et al., 1983a; Olson et al., 1983). This section will examine our current understanding of the absorption, distribu tion, metabolism and excretion of 2,3,7,8-TCDO 1n various manmallan species. Absorption The dermal and gastrointestinal absorption of 2,3,7,B-TCDD have been Investigated 1n several species. No studies are available on the toxico kinetics of 2,3,7,8-TCDD through the Inhalation route of exposure. Absorption From the Gastrointestinal Tract. Experimentally, 2,3,7,8TCDD Is generally administered 1n the diet or by gavage 1n an oil vehicle. In Sprague-Dawley rats given a single oral dose of 1.0 pg [14C)2,3,7,8TCDO/kg bw, absorption from the Intestinal tract was estimated at -83% (Rose et al., 1976). With repeated oral dosing at 1.0 pg/kg/day (5 days/week x 7 weeks), absorption was observed to be approximately that observed for the single oral dose. With a much larger single oral dose, 50 pg/kg bw, -70% of the dose was absorbed by Sprague-Dawley rats (Piper et al., 1973). In these studies, the chemical was administered by gavage 1n acetone:corn oil (1:25 or 1:9). One study In the guinea pig reported that -50% of a single oral dose (quantity not mentioned) of 2,3,7,8-TCDD 1n acetone'.corn oil was absorbed (Nolan et al., 197f9). The gastrointestinal absorption of 2,3,7,8-TCDD was also examined In the hamster, the species most resistant to the acute toxicity of this toxin (Olson et al., 1980a), Olson et al. (1980a) administered hamsters a single, sublethal, oral dose of 01310 III-l 09/18/84 [1,6-*H]-2,3,7,8-TC0D In olive oil (650 yg/kg) and reported that 74% of the dose was absorbed. When 2,3t7,B-TC0D was administered to rats In the diet at 7 or 20 ppb (0.5 or 1.4 yg/kg/day) for 42 days, 50-60% of the consumed dose was absorbed (Fries and Narrow, 1975). These findings Indicate that over a wide range of doses and under these experimental condi tions, 2,3,7,8-TCDD 1s generally well absorbed from the gastrointestinal tract of the three species that have been examined. Contact with 2,3,7,8-TCDD In the environment would most often Involve exposure to a complex mixture containing the toxin, as opposed to the above experimental situation, where 2,3,7,8-TCDO was administered In the diet or through an oil vehicle. The Influence of dose and vehicle or adsorbent on gastrointestinal absorption has been Investigated 1n rats by Polger and Schlatter (1980), using hepatic concentrations 24 hours after dosing as an Indicator of the amount absorbed. They found a linear relationship between ng 2,3,7,8-TCDD administered In 50% ethanol (for doses of 12-280 ng, equivalent to 0.06-1.4 vg/kg) and the percentage of the dose 1n hepatic tissues (36.7-51.5%). At the next higher dose of 1070 ng, however, the percentage fell off to about 42%. Their results regarding the Influence of vehicle or adsorbent on gastrointestinal absorption have been summarized In Table II1-1. Admin istration of 2,3,7,8-TCDO In an aqueous suspension of soil resulted In a decrease In the hepatic levels of 2,3,7,8-TCDD as compared with hepatic levels resulting from administration of 2,3,7,8-TCDD In 50% ethanol. The extent of the decrease was directly proportional to the length of time the 2,3,7,8-TCDD had been 1n contact with the soil. When 2,3,7,8-TCDD was mixed 01310 111-2 08/10/84 I TABLE III-l Percentage of 2,3,7,8-TCDO 1n the Liver of Rats 24 Hours After Oral Administration of 0.5 mi of Various Formulations Containing TCDD* Formulation TC00 Dose (ng) No. of Animals 50X Ethanol Aqueous suspension of soil (37%, w/w) that had been In contact with TC00 for: 10-15 hours 8 days Aqueous suspension of activated carbon 125%, w/w) 14.7 12.7, 22.9 21.2, 22.7 14.7 7 17 10 6 Source: Adapted from Polger and Schlatter, 1980 Percentage of Dose 1n the Liver 36.7 * 1.2 24.1 * 4.8 16.0 + 2.2 <0.07 01310 I I 1-3 08/10/84 in an aqueous suspension of activated carbon, absorption was almost totally eliminated (<0.0?% of the dose in hepatic tissues). Philippi et al. (1981) and Hutter and Philippi (1982) have shown that *' radiolabeled 2,3,7,8-TCDD becomes progressively more resistant with time to extraction from soil. Similarly, the feeding of fly ash, which contains PCDDs, to rats in the diet for 19 days resulted in considerably lower hepatic levels of PCDDs than did the feeding of an extract of the fly ash at comparable dietary concentrations of PCDDs (van den Berg et al., 1983). The PCDDs were tentatively identified as 2,3,7,8-TCDD, 1,2,3,7,8-PeCD0, 1,2,3,6,7,8-HxCDD and 1,2,3,7,8,9-HxCOO and the difference In hepatic levels noted between fly ash-treated and extract-treated rats was greater for the more highly chlorinated isomers than 1t was for 2,3,7,8-TCOO. These results indicate the importance of the formulation or vehicle containing the tox1n(s) on the relative bloavailabillty of 2,3,7,8-TCDD, PeCDO and HxCDDs following oral exposure. Information on the absorption of 2,3,7,8-TCDD through the skin is found only in a study by Poiger and Schlatter (1980). The authors administered 26 ng 2,3,7,8-TCDD In 50 yl methanol to the skin of six rats. After 24 hours, the liver contained 14.8+2.6% of the dose. By comparing to the hepatic levels obtained after oral administration in 50% ethanol (In the same study), the amount absorbed from a dermal application can be estimated at "40% of the amount absorbed from an equivalent oral dose. This compari son assumes that hepatic levels are valid estimates of the amount absorbed from both oral and dermal routes and that absorption from methanol is equiv alent to absorption from 50% ethanol. As compared with dermal application 01310 111--4 08/10/84 in m e th a n ol , dermal a p p l i c a t i o n of 2 , 3, 7 , 8- T C DD to rats In vaseline or p o l y e t hy l e ne glycol reduced the p e r c e n t a g e of the dose In hepatic tissue to 1.4 and 9.3%, respectively, but had no observable effect on the dose of 2,3,7,8TCDD required to Induce skin lesions (~1 yg/ear) in the rabbit ear assay. Appli c at i o n of 2 , 3 , 7 , 8-T C DD in a soil/water paste decreased hepatic 2,3,7,8T C DD to ~ 2% of the a d m i n i s t e r e d dose and i ncreased the amou n t requ i r ed to produce skin lesions to 2-3 yg in rats and rabbits, respectively. A p p l i cation in an activ a ted carbo n /water paste e s sentially e l iminated absorption, as measured by percent of dose in the liver, and increased the amount of 2.3.7.8- TCDD required to produce skin lesions to -160 yg, These results suggest that the dermal absorption and acnegenic potency of 2,3,7,8-TCDD are dependent on the formulation (vehicle or adsorbent) containing the toxin. Since 2 , 3 , 7 , 8 - T C D D in the e n v i r o n m e n t is likely to be a b so r b e d to soil, McConnel et al. (1984) and Lucier et al. (1986) compared the absorption of 2.3.7.8- TCDD from contaminated soil to that from 2,3,7,8-TCDD administered in corn oil. As indi c a te d byb i ological effects and the amount of 2 . 3 . 7 . 8 - TCDD in the liver, the absor p ti o n from soil was - 5 0 % less than from corn oil. Umbreit et al. (1986a) showed that 2,3,7,8-TCDD contaminated soil was less toxic than an equivalent amount of 2,3,7,8-TCDD, suggesting that binding to soil had an influence on bioavaliability. Although these data indicate that substantial absorption occurs from contaminated soil, soil type and duration of contact, as suggested from the data which demonstrated decreased extraction efficiency with increasing contact time between soil and 2,3,7,8-TCDD (Phillipi et al., 1981; Huetter and Phlllipi, 1982), may substantially affect the absorption of 2,3,7,8-TCDD from soils obtained from different contaminated sites. 01310 I I I -5 04/12/88 Poiger and Schlatter (1986) Investigated the absorption of 2,3,7,8-TCDD in a 42 year old man after injestion of 105 ng 9H-2,3,7 ,8-TCDD in 6 mi. corn oil and found that greater than 87% of the oral dose was absorbed from the intestine. Following absorption, the half-life for elimination was estimated to be 2,120 days. Distribution The tissue d i s t r i b u t i o n of 2 , 3 , 7 , 8 - T C D D in a number of species is s u m m a rized in T a ble 1 1 1 -2. From these data it is a p pa r e n t that 2 , 3 , 7 , 8 - T C D D d i s tributes preferentially to the liver and adipose tissue of most species that have been examined. Piper et al. (1973) used a single oral dose of [1 4 C ] 2 , 3 , 7 , 8 - T C D D to study d i s t r i b u t i o n and e x c r e t i o n in male S p r a g u e Dawley rats. Host of the radioactivity (53.2%) was excreted via the feces, but the urine and expired air accounted for 13.2 and 3.2%, respectively. Analysis of the tissues after 3 days showed liver and adipose tissue to contain the highest percent of the dose per gram of tissue, with 3.18 and 2.60%, respectively. Rose et al. (1976) also examined the distribution of [14C]2,3,7,8-TCDD in the rat. T w e n t y - t w o days after a single oral dose of 1.0 ^g/kg, liver and adipose tissue had retained most of the 14C activity, with 1.26 and 1.25% of the label retained per gram of tissue, respectively. With repeated oral doses, the a c t i v i t y was again loca l i ze d m a i n l y in the liver and adip o s e tissue, but the liver had five'times as much radioactivity as did the fat. With the single oral dose, no radioactivity was detected in either the urine or expi r e d air, I n d i c a t i n g that most if not all of the e l i m i n a t i o n of 2,3,7,8-TCDD and/or Its metabolites was through the feces. With repeated oral doses, the 14C activity was also excreted primarily through the 01310 1 1 1-6 04/12/88 01310 TABLE 111-2 Tissue Distribution of 2,3,7,8-TCDD 1 1 1 -7 Spec ies Rat Rat Rat Rat Rat Rat House House Rhesus monkey Golden Syrian hamster Guinea pig Guinea pig Route of Administration oral oral oral oral oral l.p. oral l.p. l.p. l.p. or oral oral l.p. Tissues with the Highest Concentration of 2,3,7,8-TCDD liver liver > fat liver > fat 11ver > fat liver > fat liver > fat liver > fat > kidney > lung liver > fat > kidney > lung > spleen fat > skin > liver > adrenal = thymus liver > fat fat > liver > adrenals > thymus > skin fat > 11ver > skin > adrenals References Fries and Harrow, 1975 Rose et al., 1976 Piper et al., 1973 Koclba et al., 1978a,b Allen et al., 1975 Van Hiller et al., 1976 Hanara et al., 1982 Hanara et al., 1982 Van Hiller et al., 1976 Olson et al., 1980a Nolan et al., 1979 Gaslewlcz and Neal, 1979 04/08/88 feces, but significant amounts were found in the urine, e s p e c i a l l y of the female rats. Male rats given 1.0 yg/kg/day of 2,3,7,8-TCDD for 7 weeks e x cr e t e d an aver a g e of 3 . 1% of the c u m u l a t i v e dose in the urine w h ile the female rats excr e t ed an a v e r a g e of 1 2 .5 % in the urine (Rose et al., 1976). Fries and Marr o w (1975) have also reported e v id e n ce of sex d i f f e r en c e s in tissue dist r i but i on in rats. During 42 days of administ r a ti o n of 2,3,7,8TCDD, - 8 5% of the total body residue of male rats was located in the liver, w h il e 7 0% of the total body re si d u e of female rats was located in this organ. Studies performed by Van Miller et al. (1976) on rhesus monkeys and rats using single i.p. doses of triti a ted 2,3,7,8-TCDD (400 yg/kg bw) showed that while rats had over 4 0% of the 2 , 3 , 7 , 8 - T C D D in the liver 7 days after dosing, the monkeys had only about 10% in the same organ at that time. In two strains of mice, the liver contained -35% of an administered dose of 2,3,7,8-TCDD 1 day after oral or i.p. administration (Manara et al., 1982). The liver was also found to be the major site of accumulation of 2,3,7,8TCDD in the hamster, with 2 0 % of the dose localized in the liver (5.3% of dose/g liver) at 3 days following a sublethal dose of 650 yg 3H-2,3,7,8TCDD/kg (Olson et al., 1980a). In all three species, 1-22 days after s i n g l e - d o s e oral or I.p. a d m l n l s t r a t i p n , levels of 2 , 3 , 7 , 8 - T C D D in ad ip o s e tissue were g e nerally slightly lower than levels in the liver, and w ere c o n s i de r a bl y higher than c o n c e n t r a t i o n s In other tissues (Piper et al., 1973; Rose et al., 1976; Van Miller et al., 1976; Olson et al., 1980a; Manara et al., 1982), Including the thymus (Rose et al., 1976; Van Miller et al., 1976; Olson et al., 1980a). Kociba et al. (1978a,b) found that female rats maintained on a daily dietary 2,3,7,8-TCDD Intake of 0.1 yg/kg/day for 2 years had an average 01310 111 -8 04/08/88 2 , 3, 7 ,8-TCDD content of 8100 ppt in fat and 24,000 ppt in the liver. Rats given 0.01 v g / kg/day had an a v er a g e of 1700 ppt of 2 , 3 , 7 , 8 - T C D D in the fat and 5100 ppt in the liver. For both of these d a ily dosages the liver-.body fat ratio of 2,3,7,8-TCDD was 3:1. At the lowest dose level of 0.001 yg/kg/day, both fat and liver contained an average of 540 ppt 2,3,7,8TCDD. Kociba et al. (1976) presented evidence that steady-state had been reached after <13 weeks of feeding of 2,3,7,8-TCDD. McNulty et al. (1982) reported that 2 years after a d ministration of a single oral dose of 1 yg/kg of 2,3,7,8-TCDD to an adult rhesus macaque monkey, tissue levels of the compound were 100 ppt in adipose tissue and 15 ppt In liver. These results Indicate that prolonged retention of 2,3,7,8TCDD may occur in this species. The tissue d i s t r i b u t i o n of 2 , 3 , 7 , 8 - T C D D in the guinea pig appears to be similar to the monkey, with the highest c o n c e n tration of the toxin being found in adipose tissue (Gaslewlcz and Neal, 1979; Nolan et al., 1979). The Interspecies d i fference In the tissue d i s tribution of 2,3,7,8-TCDD may be related to the relative adipose tissue content of a given species and/or the affinity of 2,3,7,8-TCDD for the hepatic microsomal fraction; however, the significance of these differences remains 1n doubt. For example, the h e p a t o t o x l c 1ty of 2 , 3 , 7 , 8- T C DD in a given species does not appear to be related to the hepatic concentration of the toxin (Neal et al., 1982). 2 , 3 , 7 , 8 - T C D D has been d e m o n s t r a t e d to be t e r a t o ge n i c and fetotoxlc 1n the rat (see Teratogenicity section); the ability of 2,3,7,8-TCDD to gain access to the developing fetus of Fischer 344 rats following a single oral dose of [14C]2,3,7,8-TCDD was Investigated by Moore et al. (1976). They found low concentrations of 2,3,7,8-TCDD In the fetus at gestation days 14, 01310 111 -9 04/08/88 18 and 21. The r a d i o a c t i v i t y appeared to be e v enly d i st r i bu t e d throughout the fetus on days 14 and 18; however, increased levels of r a d i o a ct i v it y were dete c t ed in fetal liver on day 21. Nau and Bass {1981 ) (more rece n t ly reported by Nau et al., 1982) investigated the fetal uptake of 2 , 3 , 7 ,8-lCDD in NMRI mice f o ll o w in g oral, i.p. or s.c. a d m i n i s t r a t i o n of the c o mp o u nd at dose levels of 5, 12.5 or 25 y g /kg in D M SQ : c or n oil or a c e t o n e : c o r n oil. The chemical was usually administered as a single dose 2 days prior to sacrifice. All three modes of administration produced similar maternal and embryonic or fetal levels of 2 , 3, 7 , 8 - T C D D at 5 and 12.5 yg/kg. At 25 ug/kg, higher maternal and fetal tissue levels were obtained with s.c. administration, and much higher levels were obtained with i.p. administra tion, than were obtained with oral administration. Embryonic 2,3,7,8-TCDD concentrations were maximal on gestational days 9 and 10; however, low levels were found in the embr y o and fetus betw e e n g e st a tional days 11 and 18. This sharp decrease in 2 ,3,7,8-TCDD c o nc entration coincides with placentation. 2 , 3 , 7 , 8- T C DD concentr a t io n s in the placenta were an order of m a g n i t u d e greater than in the fetus itself. The a f f i n i ty of fetal liver for 2.3.7.8- TCDD was relatively low, as compared to maternal U v e r ; however, 2 . 3 . 7 . 8 - TCDD levels in fetal livers w e re 2-4 times higher than the levels in other fetal organs. An a t t e m p t was made to c o r r e l a t e 2 , 3 , 7 , 8 - T C D D levels in the fetuses with the observed Incidence of cleft palate, but no clear rela tionship was observed. Autoradiographic studies of tissue localization following l.v. a d m i n i s tration of [14C ] 2 , 3 , 7 , 8 - T C D D in DMSO to three strains of m i ce indicated that the liver had the highest concentration and longest retention of radio a c t i v i t y in the body, f o ll o w ed by the nasal m u co s a ( A ppelgren et al., 1983). In pregnant mice, the c o n c e n t r a t i o n of r a d i o a ct i v it y in the fetuses was 01310 III-10 04/08/88 lower than in the dams, but a similar, s elective labelling of the liver and the nasal m u co s a was seen in the fetuses at day 17 of gestation. In the adult animals, labelling of the adrenal cortex was about equal to that of the liver at 1 hour after dosing, but t h ereafter was much lower than in the liver. Labelling of the thymus, lymph nodes, bone marrow and prostate were low at all o b s e r v a t i o n times (l.e., 5 m inutes to 61 days after injection). Very few data are available on the tissue distribution of 2 , 3 , 7 ,8-TCDD in humans. Face he11 i et a l . (1980) reported tissue concen tr a t io n of 2 , 3,7,8-TCDD at levels of 1-2 ng/g in liver and <0.1 ng/g in thyroid, brain, lung, kidney and blood in a w oman who died 7 months after potential exposure to 2,3,7,8-TCQD from the Seveso accident. This pattern of 2,3,7,8-TCOO distribution, however, may not be representative for humans since the woman at the time of death had an adenocarcinoma (which was not considered related to the accident) involving the pancreas, liver and lung. In addition Young et a l . (1983) reported preliminary results of the analysis of adipose tissue from soldiers exposed to Agent Orange. Two analyses were performed, one using the exact mass of 321.8936 and the other the signal profile at masses 321.8936 and 319.8965. Three groups were studied consisting of 20 veterans claiming health problems related to Agent Orange exposure, 3 Air Force officers with known heavy exposure to Agent Orange during disposal operations, and 10 control veterans with no known herbicide exposure. In the first group, 10 of the 20 had measurable levels of 2 , 3 , 7 ,8-TCDD (5 w i th 5-7 ppt, 3 w i t h 9 - 13 ppt and 1 with 23 and 35 ppt and another with 63 and 99 ppt). In the second group only two officers had measurable 2,3,7,8-TCDD levels and these did not exceed 3 ppt. In the 10 control veterans, 4 had 2,3,7,8-TCDD levels between 7 and 14 ppt. Levels of 01310 III-ll 04/08/88 2.3. 7 . 8- TCDD in adipose tissue did not appear to be associated in this study with ill health or any part i c ul a r symptom. However, it was c o n s i d e r e d that i n fo r mation on b a c k g r o u n d levels of 2 , 3 , 7 , 8 - T C D D in a d i p o s e tissue was too limited to draw any firm conclusions. Following the analytical techniques described by Albro et al. (1985), Ryan et a l . (1985) analysed seventy-two autopsy samples of human adipose tissue from North America and observed the presence of PCDDs and PCDFs at low ppt levels. Distr i bu t i on of 2 , 3 , 7 , 8- T C DD has been reported in the general population by Nygren et al. (1986); in Viet n a m and veterans by Schecter et al. (1986, 1987); in mothers milk by Nygren et al. (1986), Rappe et al. (1984), and Fuerst et a l . (1987). Although monitoring of adipose tissue may provide some qualitative indication that exposure has occurred, there are no good correlations available between adipose tissue levels and the extent of exposure. In addition, the background level of 2,3,7,8-TCDD 1n the adipose tissue of individuals with no known history of exposure to 2,3,7,8-TCDD generally are in the r ange of 5-18 ppt. This w o uld suggest an ubiq u i to u s e x po s u re to 2 . 3 . 7 . 8 - TCDD, w h ich m a ke s it d i f f i c u l t to assess the c o n t r i b u t i o n to body burden from any particular small additional exposure. Similar lack of correlation between estimated exposure to 2,3,7,8-TCDD and sera levels of 2 . 3 . 7 . 8 - TCDD w e r e r e po r t e d In a p r e l i m i n a r y report in the M M WR (1987) w h ich compared Vietnam veterans with military histories Indicating exposure to herbicides containing 2,3,7,8-TCDD and non-Vietnam veterans with presumably no unusual exposure to 2,3,7,8-TCDD. At least In these preliminary results 01310 111-12 04/12/88 there was no d i ff e r en c e in the range of 2,3, 7 , 8- T C DD levels (1-9 ppt based on lipid weight) or the m e dian 2,3,7,8-TCDD level (3.9 ppt for the p r e s u m ably exposed group and 3.8 ppt for the non-exposed group). Biological m o n i t o ri n g , such as m o n i t o r i n g levels in breast milk, only p r ovides poss i b le qualitative indications of exposure. With commonly available analytical techniques, 2 , 3 , 7 , 8 - T C D D is not d e te c t ed in body fluids, such as b lood or urine, although a recent method with ppq (parts per quadrillion) sensitivity has detected 2,3,7,8-TCDD in human serum (Patterson et a!., 1987b). Metabolism Vinopal and Casida (1973) found no evidence of water soluble metabolites of 2,3,7,8-TCDD following incubation with ma mm a l ia n liver mlcrosomes or i.p. injection into mice. In the same experiment, only unmetabolized 2,3,7,8TCDD was extractable from mouse liver 11-20 days after treatment. Van Miller et al. (197b) claimed that the slow elimination of 2,3,7,8-TCDD they obse r v ed in both rats and monk e y s after i.p. injections suggested that 2,3,7,8-TCDD was not readily metabolized. Metabolites of 2,3,7,8-TCDD have been d e te c t e d in the b i le and urine of Syri a n Gold e n hamsters after single oral or i.p. doses (Olson et al., 1980a) and in the bile of dogs following repeated direct introduction of the chemical into the duodenal lumen (Polger et al., 1982a). Polger and Schlatter (1979), Ramsey et al. (1979) and Ramsey et a l . (1982) demonstrated biliary excretion of several metabolites of [14C]2,3,7,8-TCDD by rats after repeated oral dosing. The metabolites were tentatively identified as glucuronides of hydroxylated 2,3,7,8-TCDD. The am ou n t s of m e t a b o l i t e s found w e re small, i n di c a ti n g that 2 , 3 , 7 , 8 - T C D D is only slowly m e t a b o l i z e d in the liver. Previous work by Piper et al. (1973) 01310 III-13 04/08/88 using single oral doses of 2 , 3 , 7 ,8-TCDD concluded that, since small amounts of r a d i o a c t i v i t y were found in the urine and expired air of m a le rats duri n g the first 10 days, metabolic alteration or breakdown must occur. The study by Rose et al. (1976 ) using oral doses stated that while the 14C activity in the rat livers a p pe a r e d to be p resent as u n c h a n g e d 2 , 3 , 7 , 8 - T C D D , a s i gn i ficant amount of r a d i o a c t i v i t y found in the feces a p pe a r e d to come from s u bstances other than 2 , 3 , 7 ,8-TCDD; the e x c r e t i o n of 1 4 C in the u rine also indicated that metabolism had occurred. Poiger et al. (1982a) investigated the toxicity of 2,3,7,8-TCDD m e t a b o lites by administering extracts of bile from 2 , 3 , 7 ,8-TCDD-treated dogs to male guinea pigs in single oral doses equivalent to 0.6, 6.0 and 60 yg/kg of parent compound. Other groups of guinea pigs received bile extract from untreated dogs or 2,3,7,8-TCDD Itself. A comparison of the mortality data at 5 weeks after dosing Indicated that the acute toxicity of 2,3,7,8-TCDD to guinea pigs was at least 100 times higher than was the acute toxicity of Its metabol1tes. More recently, Olson et al. (1983) reported that all of the radioactiv ity In urine and bile from 1 4 C - 2 , 3 , 7 ,8-TCDD-treated rats, hamsters and guinea pigs corresponded to metabolites of 2,3,7,8-TCDD. The enzymatic hydrolysis of the 2,3,7,8-TCDD metabolites from the rat and hamster altered the chromatographic profile of the metabolites. Indicating the presence of glucu r on l d e conjugates in bile and sulfate conjugates in urine (Olson and Bittner, 1983). The apparent absence of these metabolites In extracts of hamster and rat liver suggest that once formed, the metabolites of 2,3,7,8TCDD are readily excreted (Olson et al., 1980a; Rose et al., 1976). These results also Indicate that urinary and biliary elimination of 2,3,7,8-TCDD 01310 111-14 04/08/88 is dependent upon m e t a b o l i s m of the toxin. Alth o u gh urine and bile appear to be free of unmetabolized T C D D , data from the hamster and rat indicate that from 10 to AO% of the 2 , 3 , 7 , 8-TCDD-d e r iv e d r a dioactivity in feces represents unchanged 2,3,7,8-lCDD (Olson et al., 1983; Olson and Bittner, 1983). The daily p r e s e n c e of u n ch a n ge d 2 , 3 , 7 , 8 - T C D D in feces and its absence in bile suggests that direct intestinal e l im i n a t i o n may be the source for the fecal excretion of 2,3,7,8-TCDD. This finding demonstrates that the half-life for elimination of 2,3,7,8-TCDD may not directly reflect the i_n vivo rate of 2 , 3 , 7 , 8 - T C D D m e t a b o l i s m in a given animal. N e v e r t h e less, the m e t a b o l i s m of 2,3,7 , 8- T C DD does in part regulate U s elimi n at i o n or relative persistence in a given animal. Several metabolites of 2,3,7,8-TCDD have recently been identified. S a wa h a t a et al. (1982) i n v e s t ig a t ed the Vn vitro m e t a b o l i s m of 2 , 3 , 7 , 8 - T C D D in isolated rat hepatocytes. The major product was deconjugated with B-glucuronidase, derivatized with dlazomethane, and separated into two com pounds by high performance liquid chromatography (HPLC). These metabolites were subsequently identified as 1 -hydroxy-2,3,7,8-TCDD and B-hydroxy-2,3,7trichlorodibenzo--dioxin. Poiger et al. (1982a) identified six metabolites in the bile of dogs that were given a lethal dose of [ 3 H] 2 , 3, 7 ,8-TCDD. The major metabolite was 1,3,7,8-tetrachloro-2-hydroxydibenzo--dioxin; 3,7,8trichloro-3-hydroxydlbenzo--d1oxln and 1 ,2-dichloro-4,5-hydroxybenzene were also identified as minor metabolites. The structures of the three remaining metabolites were not determined; however, two appeared to be trichlorohydroxydibenzo-p-dloxlns and the third was apparently a chlorinated 2-hydroxydiphenyl ether. 01310 111-15 04/ 08/88 Data on the metabolism of 2,3,7,8-TCDD suggests that reactive epoxide intermediates may be formed. Poland and Glover (1979) have investigated the i_n v ivo b i n d i n g of [ 1 ,6-3FI]-2,3,7,8-TCDD derived r a d i o a c t i v i t y to rat hepatic macromolecules. They found m a ximum levels equivalent to 60 pmol 2 . 3 . 7 .8- T C DD / m o l e of a m in o acids in protein, 12 pmol 2 , 3 , 7 , 8 - T C D D / m o l e of n u cl e o t i d e in rRNA, and 6 pmol of 2 , 3 , 7 , 8 - T C D D / m o l e of n u cl e o ti d e in DNA. This corresponds to one 2,3,7,8-TCDD-DNA adduct/35 cells. Poland and Glover (1979) suggest that it is u n li k e l y that 2 , 3 , 7 , 8 - T C D D- 1 n du c e d onco g e ne s i s is through a mechanism of covalent binding to DNA and somatic mutation. Further studies in other species, p o ssibly with [14C ] - 2 , 3 , 7 ,8-TCDD, are needed to confirm these results and assess the relationship between covalent binding and the short and long-term toxicity of 2,3,7,8-TCDD. Isolated rat h e p a t o cy t e s in s u sp e n s i o n have been used as an ijn vitro system for assessing 2,3,7,8-TCDD metabolism under various conditions (Olson et a 1., 1981). Data i n dicate that the rate of 2 , 3 , 7 , 8 - T C D D m e t a b o l i s m in rat h e p a t o cy t e s c o rr e l at e s d i r e c t l y w i th d r ug Induced changes in hepatic c y t o c h r o m e P-450 m o n o o x y g e n a s e a ctivity, s u gg e s t i n g that 2 , 3 , 7 , 8 - T C D D is metabolized by this enzyme (Neal et al., 1982). Pretreatment of rats with 2.3.7.8- TCDD has been shown to enhance the rate of 2,3,7,8-TCDD metabolism in isolated hepatocytes, d e m o n s tr a t in g that 2,3,7 , 8- T C DD can Induce its own rate of metabolism. Beatty et al (1978) also found a correlation between hepatic mixed-function oxidase (MFO) activity and the toxicity of 2,3,7,8TCDD In rats. In both naturally, o c cu r r i n g age and s e x - r e la t e d d i f f e r en c e s in MFO activity, and follo w ing a d m i n i st r a ti o n of inducers and inhibitors of MFO enzyme systems, hepatic MFO activity was directly correlated with the 20-day LD^q * 01310 III-16 04/08/88 Olson and Bittner (1983) reported that the rate of 2, 3, 7 ,8-TCOD m e t a b o lite formation j_n vitro was higher in h e p a t o cy t e s from the hamster than in h e pa t ocytes from the rat. Q u a l i t a t i v e e v al u a t i o n of j_n v 1vo and jjn vitro metabolites by HPLC also suggested significant interspecies variability. The authors sugg e s te d that such di ff e r en c e s in m e t a b o l i s m may p a rt i a l l y explain the diffe r enc e s in toxicity among species. Excretion The f o ll o w in g d i s c u s s i o n assumes that e l im i n a t i o n is a first order process. With the exception of the guinea pig, which may follow zero order kinetics (Gasiewicz and Neal, 1979), elimination data yield a straight line on a s e m i l o g a r 1thmic plot, indi c a ti n g that e l i m i n a t i o n is a single, first order process. Hiles and Bruce (1976) have pointed out that the studies of Allen et al. (1975) and Piper et al. (1973) can be interpreted equally well by either zero or first order kinetics. The majority of the data, however, seem to support the assumption of a first order elimination process. The excretion of 2,3,7,8-TCDD and U s metabolites has been investigated in a number of species. Table I I I -3 summarizes results on the elimination of 2 , 3 , 7 ,8-TCDD-derived radioactivity, following a single exposure to 3H- or [ 14C]-2,3,7,8-TCDD. These studies show that 2,3,7,8-TCDD was slowly excr e t ed from the bodies of all species tested, w ith a h a l f - l i f e in the body of 10-43 days. In the Syrian Golden hamster, the least sensitive mammalian species to the acute toxicity of 2,3,7,8-TCDD, excretion occurred readily through both the urine (35% of administered dose, 41% of total excreted radioactivity) and feces (50% of the administered dose, 59% of total excreted radioactivity) (Olson et al., 1980b; Gasiewicz et al., 1983a). The high levels found in the urine of infant monkeys were probably 01310 1 11-17 04/08/88 01310 TABLF 111-3 Elimination of 2,3,7,8-TCDD 111-18 Species Single Treatment iig/kg (route) Half-Life for Elimination (days) Relative % of TCDD-Derlved Radioactivity Feces Urine Reference Guinea pig Guinea pig Rat Rat Rat Rat Monkey (adult) Monkey (Infant) Mouse C57B1/65 DBA/2J B6D2F!/J* Hamster Hamster 2 (i-p.) 1.45 (oral) 1.0 (oral) '50 (oral) 50 (oral) 400 (l.p.) 400 (l.p.) 400 (l.p.) 10 (l.p.) 10 (l.p.) 10 (l.p.) 650 (l.p.) 650 (oral) 30.2 c 5.8 22 - 43 31 I 6 17.4 c 5.6 21.3 i 2.9 NT NT NT 11.0 v 1.2 24.4 c 1.0 12.6 0.8 10.8 2.4 15.0 c 2.5 94.0 NT >99 80.0 95.5 91.0 78.0 39.0 6.0 NT <1 20.0 4.5 9.0 22.0 61.0 72.0 54.0 72.0 59.0 NT 28.0 46.0 28.0 41.0 Nl Gaslewlcz and Neal, 1979 Nolan et al., 1979 Rose et al., 1976 Piper et al., 1973 Allen et al., 1975 Van Miller et al., 1976 Van Miller et al., 1976 Van Miller et al.. 1976 Gaslewlcz et al., 1983a,b Gaslewlcz et al., 1983a,b Gaslewlcz et al., 1983a,b Oison et al., 1980a Oison et al., 1980a `Offspring of C57B1/6J and DBA/2J which are heterozygous at the Ah locus NT = Not tested 04/08/88 due to the incomplete separation of urine and feces (Van Miller et al., 1976). In all the other species tested so far, e x cretion occu r r ed mainly through the feces (80-100% of total urinary and fecal radioactivity) with only minor amounts of 2,3,7,8-TCDD metabolites found 1n the urine (Piper et al., 1973; Allen et a l ., 1975 ; Rose et a l ., 1976; G a sl e w ic z and Neal, 1979). Only Piper et al. (1973) r e po r t ed the e x c r e t i o n of m e t a b o l i t e s in the expired air. During 21 days foll o w in g a d m i n i s t r a t i o n of a single oral dose of [14C]2,3,7,8-TCDQ to rats, 3.2% of the administered radioactivity (4.6% of the excreted radioactivity) was recovered 1n the expired air. Rose et al. (1976) investigated the elimination of [l*C]2,3,7,8-TCDD In rats given repeated oral doses of 0.01, 0.1 or 1.0 ug/kg/day Monday through Friday for 7 weeks, or a single dose of 1.0 yig/kg. In the s i n g l e dose study, no 14 C was excreted In the urine or expired air; In the r e p e a t e d - d o s e study, however, 3 - 1 8 % of the c u m u l a t i v e dose was e x cr e t ed In the urine by 7 weeks. This study Indicated that steady-state concentrations will be r eached In the bodies of rats in -13 weeks. The rate cons t a nt defining the approach to steady-state concentrations was Independent of the dosa g e of 2 , 3 , 7 , 8 - T C D D over the range studied. This Is c o n s i s t e n t with the observations of Fries and Marrow (1975) who found that the total retention in the bodies of rats was proportional to total Intake. When rats were maintained on a diet containing either 7 or20 ppb 2,3,7,8-TCDD, the amount of 2 , 3,7 ,8-T C DD retai n ed In the body was 5.5 times the dally Intake of 2,3,7,8-TCDD at 14 days, 7.5 times the dally Intake at 28 days, and 10.0 times the dally intake at 42 days. The data In T a bl e I I I -3 suggest some Inter s pe c i es d i f f e r en c e s in the h a l f - l i f e for e l i m i n a t i o n (t 1/2) of 2,3, 7 , 8- T C DD . In the hamster, the 01310 III-19 04/08/88 least sensitive species to the acute toxicity of 2 ,3 , 7 . 8 -TC D O , a mean t 1/2 1 of 10.8 days was o b served (Olson et al., 1980a,b), and in the guinea pig, the most sens i t iv e species to the acute toxicity of 2 , 3 , 7 , 8 - T C D O , the mean t 1/2 was 30.2 days (Gasiewicz and Neal, 1979). The observed interspecies d i f f e r e n c e s in the t 1/2 of 2 , 3 , 7 , 8 - T C D D may in part be r elated to the relative s e ns i t iv i t y of a given species to the acute toxicity of 2,3,7,8-TCOD. ' The I n traspecies d i f f e r e n c e s in the t 1/2 of 2 , 3 , 7 , 8 - T C D D in three m o us e strains may be due to the finding that the DBA/2J strain possesses ~2-fold greater adipose tissue stores than the C57B1/6J and B6D2F ^/J strains ( G asiewicz et a l . f 1983b). The s e q u e s t e r i n g of the l i pophilic toxin in adipose tissue stores of the DBA/2J mouse may contribute to the greater p e rs i s te n c e of 2 , 3, 7 , 8- T C DD in this strain. In all of the rat studies shown in Table 1 1 1 -3, urinary and fecal e l i m i nation were monitored for a period of only 20-22 days, and from these data it was assumed that elimination followed a single component, first order kinetic model. Recently, Olson and Bittner (1983) examined the elimination of 2 , 3,7,8-TCDD-derlved radioactivity in rats over a 35-day period following a sing l e i.p. e x p o s u r e at 1 jig 3H - 2 , 3 , 7 , 8 - T C D D / k g . They o b s e r v e d first order kinetics for elimination, with a fast component having a t 1/2 of 7 days (representing 13% of total elimination) and a slow component having a t 1/2 of 75 days (87% of total). The second, slow component for elimination was evident only when urinary and fecal elimination were monitored for >30 days. This study suggests that 2,3,7,8-TCDD may be more persistent than earlier studies s u ggested. A p r e l i m i n a r y study in the rhesus m o n k e y i n d i cates that 2,3,7,8-TCDD may be exceptionally persistent in adipose tissue. 01310 III-20 04/08/88 M c N u l t y et al. (1982) e s ti m a t e d the appa r e nt half- l if e of 2 , 3 , 7 , 8 - T C D D in the fat of a m o nke y to be ~1 year. Studies in the rat, guinea pig, hamster and m o use have found that all of the 2,3,7,8-TCDD derived radioactivity excreted in the urine and bile c o r r e sponds to metabolites of 2,3,7,8-TCDD (Olson et al., 1983). The apparent absence of 2,3,7,8-TCDD metabolites in liver and fat suggests that, once formed, the metabolites of 2,3,7,8-TCDD are readily excreted. Thus, urinary and biliary e l im i n a t i o n of 2 , 3 , 7 , 8 - T C D D is d e pe n d en t upon m e t a b o l i s m of the toxin. Although urine and bile appear to be free of unmetabolized 2,3,7,8TCDD, data from the hamster and rat indicate that a significant amount (10-40%) of unchanged 2,3,7,8-TCDD may be excreted into the feces (Olson et al., 1983). Unmetabol1zed 2,3,7,8-TCDD thus appears to enter the intestinal lumen by some route other than bile (direct intestinal elimination) for a number of days following treatment. Studies in lactating rats have also found that unchanged 2,3,7 , 8- T C DD may be excreted in the milk of lactating animals (Moore et al., 1976; Lucier et al., 1975). Lactation, direct intes tinal elimination, and perhaps sebum may serve as routes for excretion of 2.3.7.8-TCDD, which are not dependent upon metabolism of the toxin. These data suggest that the i_n vivo h a l f - l i f e for e l i m i n a t i o n of 2 , 3, 7 , 8- TCDD may not directly reflect the rate of 2 , 3, 7 , 8- T C DD m e t a b o l i s m in a given animal (Neal et al., 1982). Due to the lipophilic nature' of milk, secretion of milk can provide a relatively efficient m e c h a n i s m for decreasing the body burden of 2.3.7.8-TCDD in females. As discussed by Graham et al. (1986), this e l im i n a t i o n of 2 , 3 , 7 , 8 - T C D D through m o t h e r 's m i lk can result in large exposures of the infant. Since both milk and the fatty tissues of fish are 01310 111-21 04/12/88 e s s e n t i a l l y p r ov i d i n g an oily vehicle, it w o uld be likely that these sources w o uld prov i d e 2 , 3 , 7 , 8 - T C D D in a form that is r e a d i l y b i o a v a l l a b l e . Several investigators have recently quantified the levels of 2,3,7,8- TCDD in human milk samples. Many of the milk samples were pooled (Jensen, 1987). Rappe et al. (1984) reported levels of 1-3 ppt 2 , 3, 7 , 8 - T C D D in milk fat from five v o lunteers in W. Germany and in a later report Rappe et al. (1985) r e po r t ed an a v e r a g e level of 0.6 ppt 2 , 3 , 7 , 8 - T C D D in milk fat from four volunteers in northern Sweden. Furst et al. (1986) reported an average level of 9.7 ppt 2,3,7,8-TCDD in milk fat from 3 individuals in the N e th e r lands and <1.0 ppt 2,3,7 , 8- T C DD in milk fat from 2 individuals in Y u g o slavia. Nygren et al. (1986) r e po r t e d a v e r a g e levels of 2 , 3 , 7 , 8 - T C D D in human milk samples from 4 subjects 1n Sweden to be 0.6 pg/g In milk fat, in 5 subjects from W. G e r m a n y to be 1.9 pg/g in milk fat, and in 4 subjects from V i e t n a m to be <0.5 pg/g In m i l k fat. . High levels of 2,3,7,8- TCDD have been reported in the milk of mothers exposed to high levels of 2,3,7,8-TCDD in the environment. Reggiani et al. (1980) reported levels between 2.3 and 28.0 ppt 2,3,7,8-TCDD in whole milk from m o th e r s in Seveso. B a ug h m an (1976) repo r t ed levels betw e e n 40.0 and 50.0 ppt 2 , 3, 7 , 8- T C DD in w h ol e milk form mothers in South Vietnam. Schecter et al. (1987), also, found high ppt levels of 2,3,7,8-TCDD in human milk samples f r om Vietnam. T h es e auth o r s found the samples taken in 1985 from Vletnameese mothers were comparable to the level of 2,3,7,8-TCDD presently found in North A m e r i c a n human m i lk samples (5 ppt). 01310 111-22 04/12/88 Summary The t o xi c o ki n e ti c s of 2 , 3 , 7 , 8 - T C D D has been i n vestigated in a number of labo r a to r y animals; the reader is r e ferred to recent reviews (Neal et al., 1982; Gasiewicz et al., 1983a; Olson et al., 1983} for Indepth discussions of this subject. B ecause 2 , 3 , 7 , 8 - T C D D is a stro n g ly lipophilic c o m p o u n d (Crummett and Stehl, 1973), gavage treatment with single or repeated doses of the compound in oil has r e sulted in a b s o r p t i o n of - 5 0 % of the dose a d m i n i s t e r e d to guinea pigs (Nolan et al., 1979), -70-83% of the dose administered to rats (Rose et al., 1976; Piper et al., 1973) or to hamsters (Olson et al., 1980a). Dietary a d m i n i s t r a t i o n of c o m p a r a b l e d o s e - r an g e s to rats r e sulted in s o m e what reduced GI absorption (-50-60% of administered dose was absorbed) (Fries and Marrow, 1975). Using hepatic concentration of 2,3,7,8-TCDD as a endpoint, Poiger and Schla t te r (1980) d e m o n s t r a t e d a linear r e l a t i o n s h i p in rats b etween the magnitude of an oral dose of 2,3,7,8-TCDD and absorption, up to a dose of -1.0 yg. By treating rats with aqueous suspensions of soil treated with 2,3,7,8-TCDD these researchers (Poiger and Schlatter, 1980) were able to show a decrease in Gl absorption of 2,3,7,8-TCDD directly proportional to the length of time the co mp o u nd had been in contact with soil before the suspension was made and the rats were treated. Mixing 2,3,7,8-TCDD with a aqueous suspension of activated charcoal essentially eliminated absorption as measured by hepatic concentrations of 2,3,7,8-TCDD (Poiger and Schlatter, 1980). That adsorbant materials may reduce the GI absorption of 2,3,7,8- 01310 111-23 04/12/88 TCDD was observed by Van den Berg et al. (1983), who demonstrated that absorption of PCDDs was less from fly ash (naturally containing PCDDs) than from comparable doses of PCDDs from extracts of fly ash. Percutaneous absor p ti o n of 2 , 3,7,8-TCDD has been estimated in rats to be -40% of the absorption of an equivalent dose orally administered (Poiger and Schlatter, 1980}. Dermal a p plication of the compound in vaseline, p o l y ethylene glycol or soll/water paste substantially reduced dermal- a b s o r p tion. A p plication of the comp o u nd In a c tivated c a rbon/water paste virtually eliminated absorption. Tissue distribution following oral or 1.p. administration of 2,3,7,8TCDD to rats appears to be preferentially to the liver and adipose tissue (Fries and Harrow, 1975; Rose et al., 1976; Van Miller et al., 1976; K o d b a et al., 1978a). Other tissues showed substantially lower concentrations of 2,3,7,8-TCDD. Soon after treatment the liver may have concentrations =3 (Kociba et al., 1978a) to 5 (Rose et a l ., 1976) times that in a d i p o s e t i s sue. It was suggested that m ale rats a c cu m u la t e 2,3,7 , 8- T C DD in the liver more efficiently than female rats (Fries and Marrow, 1975). Tissue distri bution in m i ce (Manara et al., 1982) and hams t e rs (Olson et al., 1980a) seems to be similar to that in rats. Monkeys, however, appear to a c c u m u l a t e 2 , 3 , 7 , 8 - T C D D p r e f e r e n t i a l l y in adipose tissue > liver (Van Miller et al., 1976; McNulty et al., 1982). Two years after a single oral dose to a monkey, the fat contained 100 ppt and the liver 15 ppt 2,3,7,8-TCDD (McNulty et al., 1982). Prolonged tissue 01310 I I I -24 04/12/88 rete n t io n of the comp o u nd was thus d e mo n strated. Tissue d i s t r i b u t i o n in guinea pigs appears similar to that in monkeys (Gasiewicz and Neal, 1979; Nolan et al., 1979) in that tissue levels in fat exceed those in the liver. Evidence that 2,3,7,8-TCDD accumulates in the adipose tissue of exposed humans was presented by Young et al. (1983) who reported levels of 3-99 ppt in the a d i p o s e tissue of a rmed forces veterans c l a i m i n g health problems related to Agent Orange. Distribution of 2,3,7, 8- T C DD to the fetus has been studied in rats (Moore et al., 1976) and mice (Nau and Bass, 1981; Nau et al., 1982). Levels of 2,3,7,8-TCDD were low in rat fetuses on gestation days 14 and 18 and appeared to be evenly d istributed In all fetal tissues. At gestation day 21, the fetal liver showed a marked affinity for 2,3,7,8-TCDD (Moore et al., 1976). 2,3,7,8-TCDD was distributed to the fetuses of mice following oral, l.p. or s.c. administration (Nau et al., 1982). Maximum fetal c o nc en trations occurred on gestation days 9 and 10; lower fetal concentrations were observed on gestation days 11-18, coincident with placentation. The fetal liver had less affinity for the compound that did the maternal liver. In an e arly m e t a b o l i s m study, Vinopal and Casida (1973) r e ported that .in vivo or jn. vitro studies w i th m i c e showed that polar m e t a b o l i t e s of 2,3,7,8-TCDD were not produced by this species. In rats, however, hydr o x y lation and conjugation with glucuronlde and sulfate have been demonstrated (Poiger and Schlatter, 1979; Olson et al., 1983; Polger et al., 1982a. G l u curonlde conjugates tended to predominate In the bile (Polger and Schlatter, 1979) and sulfate conjugates were located In the urine (Olson et al., 1983). 01310 111-25 04/12/88 Poiger and Schlatter (1979) stated that liver metab o li s m of 2 , 3 , 7 ,8-TCDD p r oceeds slowly in the liver, Neal et al. (1982) d e m o n s t r a t e d that the rate of hepatic metabolism was enhanced by activated cytochrome P-450 m o no oxygenase. It was suggested that metabolism of 2,3,7,8-TCDD proceeds by the formation of reactive epoxide intermediates (Poland and Glover, 1979). That dechlorination also occurs was demonstrated by Olson et al. (1983) and Sawahata et a l . (1982) who Identified tri- and dlchlorodibenzo--dioxins as m e ta b o l i t e s in in vitro rat h e p a t o c y t e systems. F r om the bile of dogs, six major m e t a b o l i t e s have been iden t i fi e d (Poiger et a l ., 1982a); h y d r o x y l a t e d conjugates of tetra-, tri- and dichlorodlbenzo--d1ox1n predominated. When the excretion data are subjected to a semi-logarithmic plot, a straight line results, s u gg e s t i n g that e l i m i n a t i o n of 2 , 3, 7 , 8 - T C D D is a first-order phenomenon, particularly for rats. Excretion in the guinea pig may be a zero-order process (Gasiewicz and Neal, 1979). The half-life for body e l i m i n a t i o n varied c o n s i d e r a b l y with ranges of -10- 1 5 days in the hamster (Olson et al., 1980a), the species least sensitive to the toxic effects of 2 , 3, 7 , 8 - T C D D , to estim a te s of - 1 1 - 2 4 days in the m o u s e (Gasiewicz et al., 1983a,b), -17-31 days in the rat (Rose et al., 1976; Piper et al., 1973; Allen et al., 1975) and -22-30 days in the guinea pig (Gasiewicz and Neal, 1979; Nolan et al., 1979). One strain of mice, DBA/23, had a half life for elimination (-24 days) about twice as high as other strains tested by Gasiewicz et al. (1983a,b). These authors also noted that this strain of mice accumulated 2,3,7,8-TCDD in adipose tissue more strongly than other strains and that this phenomenon probably resulted in slowed body e l i m i n a tion. Half-lives for body elimination of 2,3,7,8-TCDD have not been calcu- 01310 I I I -26 04/12/88 lated for the monkey, but it was s uggested that the tendency of this species to a c c u m u l a t e 2 , 3 , 7 ,8-TCDD in body fat may well result in slowed body e l i m i nation (Van Miller et al., 1976). Recently, Olson and Bittner (1983) examined the elimination of 2,3,7,8TGDD in rats over a longer period than the studies previously summarize d and determined that a biphasic elimination occurred. They suggested a half-life of ~7 days for the initial rapid phase and a half-life of -75 days for the slower phase, probably related to release from stores of body fat. McNulty et al. (1982) estimated the half-life for elimination from the fat of m o n keys to be -1 year. The 'fecal route seems to be the major pathway for the elimination of 2,3,7,8-TCDD-derlved radioactivity from rats (Rose et al., 1976; Piper et al., 1973; Allen et al., 1975; Van Miller et al., 1976), guinea pigs (Gasiewicz and Neal, 1979) and mice (Gasiewicz et al., 1983a,b). Urinary excretion played less of a role in these species, accounting for <1-28% of total excreted radioactivity while fecal excretion accounted for 72->99% of the eliminated radioactivity. Urinary excretion accounted for a more sub stantial p r o p o r t i o n of body e l i m i n a t i o n in hams t e rs (41% c o mp a r e d to 59% by feces) (Olson et al., 1980a) and that strain of mice (DBA/2J) which pr efer e n ti a l l y a c c u m u l a t e d 2 , 3 , 7 , 8 - T C D D in body fat ( G asiewicz et a l ., 1983a,b). The failure to detect metabolites of 2,3,7,8-TCDD in liver and fat (Olson et al., 1983) Indicates that elimination of the metabolites occurs rapidly and that the rate of e l i m i n a t i o n is g o v e r n e d p r i m a r i l y by the rate of hepatic metabolism. 01310 111-27 04/ 12/88 I I IV. HUMAN EXPOSURE Text to be provided by the Office of Drinking Water. t 01320 IV-1 03/16/84 Experimental Animals V. HEALTH EFFECTS IN ANIMALS Acute Toxicity. Lethal Effects -- There have been studies 1n a variety of species ij defining the doses necessary to cause death after acute exposure to 2,3,7,8- TCDD. A summary of the single dose LD^^ data for 2,3,7,8-TCDD Is present ed 1n Table V-l. The dose that results In death varies extensively with species, with the male guinea pig the most sensitive species tested (L0g0 of 0.6 yg/kg) (Schwetz et al., 1973), and the male hamster the least sensitive species tested (LD^ of 5051 yg/kg) (Henck et al., 1981). The rat and monkey appear to be the second most sensitive species, with LD5q S between 22 and 70 yg/kg (Schwetz et al., 1973; McConnell et al,, 1978a), while other species tested (rabbit and mouse) had L O ^ s between 114 and 283 yg/kg (Schwetz et al., 1973; McConnell et al., 1978b; Vos et al., 1974). Schwetz et al. (1973) found male rats more sensitive to 2,3.7,8- TCDO, while Beatty et al. (1978) found adult female and weanling male rats more sensitive than adult male rats (see Table V-l). In C57B1/10 mice, Smith et al. (1981) reported adult males to be far more sensitive to the acute toxicity of 2,3,7,8-TCDD than adult females. Thus, data on sex differences 1n sensitivity to the acute toxicity of 2,3,7,8-TCDD are con flicting and may depend on the species examined. Harris et al. (1973) studied the toxic effects of 2,3,7,8-TCDD 1n rats, mice and guinea pigs with regafd to single or multiple exposures. Similar effects were observed after a single exposure to 2,3,7,8-TCDD as were observed when multiple exposures totaled the same dose as received In the single exposure. As Illustrated most clearly 1n rats, a single dose of 00110 V-l 09/18/84 tABLE -1 Lethal Doses of 2,3,7,8-TCDD Following Acute Exposure 00110 V-2 08/11/84 Specles/Strain Sex/No./Group Guinea pigs/ Hartley Guinea pigs/ Hartley Guinea pigs/ Hartley M/NR H/NR H/9 Route/ Vehlcle gavage/corn oll-acetone (9:1) goa1vla-gaec/ectoornen <9:1| gavage/ corn ol Dose Tested (vg/kg) NR NR NR Duration of Observation 2 -0 weeks 2 -0 weeks 30 days Guinea pigs/ Hartley Guinea pigs/ Hartley Rats/ Sherman Rats/ Sherman r Rats/SpragueDawley Rats/SpragueDawley F/ 6 gavage/ corn ol * F/ 6 gavage/ methyl cellulose H/5-10 F/NR H/ 6 F/ 6 gavage/corn oll-acetone (9:1) gavage/corn oll-acetone (9:1) l.p./olive ol l.p./olive ol 0.1 0.5 2.5 12 20.. 05 0.1 0.5 2.5 2120.. 05 6 16 32 63 NR NR NR 42 days 12 days 2 - 0 weeks 2 - 0 weeks 20 days 20 days LD50 (vg/kg) 0 .6 (0.4-0.9)* 2.1 (1-5-31* 2 ^ 2.5 (1.2-5.4. 95 confidence) Comvents Time to death was 5-34 days, the 2,3,7,0-TCDO was 91 pure Time to death was 9-42 days, the 2,3,7,8-TCDD was 99 pure NedIan time to death was 17-20 days. marked weight loss, thymus atrophy. Intestinal hemorrhage, no porphyria and only mild liver Injury Time to first death was 32 days In the 2.5 vg/kg group, with 50 mortality by day 42 Reference Schwetz et al., 1973 Schwetz et al., 1973 HcConnell et al.. 1978b Sllkworth et al., 1982 19 (15-23. 95 confidence) 22 Time to first death was 12 days In the 20.0 v9/kg group, with 67 mortality by day 42 Time to death was 9-27 days, the 2,3,7,B-TC00 was 91 pure Sllkworth et al., 1982 Schwetz et al.. 1973 45 (30-66)* 60 25 Time to death was 13-43 days, the 2,3,7,8-TCOO was 91 pure LDcq (vg/kg, mean SE) adult male, 60.2 7.8; weanling male, 25.2 1.4 Adult female had a mean _ SE of 24.6 2.0 vg/kg Schwetz et al.. 1973 Beatty et al., 1978 Beatty et al., 1978 OOnO V-3 08/11/84 TABLE V-1 (c o n t.) i Specles/Straln Sex/No./Group Route/ Vehicle Dose Tested (ugAg) Duration of Observation Monkey/rhesus M1ce/C57B1 F/3 M/14 gavage/ corn oil gavage/corn oil-acetone 0 :1) 0 70 350 0 100 150 200 . >35 days 60 days L05O (ug/kg) <70 114 M1ce/C57B1 M/9 gavage/ corn oil NR 30 days 283.7 H1ce/CS7B1/10 M/5 M1ce/C57B1/10 F/5 M1ce/C57B1/6J M1ce/0BA/2J M1ce/B6D2F,/J M/NR M/NR M/NR gauge/ arachls oil gauge/ arachls oil 1.p./olive oil l.p./olive oil 1.p./olive oil 85 107 135 170 213 B5 107 135 170 213 269 33B 426 536 NR NR NR 45 days 45 days 30 days 30 days 30 days 146 >450 132 620 300 Comnents Reference Weight loss, edema, severe thymus atrophy, loss of hair, mild liver damage Time to death In the high dose group was 15-20 days, bw loss, edema In 25% of treated animals, severe thymic and spleen atrophy, hemor rhage In the region of the eye and small Intestine, liver necrosis in the centrllobular region Median time to death was 22-25 days, dose-related bw loss, thymic atrophy. Increased liver weight and porphyria, gross and historic liver alterations, subcutaneous edema. Intestinal hemorrhage 95% confidence limits of 111-211 pg/kg. Host deaths occurred from 22-26 days after dosing. Signs of porphyria, edema, hemorrhage. McConnell et al., 1978a Vos et al., 1974 McConnell et al.. 1978b Smith et al., 1981 1 of 4 animals died at dose of 426 pg/kg Smith et al., 1981 BGD2Fi/J mice are the offspring of C57B1/6J and DBA/2J. The BGD2j/J mice are heteroiygous at the Ah locus. No comnent 6as1ew1cz et al., 1983a,b Gaslewlcz et al., 1983a,b Gaslewlcz et al., 1983a,b TABLE V-l (coni.) 00110 V-4 09/18/84 Specles/Straln Sex/No./Group Route/ Vehicle Dose Tested (Mg/kg) Duration of Observation lo50 (wg/kg) Conmenls Reference Rabbits/ New Zealand Rabbits/ New Zealand Rabbits/ New Zealand Hamster/ golden Syrian Hamster/ golden Syrian Hamster/ golden Syrian Dogs/beagle Dogs/beagle NAT/NR NAF/5 HAF/NR H/6 HAF/5-6 N/5 n/2 Fn gavage/corn ol1-acetone (9:1) I.p./ corn oil dermal/ acetone gavage/corn oil-acetone (91) I.p./ olive oil gavage/ olive oil gavage/corn oil-acetone (9:1) gavage/corn oil-acetone (9:1) NR 32 63 126 252 500 31.6 63 126 252 500 0 300 600 1000 3000 6000 0 500 1000 2000 3000 500 1000 2000 3000 3000 30 100 2-8 weeks 4 weeks 115 (38-345)* NR 3 weeks 275 (142-531)* Time to death was 6-39 days, the 2,3,7.8-TCDO was 91% pure Time to death was 6-23 days. 2-3 anlmals/group died In all but the low exposure group Time to death was 12-22 days Schweiz et al., 1973 Schwetz el al., 1973 Schweiz et al.. 1973 55 days 50 days 50 days 2-8 weeks 2-8 weeks 5051 (3076-18,487, 95% confidence) Time to death was 26-43 days, the liver and thymus appeared to be the primary target organs, only 1 death occurred In the 300 and 3000 wg/kg group Henck et al., 1981 >3000 1157 NA Significant, dose-related decrease In thymus weight starling at 500 wg/kg, only 2 deaths occurred out of 11 hamsters In the 3000 wg/kg group. Death generally occurred between 24 and 45 days, decrease In bw above 2000 w9/kg, proliferative Ileitis with mild to severe inflanmatlon All animals died Olson et al.. 1900b Oison et al., 1960b Schwetz et al., 1973 NA All animals survived Schwetz et al.. 1973 `The number In parentheses appears to Indicate the range of lethal doses; however, the article did not specify what these numbers represented. I.p. = Intraperitoneal; NR = Not reported; NA - Not applicable i 25 yg/kg, 6 weekly doses of 5 yg/kg, or 30 dally doses of 1 yg/kg were all the threshold dose for observing a decrease In body weight. Other endpoints, Including lethality, decrease 1n thymus weight, and a no effect level for body weight change In rats, mice and guinea pigs 1n general t .. appeared likewise to require a specific threshold level regardless of whether this level was achieved through a single exposure or a small number of multiple exposures. Although 2,3,7,8-TCDD has over a 10*-fold difference In toxicity depending upon the species tested, some of the signs of lethal toxicity were the same regardless of species. One of the most characteristic observations after acute lethal exposure to 2,3,7,8-TCDD was the protracted time between exposure and death (see Table V-l). In determining the LD^q In the least sensitive animal, the hamster, the test animals died between 24 and 45 days after a single acute exposure (Olson et al., 1980b), and similar observa tions were made 1n all other species, tested Including the most sensitive species, the guinea pig, 1n which animals died up to 42 days after treatment (Schwetz et al., 1973). During this extended period between treatment and death the animals had poor weight gain or loss of weight and appeared to be "wasting away." In female Wlstar rats Intubated with 2,3,7,8-TCDD at a dose of 100 yg/kg, the weight loss was blphasle (Courtney et al., 1978). The Initial weight loss occurred rapidly during the first 7-10 days after treatment and was asso ciated with decreased food and water consumption. This Initial phase of weight loss was reversed with the resumption of normal food Intake for 4 or 5 days, only to be followed by a second, more gradual, decline In food and 00110 V-5 08/11/84 water Intake and weight until death. Providing animals with an adequately nutritious liquid diet by Intubation did not appreciably alter the pattern of weight loss nor affect survival. In contrast, Gaslewlcz et al. (1980) observed that providing rats with total parenteral nutrition would prevent t ... some of the weight loss Induced by 2,3,7,8-TCDO; however, there was no protection from the lethal effects of 2,3,7,8-TCDO. In yet another study, Seefeld and Peterson (1983) suggest that a reduction In food Intake caused by 2,3,7,8-TCDD 1s primarily responsible for the loss of body weight or depressed growth rate of rats. Pair-fed control rats lost weight at the same rate and to the same extent as their weight-matched 2,3,7,8-TCDDtreated partners (25 or 50 yg/kg) until day 10 after treatment. At 20-35 days after treatment, the body weight of the two groups began to diverge, with the pair-fed control group having body weights that were 20-30 g higher than the corresponding 2,3,7,8-TCDO groups. The mortality in the 25 and 50 yg/kg groups was 33 and 75X, respectively, while In the corresponding pair-fed groups the mortality was 0 and 15X. The authors proposed a hypoth esis that 2,3,7,8-TCDD lowers a regulated level or "set-point* for body weight control In the rat. The ensuing change In food Intake was thought to occur secondarily to the change 1n set-point. Also, severe thymic atrophy 1s universally observed In all species given lethal doses of 2,3,7,8-TCDD, and since weight loss and thymic atrophy are both associated with malnutrition, van Logten et al. (1981) Investigated the effects of dietary protein on 'the toxicity of 2,3,7,8-TCDO. Groups of female Fischer 344 rats administered 2,3,7,8-TCDD (20 yg/kg) and main tained on low (3.5X), normal (26X) or high (55X) protein diets maintained approximately the same amount of weight (-0.2+3, 7+6 and 7+3 g for each 00110 V-6 08/11/84 dietary group, respectively) during the subsequent 10-day period. The weight gain 1n treated animals was 10-10 g less than that 1n the respective control rats. Dietary protein also had no effect on preventing or enhancing the 2,3,7,6-1000 Induced thymic atrophy. Although weight loss and thymic I atrophy were present In most species tested, there were other symptoms which were characteristic of toxicity 1n only some species. In the guinea pig, besides thymic atrophy, no gross changes were observed 1n Internal organs after a lethal oral or 1.p. dose of 2,3,7,8-TCDD (Grelg et al., 1973, Gupta et al., 1973). Hemorrhages were observed 1n a number of organs Including the adrenal gland, urinary bladder, 61 tract and mesenteric lymph nodes; however, these were considered unremarkable changes by Gupta et al. (1973). Histologic examination confirmed the gross observa tions with atrophy and lymphoid cell depletion 1n the thymus, spleen and lymph nodes, and hemorrhages observed 1n many organs. In addition, marked hyperplasia of the urinary bladder was observed. Of particular Interest was the absence of severe toxic effects on the liver. Gross observation under UV light Indicated no excess of porphyrin, while histologic examinations revealed diffuse single cell necrosis. Identical observations were made by McConnell et al. (1978b) 1n guinea pigs administered lethal doses of 2,3,7,B-TCDD, with the additional observation that the sternal bone marrow was hypocellular 1n all types of blood-forming cells. Turner and Collins (1983) described some histologic changes In the liver of guinea pigs treated with 2,3,7,8-TCDD. Groups consisting of 4-6 female Hartley guinea pigs were treated with 2,3,7,8-TCDD at doses of 0.0, 0.1, 0.S, 2.5, 12.5 or 20 pg/kg, and 1 male guinea pig each was treated with a 00110 V-7 08/11/84 dose of 0.1 or 0.5 yg/kg. The 2,3,7,8-TCDD was administered by gavage as an aqueous suspension 1n 0.75% methyl cellulose and surviving animals were killed 42 days after treatment. A second group of guinea pigs {6 males and 5 females/dose} were administered soot generated from a fire In a trans former cooled by polychlorinated biphenyls and chlorinated benzenes (1, 10, 100 and 500 mg/kg). The histologic observations as described were applied 1n general to both treatment groups and there was no apparent relationship between dose and response. The authors reported that, compared to controls, all experimental groups showed liver alterations but that qualitative dif ferences among the dosage groups were not detectable by light microscopy. At the light microscope level, hepatocellular hypertrophy, steatosis, focal necrosis, cytoplasmic degeneration and acidophilic hyalln-Hke cytoplasmic Inclusion bodies were observed. Even though there was no dose-response relationship for these liver lesions, the doses spanned a range that result ed In the lowest dose being nonlethal (none of the 4 female guinea pigs died during the study), while 1n the high dose group 4 of 6 animals died before 42 days post-treatment. The LD^g for female guinea pigs was determined 1n this study, and reported by Sllkworth et al. (1982), to be 2.5 or 19 yg/kg bw depending on whether the compound was administered by gavage In corn oil or 1n aqueous methyl cellulose. The greatest difference at necropsy 1n the gross and histologic effects 1n rats and mice of exposure to lethal doses of 2,3,7,8-TCDD was pathologic alterations In the liver, as compared with guinea pigs. An early report by Buu-Ho1 et al. (1972) described alterations In the architecture of the liver of rats within 5 days of receiving a low dose of 2,3,7,8-TCDD (10 yg/kg by l.p. Injection). At higher oral doses of 100 or 50 yg/kg, which killed 43 00110 V-8 09/18/84 and 1% of the animals, respectively, Gupta et al. (1973) also observed marked distortion of liver architecture 1n rats; however, only mild regener ative changes of the liver were observed at the sublethal dose of 5 yg/kg administered weekly for 6 weeks. Liver toxicity appeared to develop slowly 1n The rat with no change fn liver function, as indicated by plasma protein and bilirubin levels, or alkaline phosphatase, glutamlc-oxalacetlc trans aminase (GOT) and glutamic-pyruvic transaminase (GPT) activity being detect ed 3 days after Intubation with 2,3,7,8-TCDD at a dose of 200 yg/kg (Grelg et al., 1973). Bilirubin levels were, however, markedly elevated from 0.33 yg/100 mi 1n control animals to 10.97 yg/100 ml 1n treated animals 21 days after exposure (the other parameters were not measured at this time, although plasma protein was slightly but significantly decreased when determined 9 days post-rtreatment). As 1n rats, the livers of mice exposed to lethal levels of 2,3,7,8-TCDD had signs of necrotic changes (Vos et al., 1974); however, Jones and Grieg (1975) reported that the centrllobular necrosis, bile duct proliferation and lipid accumulation were more extreme 1n mice than 1n rats. Examination of mouse livers using long wave UV light showed fluorescence suggestive of excess porphyrin accumulation (McConnell et al., 1978b). Although excess porphyrins may be present 1n the livers from 2,3,7,8-TCDD-exposed rats, fluorescence 1s not usually observed. Besides effects on the liver, 2,3,7,8-TCDD exposure produced other toxic effects 1n rats and mice that were not observed or were observed to a lesser extent 1n guinea pigs. In rats that died from 2,3,7,8-TCDD exposure, there were extensive hemorrhages of the heart, Uver, brain, adrenal gland and GI tract along with ulcers and necrosis of the glandular stomach, and 1n females, atrophy of the uterus (Gupta et al., 1973). In mice, facial edema 00110 V-9 09/18/84 was severe and the testicles of males appeared degenerated with necrotic spermatocytes and spermatozoa present (McConnell et al., 1978b; Vos et a1., 1974). Death In mice was frequently attributed to terminal hermorrhages (Vos et al., 1974). t , In monkeys exposed to lethal levals of 2,3,7,8-TCDD, McConnell et al. (1978a) reported clinical and histologic signs of toxicity, some of which were similar to those already described for other species. Severe thymic atrophy and edema occurred in treated animals, as well as extensive weight loss that could account for up to 38% of the body mass. As 1n guinea pigs, Uver Injury appeared to be mild; however, Increased serum GOT and aldolase activity and decreased albumin levels Indicative of liver pathology occurred near the time of death. As observed 1n mice, the bone marrow of monkeys was hypocellular. In addition to the above signs of toxicity, which were observed 1n other species as well, monkeys had progressive loss of hair, toenails and fingernails, with associated dermatitis consisting of the development of a crusty texture to the skin, squamous metaplasia of sebace ous glands and gastric mucosal dysplasia. As with most other species, a specific cause of death could not be determined for monkeys. Poland and Knutson (1982) have sunmarlzed the toxic response of various species to 2,3,7,8-TCDD (Table V-2). There was very little Information on the lethal effects of PCDD congeners other than 2,3,7,8-TCDD. McConnell et al. (1978b) determined the LOgp for nine congeners of PCDD following a single treatment by gavage 1n mice and guinea pigs. A comparison of the LDjq expressed as pmol/kg body weight 1s presented 1n Table V-3. The limited data suggest that con geners containing chlorine In the 2,3,7,8 positions were more biologically 00110 V-10 09/18/84 TABLE V-2 Toxic Responses Following Exposure to 2,3,7,8-TCDD: Species Differences3 00110 V-11 Monkey Guinea Pig Cow*5 Rat Mouse Rabbit** Chicken*5 Hamster Hyperplasia and/or metaplasia Gastric mucus Intestinal mucosa Urinary tract B1le duct and/or gall bladder Lung: focal alveolar Skin Hypoplasia, Atrophy or Necrosis Thymus Bone marrow Testicle Other Liver lesions Porphyria Edema + +C 4- ++ 44 44 4 4 4 0 4 0 400 4 + 44 00 04 44 44 0 *d 0 0 444 4- 4 4- 4 4- 4 4 4 40 4- + 40 0 4- 4+ 44 0 + 4- 0 0 4 444- 4 4- 4- 0 4+ 4 References: Monkey (McConnell et al., 1978b; Norback and Allen, 1973; Allen et al., 1977); guinea pig (McConnell et al.. 1978b; McConnell, 1980; Moore et al., 1979; Turner and Collins, 1983); cow (McConnell, 1980); rat (McConnell, 1980; Koclba et al., 1978a,b, 1979); mouse (Schwetz et al., 1973; McConnell et al. , 1978b; Vos et al., 1973); rabbit (K1mm1g and Schultz, 1957; Schwetz et al., 1973; Vos and Beems, 1971); chicken (Schwetz et al., 1973; Norback and Allen, 1973; Allen and Lallch, 1962 ; Vos and Koeman, 1970); hamster (Olson et al., 1980b; Henck et al., 1981). h^Responses foll. owed exposure to 2,3,7,8-TCDD or structurally related chlorinated aromatic hydrocarbons. cSymbols: 0, lesion not observed; +, lesion observed (number of "+" denote severity); observed to a very limited extent; blank, no evidence reported 1n literature. lesion ^Skln lesions 1n cattle are observed, but they differ from the skin lesions observed 1n other species. Adapted from Poland and Knutson, 1982 09/18/84 TABLE V-3 Estimated Single Oral LD50_30 Values for PCDOs3 t, Chlorination of PCDOs Guinea Pigs (ijmol/kg)b 2,8 2,3.7 2,3,7 ,B 1,2.3,7,8 1,2.4.7,8 1.2,3,4,7,8 1.2.3.6.7.8 1 .2.3.7.8.9 1 .2.3,4.6.7,8 >1180 120.41 0.006 0.009 3.15 0.185 0.178-0.255c 0.153-0.255 >1.400 aSource: Adapted from McConnel 1 et al., 1978b bSpearman-Karber method cEstimated range due to variability in replicates NR = Not reported Mice (pmol/kg)b NR >10 0.88 0.94 >14 2.11 3.19 >3.67 NR 00110 V-l 2 09/18/84 active than congeners deficient 1n a chlorine from any one of these positions. It also appears that addition of one or more chlorines to 2,3,7,8-TCDD results 1n a decrease In lethality. Although the congeners vary 1n effective dose between mice and guinea pigs, the relative order of tojridty of these congeners did not change. Also, similar effects of toxicity were observed for all congeners as described above for 2,3,7,8-TCDO when the comparison was made within a single species. Effects on the Liver -- The histological and ultrastructural changes 1n the liver Induced by oral exposure to 2,3,7,8-TCDO have been reported by Fowler et al. (1973}, Jones and Butler (1974) and Jones (1975). Fowler et al. (1973) treated groups of 30 male rats with a single dose of 2,3,7,8-TCDO at 0.0, 5 and 25 yg/kg by gavage. The animals were killed 1n groups of 5 on days 1, 3, 6, 9, 16 and 28 after treatment and the livers were prepared for histologic examination. The major ultrastructural change observed was a dose-related Increase 1n the smooth and rough endoplasmic reticulum (ER) 1n cells near the bile canallcull. The Initial Increases appeared at day 3, with the maximal response occurring on days 6 and 9. By day 16 the smooth ER was nearly absent from the parenchymal cells, although large amounts of rough ER were still present. By day 28 the cells had returned to normal appearance. These changes 1n Uver cells following 2,3,7,8-TCDD treatment would be consistent with the Induction of protein and RNA synthesis. Transmission electron microscopic observations revealed that single l.p. administration of 20 yg/kg of 2,3,7,8-TCDD 1n Sprague-Dawley male rats produces necrotizing hepatic lesions which become progressively worse up to the 16th week postexposure followed by gradual Improvement of the condition and disappearance of the lesions (Weber et al., 1983). 00110 V-13 09/18/84 At higher doses of 200 ug/kg, Jones and Butler (1974) observed necro sis and proliferative changes in the liver of rats to be the predominant lesions. After treatment by gavage, groups of 4 male and 4 female rats were killed and examined on a weekly basis for 10 weeks. By the first week, degenerating cells were observed near the central vein and these lesions progressed to areas of focal necrosis by the sixth week. Superimposed on the necrotic changes were hyperplasia of the viable cells with multlnucleated cells common by the ninth week. At week 10 central vein fibrosis and scattered necrosis remained. Fine structure observed after this large dose of 2,3,7,8-TCDD also revealed Increases 1n smooth ER; however, the most striking effect was degeneration of the plasma membrane with the resulting fusion of parenchymal cells. In a study of similar design, Jones (1975) followed the distribution with time after treatment of membrane associated ATPase activity by hlstochemlcal techniques. At 3 days after treatment, the first changes In ATPase patterns were observed, with loss of activity along the canalicular borders and some Increased activity In the sinusoids. The mldzonal and periportal zones had normal activity at this time. The loss of ATPase activity persisted for 34-42 days, and paralleled the histologic lesions described previously (Jones and Butler, 1974). In rats that survived treatment, the ATPase activity was back to normal by 9 months. Peterson et al. (1979a) further studied the effect of 2,3,7,8-TCDD at lower doses on hepatocyte plasma membrane ATPase activity. Liver surface membranes (LSM) Isolated from male Holtzman rats 2, 10, 20 or 40 days after Intubation with 2,3,7,8-TCDD at 0.0, 10 or 25 yg/kg were used for determi nation of Na+, K+-ATPase and Hg++-ATPase activity. The activity of Na+, and K+-ATPase was depressed to the same extent for both doses of 00110 V-14 09/18/84 2,3,7,8-TCDD from day 2-40 after treatment, while a similar depression of the Hg++-ATPase activity was observed only 1n the high dose group. In the low-dose group, there was a decrease 1n Mg++-ATPase at 20 days, but recovery to normal levels occurred by 40 days post-treatment. It was demon- strated that the effect of 2,3,7,8-TC0D on ATPase activity was not the result of 2,3,7,8-TCDO Induced food deprivation and l vl tro studies Indicated that the loss of activity was not due to the direct Interference of 2,3,7,8-TCDO with the enzyme. Quantitative changes (both Increases and decreases) have been reported for the protein composition of plasma membranes Isolated and analyzed by electrophoresis from Sprague-Dawley rats 10 days after an l.p. Injection of 2,3,7,8-TCDD, Indicating that exposure was actually affecting membrane components (Brewster et al., 1982). Peterson et al. (1979a) did observe a positive correlation between the levels of LSM ATPase activity and both In vivo cumulative biliary excretion of ouabain and bile flow (pi/mln/g liver). Using perfused liver, however, Peterson et al. (1979b) reported a segregation between LSM ATPase activity and biliary excretion of ouabain when 2,3,7,8-TCDD rats were exposed to the protective agents pregnenolone-l6o-carbon1tr1le or spironolactone. It was concluded that LSM ATPase did not directly participate 1n ouabain transport. Additional studies have described the effect of 2,3,7,8-TCDD on the biliary excretion of a variety of xenoblotlcs. Early studies by Hwang (1973) Investigated 2,3,7,8-TCDD Inhibition of biliary excretion In male CD rats given a single dose of 2,3,7,8-TCDD at 25 or 5 yg/kg by gavage. Animals were examined for Indocyanine green (ICG) excretion 1, 7 and 16 days after treatment. Unlike Peterson et al. (1979a), Hwang (1973) observed an 00110 V-15 / 09/18/84 Inverse relationship between 2,3,7,8-TCDD exposure and bile flow, with m a x i m u m bile flow observed 1n the 25 pg/kg dose group at 16 days. Even with this Increased bile flow, however, the cumulative biliary excretion of ICG was decreased In a dose-dependent manner with the greatest depression t observed 7 and 16 days after the exposure to 2,3,7,8-TCOD. The levels of ICG In the plasma and liver was higher In treated animals than 1n control animals, while the concentration 1n the bile was lower, reflecting the decrease 1n total excretion of ICG. Yang and Peterson (1977) compared the effect of 2,3,7,8-TCOD on the biliary excretion of the organic neutral compound, ouabain, with that of the organic anions phenol-3,6-d1bromophthaleln (DBSP) and sulfobromophthaleln (BSP) In male Holtzman rats. Animals were Intubated with 2,3,7,8-TCDD at doses of 10 or 2S vg/kg and excretion was evaluated periodically between 2-4 days postexposure. The biliary excretion of ouabain was depressed 1n a dose-related manner starting on the second day post-treatment, with maximum depression developing between 10 and 20 days, and some recovery observed by day 40. Decreases In bile flow followed a pattern similar to that observed for ouabain. The pattern of biliary excretion was different for DBSP and BSP In which only a transient small decrease was observed 10 days after exposure In the high dose group. In the low dose animals there was actually an Increase at days 10 and 25 In the excretion of the anions. The results obtained for DBSP and BSP differ sharply from those for the organic neutral ouabain or those reported by Hwang (1973) for the organic anion ICG, 1n which a dose-related decrease 1n biliary excretion was observed. The authors concluded that the effects of 2,3,7,8-TCDD on the multiple pathways Involved In biliary excretion depend on the specific compound being studied. 00110 V-16 09/18/84 In the guinea pig and rhesus monkey, which do not develop significant liver pathology after exposure to 2,3,7,8-TCDD, there was also little change 1n ICG blood clearance rates, while 1n the rabbit, which develops 2,3,7,8TCDD-Induced liver damage similar to the rat, there was reduced blood clear- l r .. ance of ICG (Seefeld et al., 1979, 1900). In the rabbit, there were In creases 1n serum sorbitol dehydrogenase and GPT activity as further Indica tions of 2,3,7,8-TCDD-produced liver damage. In the monkey, which received 2.3.7.8-TCDD by gavage at doses of 5, 25 or 75 yg/kg, there was an Initial slight Increase 1n the blood clearance of ICG at 2 days post-treatment, followed 1n the two higher dose groups by a dramatic decrease a few days before death. Although some serum enzymes (sorbitol dehydrogenase and GPT) Indicative of liver damage were elevated, the hlstopathology of the Uver was within normal limits. It appears that major effects on biliary excre tion occur only 1n species that are sensitive to the hepatotoxlc effects of 2.3.7.8- TCDD. Other gross signs of. the hepatotoxlc effects of 2,3,7,8-TCDD observed 1n some species Included fatty degeneration and porphyria. Early observations by Cunningham and Williams (1972) described*a decrease In In vivo (1 hour pulse) Incorporation of aH sodium acetate Into Uver lipids after exposure of male Wlstar rats to 2,3,7,8-TCDD. The rats (12-16 animals) were treated with 2,3,7,8-TCDD at a dose of ID yg/kg followed In either 3 or 7 days by the assessment of lipid synthesis. At 3 days Incorporation decreased from 258 to 98 dpm/mg Upld In the' control and treated animals, respectively. There was an approximately similar decrease observed 7 days postexposure. 00110 V-17 09/18/84 When individual classes of lipids were examined, there was a decrease In the synthesis of triglycerides, diglycerides and phospholipids. Although Cunningham and Williams (1972) observed that 2,3,7,8-TCDD decreased lipid synthesis, Albro et al. (1978) reported an Increase 1n total Uplds 1n the livers of rats 13 days after treatment with 2,3,7,8-TCDD at a lethal dose of 50 pg/kg. For Individual classes of lipids there was an Increase In free fatty acids and cholesterol esters, while no change occurred 1n the content of phospholipids, free cholesterol or triglycerides. The fatty changes 1n the Uver were confirmed by ultrastructural examination of liver specimens. At a sublethal dose of 10 pg/kg there was a different pattern of lipid accumulation, with triglycerides and fatty acids Increased and cholesterol esters decreased. The changes In the 11p1d profile of the ' liver was attributed to 2,3,7,8-TCDD Induced mobilization of body fat, a decrease In lysosomal acid lipase (74% decline In this enzyme 10 days after a 50 pg/kg dose of 2,3,7,8-TCDD) and an Increase In Upld peroxidation as Indicated by a sharp Increase In the production of Upofuscln pigments. Porphyria was Initially characterized quantitatively In mice by Goldstein et al. (1978). Groups of 12 male C5781 mice received 4 weekly intubations of 2,3,7,8-TCDD at doses of 0.0, 1, 5 or 25 pg/kg, or a single dose of 150 pg/kg followed 21-25 days after treatment by analysis of the Uver for porphyrins. Porphyrin levels were unchanged except In the 25 and 150 pg/kg groups where the levels were Increased 2000- and 4000-fold, respectively. The difference 1n responsiveness to the development of por phyria was studied by Smith et al. (1981) In C57B1 mice which were sensitive to, and DBA/2 mice which were Insensitive to, the toxicity of 2,3,7,8-TCDD. 00110 V-18 08/11/84 Hale and female C57B1 mice had a dose-related Increase 1n hepatic porphyrins 1n the two high dose groups 3 weeks after a single exposure to 2,3,7,8-TCOD at 0.0, 5, 15, 50 or 75 yg/kg, while only minimal nondose-related changes 1n hepatic porphyrin were observed In DBA/2 mice exposed to up to 1200 vg/kg. In the sensitive C57B1 mice there was only a small difference In hepatic porphyrin between the sexes even though males were >3 times as sen sitive to the toxic effects of 2,3,7,8-TCOO than females (see Table V-l). Results similar to those above were reported for urinary porphyrin levels 1n male C57B1 and DBA/2 mice given 6 weekly doses of 2,3,7,8-TCDD at 25 yg/kg (Jones and Sweeney, 1980). In the sensitive strain, the Initial elevation of porphyrin occurred In the second week. ' In rats Increased urinary porphyrin was observed only after subchronic exposure to 2,3,7,8-TCOD (Can ton 1 et a1., 1981). Female CD rats were orally administered a weekly dose of 2,3,7,8-TCOO at levels of 0.01, 0.1 and 1.0 yg/kg for 45 weeks. The Initial increase was observed In the high dose group at 3 months, and in the other two groups at 4 months, after the start of exposure. Not only did the absolute amount of porphyrin Increase, but the relative distribution also changed to compounds containing more carboxyl groups. Only In the high dose group did the livers, at the terminal necropsy, show signs of excess porphyrin under examination by UV light. In attempts to understand the mechanism of 2,3,7,B-TC0D Induced por phyria, the effects of 2,3,7,^-TCDD on the enzymes Involved 1n the synthesis and catabolism of porphyrin have been studied. Goldstein et al. (1978) showed that -aminolevulinic acid synthetase, a rate-limiting enzyme 1n porphyrin synthesis, was slightly Increased (2-fold) 1n male C57B1 mice 00110 V-l 9 09/18/84 given 4 weekly doses of 2,3,7,8-TCDD at 25 pg/kg. This dose of 2,3,7,8TCDO Increased liver porphyrin levels 2000-fold. Catabolism of porphyrin by uroporphyrinogen decarboxylase (UD) also appeared to be decreased 1n 2,3,7,8-TCDD treated mice. Smith et al. (19B1) reported a decrease 1n UD activity from -25-7 nmoles/hr/g Uver In male and female C57B1 mice 3 weeks after a single oral exposure to 2,3,7,8-TCOD at a dose of 75 pg/kg. No effect of 2,3,7,8-TCDD on UD activity was observed In DBA/2 mice which were Insensitive to the Induction of porphyria. A time course of changes In UD activity with length of time after exposure to 2,3,7,8-TCDD Indicated a steady decline In activity starting 3 days after exposure to 2,3,7,8-TCDD, which continued until day 21 when the study was terminated. Sweeney and Jones (1978) reported similar results after 5 weekly doses of 2,3,7,8-TCDD at 25 pg/kg. In this study the UD activity declined -48% In C57B1 mice and only 4% In OBA/2 mice. Other factors besides the Increase In 5-am1nolevullnlc acid synthetase and the decrease 1n UD activity may also partici pate In the dramatic Increase 1n Uver porphyrin 1n mice associated with exposure to near lethal doses of 2,3,7,8-TCDD. As a result of the protracted time observed between exposure to 2,3,7,8TCDD and the development of toxic effects, as well as the reported terato genic and carcinogenic potential of 2,3,7,8-TCDO, investigations have been conducted to determine the Influence of 2,3,7,8-TCDD on DNA synthesis In the liver. Grelg et al. (1974) measured the In. vivo Incorporation of *H-thymldlne (1 hour pulse) Into Uver DNA of male and female Porten strain rats after a single exposure to 2,3,7,8-TCDD at doses of 10 and 200 pg/kg. 00110 V-20 09/18/84 When the 2,3,7,8-TCDD was given either 0, 24 or 72 hours before a 3/4 partial hepatectomy there was only a slight, but not significant, decrease In thymidine Incorporation observed when DNA synthesis was measured 24 hours after the operation. j Although 2,3,7,8-TCDD had no effect on 1n_ vivo DNA synthesis, similar studies by Conway and Hatsumura (1975) and Olckens et al. (1981) demonstrat ed an Increase In thymidine Incorporation when determined In v1tro. Conway and Hatsumura (1975) administered male Sprague-Dawley rats 2,3,7,8-TCDD at a dose of 5 ug/kg followed In 10 days by removal of the liver and the l vltro determination of DNA synthesis In liver slices. Incorporation of thymidine Into the nuclei Increased from 29 cpm/mg 1n control animals to 45 cpm/mg 1n treated animals. A similar near doubling of DNA synthesis was observed by Dickens et al. (1981); however, when DNA synthesis was stimulat ed by a 1/3 partial hepatectomy, thymidine Incorporation Into liver slices was Increased 10-fold 1n rats treated 5 days earlier with 2,3,7,8-TCDD as compared with hepatectomlzed controls. The onset of DNA synthesis after partial hepatectomy (-20 hours) was the same In both 2,3,7,8-TCDD treated and control animals; however, the treated animals had a more rapid and extensive Increase 1n DNA synthesis between 20 and 32 hours after the partial hepatectomy. The rates of DNA synthesis were again the same 1n both groups 35 hours after the operation. It was shown by hydroxyurea Inhibition that the DNA synthesis In both the treated and control animals was predomi nantly sem1conservat1ve. further studies are needed to determine the reason for the difference observed between in vltro and in vivo measurements of DNA synthesis In the liver after exposure to 2,3,7,8-TCDD. 00110 V-21 08/11/84 Extensive hepatic necrosis In the rabbit may be responsible for death 1n this species (Poland and Knutson, 1982). 8es1des the effects on the liver of 2,3,7,8-TCDD exposure described above, 1t 1s known tha't 2,3,7,8-TCOD 1s a potent Inducer of microsomal enzymes. These studies will be discussed 1n the Enzyme Induction by TCDO Section, which describes the ability of this xenoblotlc to Induce microsomal enzymes In a number of tissues and organs. Effects on Other Organ Systems -- The most noticeable feature of 2,3,7,8-TCDD toxicity 1s the loss of body weight and the apparent 'wasting away* until death. Since decreased food consumption may not totally account for these findings, the effect of 2,3,7,0-TCDD on Intestinal absorption has been studied. Madge (1977) assessed the ability of the Intestine to absorb D-glucose, D-galactose, L-arg1n1ne and L-h1st1d1ne using the everted Intes tinal sac technique In CD-I mice exposed to 2,3,7,8-TCDD. In measurements made 7 days after treatment with doses of 0.0, 10, 25, 75, 150, 200 or 300 yg/kg, D-glucose was absorbed to a lesser degree at all doses than 1n control animals. The two low doses produced a dose-related decrease 1n absorption; however, at doses of >75 yg/kg the decrease was uniform. At a dose of 150 yg/kg, decreased absorption of D-g1ucose was slight 3 days after treatment, became maximally decreased by 7 days, and this depressed level was maintained for 28 days, at which time the study was terminated. Providing D-mannose to the Incubation mixture as an energy supply Increased the absorption of D-glucose to control levels; however, the amount of D-glucose on the serosal side was still lower than control levels. This suggested that Intestinal utilization of 0-glucose was taking place and might account for some of the observed malabsorption. Treatment with 00110 V-22 08/11/84 2,3,7,8-TCOO had no effect on the absorption of the other compounds Investi gated. In a similar experiment In Sprague-Oawley rats, Ball and Chhabra {1981) also observed malabsorption of D-glucose. In this study, however, absorption of leucine was also decreased. The decrease 1n leucine absorp tion took longer to manifest Itself, with a significant decrease only observed 2 weeks after treatment with 2,3,7,8-TCOO. In contrast to the results observed for 0-glucose, Intestinal Iron transport was shown to be elevated by exposure to 2,3,7,8-TCDD, Hanls and K1m (1979a) examined the effect of prior treatment of male Sprague-Oawley rats on the 30-minute transport of " Fe out of a duodenal loop created by ligating a section of the Intestine In. situ. At single 2,3,7,8-TCOO doses of between 22 and 84 yg/kg there was Increased serosal transfer of s,fe measured 48 hours after treatment. At doses >42 yg/kg the Increase was -100%. The time after treatment at which serosol transfer was greatest was 1 day, with rapid decline 1n stimulation to near the levels of controls observed on days 2-7. There was also an apparent effect of route of administration, with gavage treatment being more effective 1n Inducing Iron transport than 1.p. Injection. In similar experiments calcium transport was decreased, and galactose and proline transport were unaffected by prior exposure to 2,3,7,8-TCOO. Hanls and K1m (1979b) had Identical results when the everted Intestinal sac was used to assess Iron transport. It was Interesting to note that only duodenal sacs were stimulated, with no effect of 2,3,7,8-TCOO exposure observed 1n the adjacent distal segment of the Intestine. Increased Iron transport was also observed by Hanls and Kim (1979a) In an unidentified strain of mice. Increased Iron transport may be one of the earliest effects of 2,3,7,8-TCOO; however, at present the toxico logic relevance of this transient disturbance 1n Iron transport Is unknown. 00110 V-23 09/ 18/84 One of the common gross observations of 2,3,7,8-TCDD toxicity 1s severe edema, suggestive of a breakdown In salt and water homeostasis. These observations prompted Investigations to determine the effect of 2,3,7,8-TCOO on the function of the kidney. Pegg et al. (1976) measured renal function * in tro using renal cortical slices obtained from male Sprague-Oawley rats 3 and 7 days after Intubation with 2,3,7,8-TCOO at doses of 10 or 25 pg/kg. (These results were also described by Hook et al., 1977). Anion and cation transport were measured by the respective accumulation of p-am1noh1ppur1c acid and N-methyln1cot1nam1de Into the cortical slices. Anion accumulation was lower 1n the high dose group, while cation transport was lower at both dose levels tested. The decrease 1n anion transport was confirmed 1n an 1_n vivo study. Ammonlogenesls and gluconeogenesls were not affected 1n 2,3,7,8-TCOO treated rats, even when the animals were made acldotlc, Indicative of no effect on the kidneys' ability to maintain acid base balance. Also, sodium reabsorption was shown 1_n vivo to be within normal range. Since decreases In cation and anion transport were the only effects observed, and since these compounds are transported by a different mechanism, the authors concluded that the effects of 2,3,7,8-TCOO were merely a general decrease 1n kidney functon reflecting the poor condition of the treated animals (animals 1n all treated groups had decreased weight gain), and not a cause of debilitation. Although kidney function was onlyminimally affected by exposure to 2,3,7,8-TCOO, Grelg et al. (1974) demonstrated that pre-exposure to 2,3,7,8TCOO could reduce the ability of the rat kidney to respond to stimuli of ONA sythesls. Folate-stimulated ONA synthesis measured In vivo 1n Porten strain rats was decreased between 67 and 25% in animals receiving 2,3,7,6-TCDO at a 00110 V-24 08/11/84 dose of 10 yg/kg on day 0-9 before administration of folic add. No significant difference 1n folate-stimulated ONA synthesis was observed 1f 2,3,7,8-TCDD was given 23 hours after folic acid. The lack of effectiveness of administering 2,3,7,8-TCOD shortly after treatment with folic a d d t suggested that 2,3,7,8-TCOO did not directly Interact with cellular ONA, nor Inhibit the protein synthesis necessary to support folate-stimulated DNA synthesis. Similar Inhibitory effects of 2,3,7,8-TCOO were observed when lead acetate was used to stimulate kidney DNA synthesis. The mechanism by which 2,3,7,8-TCOO prevents the kidney from responding to proliferative stimuli Is not known, although It was demonstrated that another agent capable of Inducing microsomal enzymes, 3-methylcholanthrene (3-HC), had similar effects on the kidney. Additionally a number a hematologic and clinical chemistry changes have been observed 1n the blood of laboratory animals after exposure to 2,3,7,8TCOD. Many of these changes, as described by Z1nkl et al. (1973), reflect damage to previously described organ systems. In female CD rats given 30 dally doses of 2,3,7,8-TCOD at levels of 0.1, 1.0 or 10 yg/kg, the clini cal chemistry of the serum reflected liver damage. In the high dose group, serum GOT and serum GPT were elevated starting 13-17 days after Initial treatment. There was a marginal change 1n GPT 1n the m1d-dose group and lactic dehydrogenase (LDH) 1n the high group, but the Increases were only transitory. Serum cholesterol was Increased In the high dose animals start1ng at day 10, with a transitory Increase again observed 1n the m1d-dose group. Conversely, there was a decrease 1n serum protein from day 24 on In the high-dose animals. Along with these clinical chemistry changes Indica tive of Hver damage, the only other major effect observed 1n the blood was 00110 V-25 08/11/84 thrombocytopenia. The decrease 1n platelet count was detected early, by day 3, 1n the10 and 1yg/kg groups, while In the 0.1 vg/kg group a signifi cant decrease was not observed until day 17. Thrombocytopenia was also observed In female guinea pigs after 8 weekly oral doses of 2,3,7,8-TCDD at t 0.2 y g / k g , and 1n mice (administered a single dose of 1.0, 10 or 50 y g / k g ) . In guinea pigs lymphopenia was also observed. Other hematologic changes were attributed to hemoconcentratlon. In a more extensive Investigation of 2,3,7,8-TC00-1nduced hyperlipidemia 1n male Sprague-Oawley rats, Poll et al. (1980) treated animals with a single 1.p. Injection of 2,3,7,8-TCOO at 2 doses of 2.S, 5, 10 and 20 yg/kg. At day 21 after treatment there was a dose-related Increase In total plasma cholesterol and high density lipoprotein cholesterol, while no change wasobserved In triglycerides or very low and low density lipopro teins (VLOL and LOL, respectively). At a dose of 20 yg/kg the maximum Increase 1n HDL cholesterol and total cholesterol occurred 30 days after treatment, and a significant elevation was still present at 60 days after treatment when the study was terminated. Slight changes 1n the apoprotein of HDL from 2,3,7,8-TCDD rats and control rats were Indicative of new apoprotein synthesis. Although the increases 1n HDL cholesterol may be In response to eliminating excess lipids, the exact function has not been clearly shown. There 1s some evidence from studies of workers exposed to 2,3,7,8-TCDD that there were reduced levels of blood HDL cholesterol and raised total cholesterol as compared with a matched control group (Walker and Martin, 1979). 00110 V-26 08/11/84 In contrast to rats, male Hartley strain guinea pigs given a single 1.p. Injection of 2,3,7,8-TCDO at a dose of 2 vg/kg had Increased hyperlipid emia characterized by Increases In VLDl and IDL (Swift et al., 1981). In animals sacrificed 7 days after exposure to 2,3,7,8-TCDD, there was an * ... increase In total serum lipid, cholesterol esters, triglycerides and phospholipids, when comparison was made with pair-fed, weight-paired or ad libitum fed control groups. Serum-free fatty acids were not changed quanti tatively; however, some qualitativechanges occurred, reflecting an Increase 1n the types of fatty adds that were abundant In the adipose tissue of guinea pigs. Anaylsls of lipoproteins revealed a 19-fold Increase In VLDL and a 4-foid Increase In LQL, with no change observed In the levels of HDL. * The VLDL was also qualitatively different 1n the 2,3,7,8-TCQO treated animals, containing less cholesterol ester and an altered C apoprotein. The Importance of these qualitative changes Is unclear. The hyperlipidemia may result from the 2,3,7,8-TCDD mobilization of free fatty acids, which are then used In the synthesis of VLDL and are subsequently formed Into LDL. The relationship of the changes 1n serum lipid levels to the mechanism of 2.3.7.8- TCDD toxicity needs further study. Elovaara et al. (1977) observed some changes In biochemicals of the brain of male Hlstar and heterozygous Gunn rats given a single Intubation of 2.3.7.8- TCDD at a dose of 20 pg/kg. At 7 days post-treatment, there was a small but significant decrease as compared with vehicle treated control animals 1n both the protein anil RNA content of the Hlstar rats, while levels of acid proteinase and DT-dlaphorase (an enzyme Induced by 2,3,7,8-TCDD 1n the liver) had a small but significant Increase In the heterozygous Gunn rats. There were no significant changes observed In homozygous rats given 00110 V--27 08/11/84 2,3,7,8-TCDO at 20 pg/kg. The authors noted that acid proteinase may participate 1n chemically Induced degeneration of the brain. Imnunological Effects-- During acute toxicity studies with 2,3,7,8I TCOD, thymic atrophy was' noted as a consistent effect In all species that have been Investigated. This finding suggested that 2,3,7,8-TCOO may alter the limtune response, and Initiated Immunotoxlclty studies 1n exposed animals. In guinea pigs treated with 8 weekly oral doses of 2,3,7,8-TCOO (0, 0.008, 0.04, 0.2 or 1.0 pg/kgbw), body weight, spleen weight and thymus weight were depressed, adrenal weight was Increased and leukocyte and lymphocyte counts were elevated (Vos et al,, 1973). Upon histological examination, 2,3,7,8-TCDD-exposed rats had a severe depletion of lymphocytes from the thymic cortex (Vos and Moore, 1974). Hematological changes were noted In rats exposed to 10 and 14 dally doses of10pg/kg 2,3,7,8-TCOO (Welssberg and Zlnkl, 1973). Increased red blood cell count, decreased platelet count, Increased neutrophil count and Increased packed cell volumes were reported 1n 2,3,7,8-TCOD-exposed rats. _A summary of the data available on the Immunotoxlc effects of 2,3,7,8-TCDO 1n animals 1s presented In Table V-4. A review of Imnuno toxicity and Immunosuppression was reported by Vos (1977). Vos et al. (1973) Investigated the humoral and cell-mediated Immune response 1n Hartley guinea pigs, CO rats and B6D2F^ mice. The humoral Immune response was tested '1n 2,3,7,8-TCDD-treated hamsters by Injecting tetanus toxoid (subcutaneously) Into the footpad and later testing for the concentration of tetanus antitoxin from the serum by an Immunodiffusion technique. Cell-mediated Immunity was tested by Injecting Mycobacterium 00110 V-28 08/11/84 00110 TABLE V-4 Immunological Effects of 2,3,7,B-TCDD In Animals Species/ Strain Sex Exposure Route Dose(s) Duration of Exposure Hlnlmum Effective Oose Parameter Ef fec t Reference M1ce/B6D2F] H gavage 0. 0.2, 1.0, 5.0. 4 weeks 25.0 |ig/kg bw/week H1ce/C57Bl/6 r.N maternally administered (gavage) Nice/ C57Bl/6Jfh H gavage 0. 1.0, 2.0, 5.0, 25.0 .g/kg 4 or 6 weeks (3 or 5 administrations) 0. 0.5. 1. 5. 10. 20 pg/kg bw/week 4 weeks Htce/Swlss n gavage 0. 1-5, 5. 15. 50 pg/kg bw/week 4 weeks Hlce/B6C3E] F In vitro 0.5. 5.0, 50 ng/ml 5-60 seconds (spleen cells) Hlce/Swlss- F.H maternally Uebster administered (diet) 0. 1. 2.5. 5. 10. 20 ppb (dietary) 10 weeks (pre-gestation and 3 weeks post- parturltlon) Hlce/CO N gavage Nlce/CD N In vitro 0. 0.01, 0.1, 1.0. up to B weeks 10.0 ug/kg bw/week 10`-10~ n single NA 5.0 ug/kg bw/week 5.0 ug/kg bw/week 1.0 ug/kg bw/week 25.0 ug/kg bw/week 2.0 ug/kg bw/week 1.0 ug/kg bw/week 1.5 ug/kg bw/week 50 ug/ml 2.5 ppb 2.5 ppb 5 ppb 1 ppb NA 0.01 ug/kg bw/week 1.0 ug/kg bw/week 10 H bw thymus weight graft-versushost response thymus weight PHA response skin graft rejection Salmonella Infection endotoxin |E. coll) susceptlbl11ty protein, DNA, and RNA synthesis antigenic RBC reaction thymic cortex contact sensitivity to DNEB endotoxin (Salmonella) susceptibility Listeria Infection serum Inmunoglobln level serum Inmunoglobln level lymphocyte blasto gnie transformat Ion no change decreased decreased Vos et al.. 1973 decreased decreased prolonged Vos and Hoore, 1974 Increased mortality and decreased time to death Thlgpen et al.. 1975 Increased Vos et al.. mortality 197Ba decreased Luster et al., 1979a,b decreased atrophy decreased Thomas and Hlnsdl11, 1979 Increased mortality no change Increased decreased Sharma and Gehring, 1979 Increased Sharma and Gehrtng, 1979 V-29 09/18/84 I Species/ Strain Sex Exposure Route Oose(s) TABLE V-4 (coni.) Duration of Exposure Minimum Effective Dose Parameter m Effect Reference 00110 V-30 Mlce/SwlssMebster F oral (diet) Mice/ C57B1/6J M l.p. mcemCSF! M.F maternally administered M1ce/C57Bl/6 M l.p. M1ce/C57Bl/6 M l.p. Rat/CD F oral Rat/CD F oral 0. 10. 100 ppb 5 weeks (or more) 0. 1. 2. 6. 30 pg/kg bw single Injection 0. 1.0, 5.0, 1S.0 pg/kg bw/day 4 days during gestation and lactation 0. 0.4, 4.0, 40 pg/kg bu/ueek 4 weeks 0. 0.004. 0.04, 0.4 pg/kg bw/week 4 weeks 0. 0.2, 1.0, S.O pg/kg bw/week 6 weeks 0, 10 pg/kg bw/day 10. 14 days 10 ppb 10 ppb 10 ppb 10 ppb 10 ppb 1 pg/kg 1.0 pg/kg bw/day 1.0 pg/kg bw/day 5.0 pg/kg bw/day 4.0 pg/kg bw/week 0.4 pg/kg bw/week 0.004 pg/kg bw/week 5.0 pg/kg bw/week 5.0 pg/kg bw/week NA 10 pg/kg bw/day 10 pg/kg bw/day 10 pg/kg bw/day tetanus response antigenic RBC response sensitization to DNFB resistance to Salmonella resistance In Listeria macrophage and natura 1 kl 11er cell activity macrophage and natural killer cell number antibody production L. monocytooenes susceptibility PVBb-tumor suscep tibility bone marrow hypocellular 11y thymus atrophy cytotoxic T-cell response In vitro genera tion of cytotoxic T-cells bw thymus weight tuberculin hyper sensitivity erythrocyte count platelet count neutrophil count decreased decreased decreased Increased mortality Increased mortality -no change decreased Hlnsdl11, et al.. 1900 Mantovani el al.. 19B0 decreased Increased Increased Increased Luster et al. 1980 Increased Clark et a l ., decreased 1981 decreased Clark et a l ., 1981 decreased decreased no change Vos et a l .. 1973 Increased decreased Increased Weissberg and Zlnkl, 1973 09/18/84 TABLE V 4 (cont.) 00110 Species/ Strain Sex Exposure Route Dose(s) Ouratlon of Exposure Minimum Effective Dose Parameter Effect Reference Rat/F-344 F.M maternally administered Rat/F tscher F.M maternally administered (NR) Rat/FIscher- F.M Ulstar maternally administered (NR) Rat/Sprague- H Dawley l.v. Guinea pig/ F gavage Hartley 0. 1.0, S.O ng/kg bw/dose 4 or 6 weeks (3 or 5 administrations) NR 4-6 weeks (during gestatlon and neonatal ly) 0, 5 ng/kg bw/dose 3 or 4 applications during gestation and neonatally 0. 1 ng/kg bw single Injection 0, 0.008, 0.04, 0.?, 1.0 ng/kg bw 8 weeks 1.0 nQ/kg bw/dose S.O S.O nng9//kkgg bw/dose bw/dose S.O ng/kg bw/dose S.O ng/kg bw/dose NA NR NR S ng/kg bw/dose S ng/kg bw/dose S ng/kg bw/dose 1 ng/kg bw 0.04 ng/kg bw/week 0.04 ng/kg bw/week 0.04 ng/kg bw/week 0.2 ng/kg bw/week bw and thymus weight spleen weight PHA response graf t-versus-host response skin graft rejection pseudorabies virus infection Con A and PHA response oxatolone skin hypersensitivity antibody production to bovine ganma globulin PHA and Con A response thymus and bw thymic RNA synthesis thymic RNA polymerase activity bw thymus weight tuberculin hyper- sensitivity tetanus antitoxin decreased decreased decreased decreased prolonged no change Vos and Moore, 1974 decreased decreased Moore and Faith, 1976 no effect Faith and Luster. 1979 decreased decreased until 135 days decreased decreased Kur1 et al., 1982 decreased decreased decreased decreased Vos et al.. 1973 H . male; F = female; l.p. = Intraper1toneal l.v. ~ Intravenous; PIIA * Phytohemagglutinin; Con A = Conconavalln A; RBC - red blood cell; DNfB - 2,4-dlnltro, 1-f luorobemene; NA = Not applicable; NR Not reported V-31 09/18/84 tuberculosis (subcutaneously) Into guinea pigs on day 35 of 2,3,7,8-TCDD treatment (during a schedule of 8 weekly doses). Intradermal tuberculin hypersensitivity was determined by measurements of skin thickening on days 47 and 54. Decreased skin hypersensitivity was noted In hamsters treated i,, with 0.04 g 2,3,7,8-TCDD/kg and higher doses. Decreased tetanus antitoxin levels were evident In guinea pigs treated with 0.2 yg 2,3,7,8-TC0D/kg, but not at lower dose levels. Vos et al. (1973) also tested the cellmediated Immunity In rats exposed to 2,3,7,8-TCOD (0, 0.2, 1.0 or 5.0 yg/kg, once weekly for 6 weeks). H. tuberculosis was Injected Into rats by day 28 of the treatment period, followed by Intradermal hypersensitivity testing on day 42. No changes In the thickness of skin were noted In 2.3.7.8- TCDD-treated rats when compared with controls. Mice were used to test the effect of 2,3,7,8-TCDD on cell-mediated Immunity by use of the *graft-versus-host* experiment (Vos et al., 1973). In this test, spleen cells from 2,3,7,8-TCDD-exposed mice (0, 0.2, 1.0 or 5.0 yg/kg once weekly for 4 weeks) of the C57B1/6 strain were Injected Into the right footpad of a hybrid recipient mouse (C5781/6 x OBA-2). Donor cells possessing sufficient activity will respond to the DBA-2 antigen on the host cells, resulting 1n the enlargement of the popliteal lymph node. Host cells are tolerant of the donor cells since both have C5781/6 antigens. In this test Vos et al. (1973) noted a significant (p<0-01) dose-related decrease In the activity of 2,3,7,8-TCDD-treated spleen cells (as measured by the degree of popliteal lymph node enlargement on the site of the spleen cell Injection). Lymph node enlargement was significantly less (p<0.01) 1n hybrid recipient mice receiving spleen cells from mice treated with 5 yg 2.3.7.8- TCOD/kg/week than from donor cells of untreated mice. 00110 V-32 08/11/84 Studies continued In an attempt to Identify the mechanism of 2,3,7,8TCDD-Induced Immunodeficiency. Rats (f-344) exposed pre- and postnatally by maternal dosing (1 or 5 yg 2,3,7,8-TC00/kg administered to dams on days 11 and 18 of gestation and 0, 7 and 14 postnatally) had prolonged times until t graft rejection, decreased spleen cell graft-versus-host activity and decreased binding response to phytohemagglutinin (PHA) (Vos and Moore, 1974; Moore and Vos, 1974). Response to conconavalln A (Con A), a humoral Immune response, was actually Increased. Since thymus-derived lymphocytes {T-cel Is) play a central role In cell-mediated Immunity and host defense mechanisms, Interest turned to these areas of Immunology. The effect of 2,3,7,8-TCDO on host resistance to Infection, a vital measure of Immune response, was tested by Thigpen et al. (1975) In male pathogen-free mice (C57B1/6Jfh). 2,3,7,8-TCDO was adminis tered to mice at 0.5, 1, 5, 10 or 20 yg/kg once weekly for 4 weeks followed by Inoculation with Salmonella bern 2 days after the final 2,3,7,8TCDO administration. Mortality rates and "time until Infection* were used to determine the Immunological effect of 2,3,7,8-TCDO. A significant (p<0.05) Increase 1n mortality and decrease 1n time of Infection were noted 1n groups treated with 1 yg/kg or higher doses of 2,3,7,8-TCOO when compared with controls. 2,3,7,8-TCOO at 0.5 yg/kg did not alter these parameters and was regarded as a no effect level. The Immune-resistance of mice to S. bern Is therefore reduced by treatment with 1 yg 2,3,7,8TCD0/kg/week (for 4 weeks). * 00110 V-33 08/11/84 Pretreatment with 2,3,7,8-TCDD greatly enhances the susceptibility of mice to E. coll endotoxin {Vos et al., 1978a). Injection of 250 yg of endotoxin to mice pretreated with 0, 1.5, 5 and 15 yg 2.3,7,8-TCDD/kg resulted In 0/5, 1/5, 6/6 and 6/6 deaths, respectively. Mice pretreated with 15 and 50 yg 2,3,7,8-TCDD/kg and Injected with 10 yg of endotoxin had 1/4 and 2/4 deaths, respectively. Mice treated with lower doses of 2.3.7.8- TCDD were not susceptible to this quantity of endotoxin. Increased mortality (2/6) 1n a control group was noted only when 500 yg of endotoxin was administered, while 10 yg of endotoxin was sufficient to cause similar mortality (2/5) In mice treated with 50 y g 2.3,7,8-TCDO/kg. The Immunocomptence of 5-week-old offspring of Swiss-Webster mice fed diets containing 1, 2.5, 5, 10 or 20 ppb 2,3,7,8-TCDD was tested by several means (Thomas and H1nsd1ll, 1979). The number of cells reactive to anti genic RBC, differential white blood cell counts, organ weights, hlstopathologles, hypersensitivity to 2,4-d1n1tro-l-fluorobenzene (ONFB) and the resistance to E. col 1 lipopolysaccharide (LPS), Listeria monocytogenes and Salmonella typhlmur1um LPS were all measured for mice exposed to different levels of 2,3,7,8-TCDD. Adult female mice were exposed to 2,3,7,8-TCDD for 4 weeks before mating, throughout gestation and for 3 weeks postparturition. Young mice being tested for Immunotoxldty were therefore exposed to 2.3.7.8- TCDD only in utero and through lactation. The typical decrease In thymus weight was noted 1n mice exposed to 2.5 and 5.0 ppb but was not evident In the 1.0 ppb group. - A decrease In the number of plaque-forming cells (PFC) reactive to sheep RBCs was significantly reduced In the 2.5 and 5.0 ppb 2,3,7,8-TCDD-exposed groups. (Because of the poor survival of young 1n the 10 and 20 ppb 2,3,7,8-TCDD-exposed groups, results and comparisons 00110 V-34 08/11/84 were usually reported for the three lower dose groups). The humoral content of antl-RCD antibodies,however, was not lower 1n 2,3,7,8-TCDD-exposed groups when compared with controls. A decrease 1n the skin hypersensitivity to DNFB following sensitization was noted In all 2,3,7,8-TCOD-treated groups *^ (only the 5 ppb group wais statistically reduced from controls). 2,3,7,8TCDD caused an Increased susceptibility (Increased mortality level) to S, typhlmurlum 1n a dose-related fashion. The response to F. coll LPS and L. monocytogenes was not different from controls. 2,3,7,8-TCOO exposure did not alter the response of lymphocytes (Band T-cells) 1_n vltro to Con A, nor was mltogen-1nduced lymphocyte proliferation affected (Thomas and Hlnsdlll, 1979). Similar findings were reported In Fischer/Wlstar rats exposed to 2.3.7.8- TCDO during gestation (18th day) and neonatally, or neonatally alone (on days 0, 7 and 14) (Faith and Luster, 1979), Dams were treated with 5 g/kg 2,3,7,8-TCDD on each dose day. Typically, body weight and thymic weights were decreased 1n progeny, which lasted until 135 days of age. The thymic- and splenic-cell response to PHA and Con A was decreased 1n all 2.3.7.8- TCOD-treated animals and did not return to normal until day 270. Delayed hypersensitive reaction was also suppressed until 270 days of age. The production of antibodies to bovine gamma globulin, which requires T-helper cell function, was not affected by 2,3,7,8-TCDD exposure during rat development (Faith and Luster, 1979). 00110 V-35 08/11/84 Neonatal B6C3F^ race, exposed to prenatal (maternal dosing on day 14 of gestation) and postnatal (days 1, 7 and H after birth) doses of 0, 1.0, 5.0 or 15.0 pg/kg 2,3,7,8-TC0D, were studied for Iramunotoxlc effects and i* host susceptibility (Luster et al., 1980). At the 15.0 pg 2,3,7,8-TCOD/kg iose level, 70% of the ^neonates died with overt toxic effects (decreased body weight, liver weight, spleen weight and thymus weight). Bone marrow hypocellularlty and depressed macrophages-granulocyte progenitor cells and pleurlpotent stem cells were associated with 2,3,7,8-TCDD exposure at the 5.0 and 15.0 pg/kg dose levels. Hematological changes, such as decreased RBC count, hematocrit and hemoglobin, and lymphocyte count showed a doserelated response. Host susceptibility to L. monocytogenes and PYB6-tumor cells was tested 1n the 2,3,7,B~TC0D-exposed neonates. Death occurred 1n 73 and 40% of the L. monocytogenes Inoculated (1.2x10* viable organisms) mice 1n the 5.0 and 1.0 pg/kg dose groups, respectively, compared with 28% of controls. Tumor development occurred 1n 44, 60 and 22% of the neonates Inoculated with 5x10* tumor cells from the 5.0 pg 2,3,7,8-TCDO/kg, 1.0 vg 2,3,7,8-TCDD/kg and control groups, respectively. H1nsd1ll et al. (1980) reported that 2,3,7,8-TCDD administered In the diet Df Swlss-Webster mice at 100 ppb for 5 weeks caused a marked suppres sion of total serum protein, gamma globulin and albumin, but an Increase 1n 0-globulins. At 10 ppb In the diet, 2,3,7,8-TCDD caused decreased Immune response to tetanus toxoid, sheep RBC, S. typhlmurlum and L. monocytogenes, and lowered contact sensitivity to DNFB. This study also suggested that although young animals are more susceptible to 2,3,7,8-TCDD, older animals are still Immunosuppressed and exposure in utero and neonatally Is not more crucial than 1n other periods. Vos and Moore (1974) had previously reported 00110 V-36 09/18/84 that "[-month-old mice were more sensitive to 2,3,7,8-TCOO than were 4-monthold mice (C57B1/6). Decreased body weight and thymus weight and spleen cell response to PHA were evident at lower doses 1n 1-month-old mice than In 4-month-old mice, The effect of single 1.p. doses of 2,3,7,8-TCDD (1, 2, 6 and 30 ng/kg) on peritoneal macrophage and splenic natural killer cell function In mice (C57B1/6J) was studied by Mantovanl et al. (1980) and Vecchl et al. (1980). 2,3,7,8-TCDD treatment at all dose levels did not decrease the cytostatic and cytocldal activity of macrophages or natural killer cells on a per cell basis. The total number of macrophages and splenic natural killer cells recovered from 2,3,7,8-TCOD-treated animals, however, was reduced when compared with untreated controls. Marked hypocellularity noted 1n the bone marrow of 2,3,7,8-TCOD-treated mice may account for the decrease In periph eral cell counts (McConnell et al., 1978b). The lack of macrophages and natural killer cells was suggested as being Instrumental In the decreased resistance to Infection common to 2,3,7,8-TCDO-exposed animals (Mantovanl et al., 1980). Although 2,3,7,8-TCDD was a strong Immunosuppressant, animals given a lethal dose of 2,3,7,8-TCDD did not appear to die from Infections, nor did a germ-free environment protect them from death (Gre1g et al., 1973], The actual mechanism of 2,3,7,8-TCDD 1mriunotox1c1ty 1s unknown but several Investigators have tested various hypotheses. Vos et al. (1973, 1978a,b) attempted to address the Indirect causes for decreased thymic growth and altered T-lymphocyte activity following 2,3,7,8-TCDD treatment. 00110 V-37 08/11/84 Vos et al. (1973) measured serum cortisol and cortlcosteron levels 1n guinea pigs exposed to 2,3,7,8-TCDD to evaluate the possible Indirect Immunosup pression by these hormones. There was, however, no significant difference In the level of these hormones between treated and control animals. * Fndlrect Immunosuppression of this type was unlikely. Later studies {Vos et al., 1978a,b} Investigated the role of thymic hormones (thymosin) on the atrophy of the thymus during 2,3,7,8-TCDD treatment. Thymosin administered In conjunction with 2,3,7,8-TCDD did not protect mice from the typical 2,3,7,8-TCDD-1nduced Imnunotoxlc alterations. Thymus weight was maintained but not Increased by thymosin, and thymus-derived cells continued to show decreased responsiveness to mitogens (PHA, Con A). Thus, It Is unlikely that 2,3,7,8-TCOO affects the supply or synthesis of thymic hormones which could lead to the observed limunosuppresslon. van Logten et al. (1980) Investigated the possible Influence of the adrenal gland, hypophysis and pituitary, and growth hormone on thymic atrophy and Immunosuppression following 2,3,7,8-TCOD exposure 1n female F-344 rats. Adrenalectomy and exogenous growth hormone had no preventative action on thymic involution. Hypophysectomlzed rats showed advanced thymic atrophy. Sharma and Gehrlng (1979) noted that 2,3,7,8-TCDD caused stimulation of lymphocyte transformation to blast form cells (mltotlcally active precurs ors) when no mitogens were present 1n the culture system. This represents a phenomenon similar to actual antigenic challenge. At low doses (0.01 and 0.1 pg 2,3,7,8-TCDD/kg/week for up to 8 weeks), serum Immunoglobulin levels were elevated 1n male CD-I mice. Larger doses of 2,3,7,8-TCDD (1.0 00110 V-38 08/11/84 and 10 yg/kg/week) resulted 1n a decrease In the serumImmunoglobulin level. It was suggested that 2,3,7,8-TCDD may elicit an antigenic response either by combining with a body protein or by causing cellular or biochemi cal damage that releases antigenic proteins. Sharma and Gehrlng (1979) also * ,, noted that thymic atrophy was observed after 2 and 4 weeks of treatment but not after 8 weeks. There may be a recovery of thymic tissue, either by Immune tolerance or Immune unresponsiveness as a sort of adaptation to 2.3.7.8- TCOO-exposure and Its possible antigenic complex. Luster et al. (1979a,b) reported that 2,3,7,8-TCDD affects the Immune system directly by altering lymphocyte function. The function of T-helper cells was not altered, since no change In response to bovine gamma globulin (requires T-helper cell cooperation) was noted In Wlstar/Flscher and Fischer rats exposed to 2,3,7,8-TCDD. In. vltro, 2,3,7,8-TCDD (100 ng/mi.) sup pressed DNA, RNA and protein synthesis 1n splenic lymphoid cells from 86C3F.J (Luster et al., 1979a). 2,3,7,8-TCDD, however, did not decrease the binding of 3H-Con A to lymphocytes, Indicating that these receptors are not blocked by 2,3,7,8-TCDO. T-lymphocytes were more susceptible to 2.3.7.8-TCOO, measured by specific mitogen binding assays, than B-lymphocytes. These authors (Luster et al., 1979a) suggested that 2,3,7,8-TCDQ may bind directly to the lymphocyte cell membrane and alter Its function. Faith and Luster (1979) reported that lymphocytes from the spleen, thymus, bone marrow and lymph nodes of Fischer rats exposed to 2,3,7,8-TCOO showed abnormal homing patterns within' the body. 2,3,7,8-TCDO exposure apparently altered the cell surface markers so that spleen lymphocytes were taken up by the thymus of recipient rats. These authors (Faith and Luster, 1979) suggested that 2,3,7,8-TCDD may change cellular metabolism, which alters the 00110 V-39 08/11/84 cell membrane constituents or may Insert directly Into the membrane. Kurl et al. (1982) reported that 2,3,7,8-TCDD causes changes 1n thymic transcrip tion and RNA synthesis that may lead to cell surface changes. Cell surface changes could presumably result In altered antigen recognition and cell-toi' cell recognition, causing Immunosuppression and thymic atrophy. Clark et al. (1981) reported that 2,3,7,8-TCDD treatment (0.4, 4.0, 40 yg/kg weekly for 4 weeks by 1.p. Injection) caused functional Impairment of cytotoxic T-cells In C5781/6 male mice. The authors felt that this response was particularly sensitive to 2,3,7,8-TCDD treatment and hypothe sized that 2,3,7,8-TCDD directly Inhibits the function of these cells. Contrary to the hypothesis tested by these authors and that held by Luster et al. (1979a,b), 2,3,7,8-TCOD treatment Impaired the generation of cyto toxic T-cells by the spleen (at doses as low as 0.004 yg/kg when detected 1n yVtro) but did not appear directly toxic to the cytotoxic T-cells. At present, the mechanism of Immunosuppression caused by 2,3,7,8-TCDD is unknown and the theories available are speculative. In a later study, however, Clark et al. (1983) reported that a 10- to 100-fold greater dose of 2.3.7.8- TCOD was required to suppress cytotoxic T-cells in 08A/2 mice as compared with C56B1/6 mice. This Indicates that susceptibility to 2,3,7,8TCDD 1mmunotox1c1 ty segregates with the Ah locus which Is consistent with a receptor mediated mechanism. , The receptor mediated mechanism was further supported by the susceptibility of the C5781/6 x DBA/2J hybrid mouse to 2.3.7.8- TCDO suppression of the cytotoxic T-cells which Is again consistent with the dominant Inheritance of Ah (Nagarkattl et al., 1984). 00110 V-40 08/11/84 Few reports re available In which the Immunological effects of 2,3,7,8TCOO exposure were studied In humans. Regglanl (1980) reported that the 1mmunocapab1l1 ty of 17 people, ranging 1n age from 3-60 years, who had been exposed to 2,3,7,8-TCOO, was normal 1n all cases. In a survey of 41 workers ^exposed to 2,3,7,8-TCDD, Ward (1982) measured Immunoglobin G, A, H, D and E, as well as lymphocytes, T-cells, B-cells, PHA response and blood cell counts. These determinations were made 10 years after workers had developed 2,3,7,8-TCD0-1nduced chloracne. In this group of workers, there was a significant Increase In the proportion of cases with reduced IgO and IgM. It was suggested that the 2,3,7,8-TCOO-exposed group had a reduced Immune capability and a deficiency In T-cell and 8-cell cooperation. The Immunotoxldty of 2,3,7,8-TCOO In humans cannot be properly assessed because of the paucity of data recorded soon after exposure. The most prominent effects In animals (1.e., humoral responses) were not measured In humans. Enzyme Induction by TCOD -- In Cell Cultures. Although 2,3,7,8-TCOO has a very low toxicity to cells 'in culture (Beatty et al., 1975; Bradlaw et al., 1976; Knutson and Poland, 1980; Yang et al., 1983), It Is an extremely potent enzyme Inducer In these systems (Kourl et al., 1974; N1wa et al., 1975; Bradlaw et al., 1976; Malik and Owens, 1977; Malik et al., 1979; Bradlaw et al., 1980). This enzyme Induction Is so sensitive that 1t has been proposed as a bio assay for detecting planar polychlorinated organic compounds (Bradlaw et al., 1975, Bradlaw and Casterllne, 1979; Nlwa et al., 1975). 00110 V-41 09/18/84 Kour1 et al. (1974) found that 2,3,7,8-TCDD Induced aromatic hydrocarbon hydroxylase (AHH) activity In cultured human lymphocytes to the same extent as 3-HC; however, the concentration of 2,3,7,8-TCDD necessary for maximal enzyme Induction was 40-60 times less than that of 3-MC. N1wa et al. (1975) Compared AHH Induction by 2,3,7,8-TCDD among cell cultures (H-4-II-E, VERQ, HTC, LB82, MA, Hepa-1, TRL2, ERl-2, NRKE and Chang). ED5Q values ranged from 0.12 nM 1n the Hepa-1 cell line to >100 nM 1n the VER0 and HTC cell lines. 2,3,7,8-TCDO did not Induce AHH activity 1n LBB2 cells. The respon siveness of AHH Induction to 2,3,7,8-TCDO was 250-900 times greater than to 3-MC. In addition, cell cultures derived from C57B1/6N mice were 16 times as sensitive to 2,3,7,8-TCDD as cell cultures derived from DBA/2N mice. The responsiveness of cell cultures td enzyme Induction by 2,3,7,8-TCDD 1s thus similar to the effects seen in vivo. The Inductive effect of 2,3,7,8-TCDD was blocked by actlnomycln D and cycloheximide, Implying that Induction Involved the sythesls of new mRNA and protein. Enzyme Induction by 2,3,7,8-TCDD, therefore. Involves an Initial RNA synthesis and continuous protein synthesis (Malik and Owens, 1977; Malik et al., 1979). In all of these studies, there was no correlation between cytotoxicity and enzyme Induction. This Implies that, despite the correlation in vl vo. there may be no direct connection betweeen enzyme Induction and the toxicity of 2,3,7,8-TCDO. In Mice and Rats. The effects of 2,3,7,8-TCDO on enzyme activity In rats and mice have been Investigated extensively. 2,3,7,8-TCDD has been found to alter many enzyme activities 1n a wide variety of organ systems (vide Infra). This alteration primarily results In Increased enzyme activity, although 2,3,7,8-TCDD has been observed to Inhibit some enzymes. 00110 V-42 08/11/84 Hook et al. (1975a) reported that 2,3,7,8-TCDD supressed hepatic micro somal N-demethylatlon 1n male but not female, rats; however, cytochrome P-450 and benzpyrene hydroxylase activity were Increased. The suppression of N-demethylase activity was undetectable for 73 days following a single i oral dose of 25 pg 2,3,7,8-TCOO/kg bw. The suppression of N-demethy lase activity was seen only In adult animals. In 10-day-old rats, 2,3,7,8-TCDO had an Inductive effect on this activity. The inductive effects of 2,3,7,B-TCDD have been demonstrated to be organ specific. A1t1o and Parkkl (1978) Investigated the effects of 2,3,7,8-TCDD on the activities of AHH, ethoxycoumarIn deethylase, cytochrome C reductase, epoxide hydratase, UDP glucuronosyltransferase, and glutathione S-transferase In the liver, kidney, lung, small Intestine and testes of male Wlstar rats. Monooxygenase activity was stimulated 1n the liver, lung and kidney, but not In any other tissue Investigated. UOP glucuronosyltransferase activity Increased by a factor of 7 In the liver, by a factor of <2 1n the kidney, and not at all In any other tissue. Epoxide hydratase and glutathione S-transferase activities were not affected 1n any of the tissues studied, although stimulation of hepatic glutathione S-transferase has been reported by other Investigators (Man 1s and Apap, 1979). Enzyme Induction has also been reported 1n rat mammary gland (Rlkans et al., 1979), mouse testes (Mattlson and Thorgelrsson, 1978), and rat prostate gland (Lee and Suzuki, 1980), but the rat adrenal gland 1s apparently Insensitive to Inductive effects of 2,3,7,8-TCOD (Guenthner et al., 1979). 00110 V-43 09/18/84 In the Uver of rats and mice, 2,3,7,8-TCOQ affects a wide range of enzymatic activities, Including OT-dlaphorase (Beatty and Neal, 1976a,b), bilirubin catabolism (Kap1tuln1k and Ostrow^ 1978), ornithine decarboxylase (Potter et al.f 1982), 7-ethoxycoumar1n O-demethylase (Greenlee and Poland, 1978), glutathione S-trarisferase (Baars et al., 1978; Hanls and Apap, 1979 ), aldehyde dehydrogenase (Lindahl et al., 1978; Deltrlch et al., 1977), uroporphyrinogen decarboxylase (Jones and Sweeney, 1977), -aminolevulinic acid synthetase (Goldstein et al., 1982a; Woods, 1973), UDP-glucuronosyl transferase (Harselos et al., 1978) and a number of microsomal oxidative enzyme systems (vide Infra). 2,3,7,8-TCDD 1s four orders of magnitude more potent than 3-MC as an Inducer of hepatic AHH activity; however, the dose-response curve for the two compounds are parallel and both produce the same maximal response (Poland and Glover, 1974). Simultaneous administrations of maximally Inducing doses of both compounds produced no greater response than either alone and both produced a cytochrome with a shift 1n the absorption maximum of the carbon monoxide difference spectrum from 450 to 448 nm. In a number of studies, Increased AHH activity and cytochrome P-448 synthesis have been separated (Chhabra et al., 1976); however, other researchers report an apparent connection between cytochrome P-448 and AHH Induction (Kltchln and Woods, 1977, 1978a,b). Thus, 2,3,7,8-TCDD not only stimulates AHH activity by Inducing cytochrome P-450 formation, but may enhance AHH activity by other mechanisms as well. 00110 V-44 08/11/84 In Rabbit. The response of the rabbit 1s quite different from that observed In rats and mice (Hook et al., 1975a). The only changes In hepatic enzyme activities observed were suppression of benzpyrene hydroxylase and benzphetamlne N-demethylase. In the same study, biphenyl 4-hydroxylase was ^Induced 1n the lung and benzpyrene hydroxylase was Induced In the kidney. In a similar study, a hepatotoxlc dose of 2,3,7,8-TCDD (30 yg/kg) failed to alter prostaglandin synthetase activity In hepatic or renal tissue (Kohll and Goldstein, 1981). In a series of studies, Johnson and Muller-Eberhard (1977a,b,c,d), Johnson et al. (1979), Norman et al. (1978), Llem et al. (1980) and Dees et al. (1982) Isolated a series of cytochromes P-450 from rabbit liver mlcrosomes. These cytochromes were Irmiunologlcal ly distinct, functioned 1n dif ferent catalytic pathways, and responded differently to Induction by poly cyclic aromatic hydrocarbons. 2,3,7,8-TCDD was found to Induce two cyto chromes, designated as form 4 and form 6. Form 4 1s the major cytochrome Induced 1n adult rabbit liver by 2,3,7,8-TCDD; however, form 6 Is the major cytochrome Induced 1n newborn rabbit liver (Norman et al., 1978b), adult rabbit lung, and adult rabbit kidney (Llem et al., 1980; Dees et al., 1982). Other Species. The guinea pig, the species most sensitive to the toxic effects of 2,3,7,8-TCDD, Is similar to the rabbit In Its response to 2,3,7,8-TCDD. Biphenyl 4-hydroxylase was Induced 1n the Uver, lung and kidney, biphenyl 2-hydroxylase yas suppressed 1n the liver, and benzpyrene hydroxylase was Induced In the kidney (Hook et al., 1975b). Testicular microsomal cytochrome P-450 content was depressed following a single oral dose of 1 yg/kg, reaching 52% of controls by 1 day and remaining at this 00110 V-45 09/18/84 level for 9 days (Tofllon et al., 1980). Testicular microsomal h e m e lev el s and -aminolevulinic acid synthetase activity were unaffected by this treatment. Tn contrast to the rat, 2,3,7,8-TCDO did not Induce DT-dlaphorase 1n brain, spleen, kidney, lung, heart or liver of male guinea pigs ^Beatty and Neal, 1978). < Aryl hydrocarbon hydroxylase and -aminolevulinic acid synthetase 1n the chick embryo have been reported to be extremely sensitive to the Inductive effects of 2,3,7,8-TCDO {Poland and Glover, 1973a,b), with maximal induction occurring with 155 pmoles/egg. This Induction Is relatively long lasting, with 70% of the maximum Induced activity present 5 days following a single dose of 2,3,7,8-TCDD. Structure-activity studies demonstrated a perfect correspondence between the toxicity and Induction potency of a series of d1benzo-^-d1ox1n congeners (Poland and Glover, 1973a), Subchronic Toxicity. Four laboratory studies described the systemic toxic effects of subchronic exposure to 2,3,7,8-TCDO In rodents. Also, one semi-controlled study evaluated the toxic effects to rabbits after confine ment to an area containing soil contaminated with 2,3,7,8-TCDO. No informa tion was found 1n the literature searched on the effects of subchronic exposure to 1,2,3,7,8-PeCDD, and only one preliminary study was available describing the effects of subchronic exposure to a mixture of two HxCODs 1n rats and mice, Koclba et al. (1976) exposed Sprague-Dawley rats to 2,3,7,8-TCDD for 13 weeks. The animals 1n groups of 12 males and 12 females received the com pound suspended 1n acetone-corn oil (1:9) by gavage 5 days/week at doses of 00110 V-46 09/18/84 0.0, 0.001, 0.01, 0.1 or 1.0 yg/kg bw. At the end of the treatment period 5 rats of each sex were killed for histopathologic examination, and the remaining animals were continued for postexposure observation. This report on gross, hematologic, clinical chemistry and histopathologic (on animals i terminated at the Interim kill or killed when moribund) observations was prepared on data available 13 weeks after termination of treatment. Signs of toxicity were observed only at the two higher dose levels, and female rats appeared more sensitive to the toxic effects of 2,3,7,8-TCOO. During the study there were five treatment-related deaths In the high dose group females, with three occurring during treatment and two In the post-treatment period. In male animals only two deaths occurred In the post-treatment period in the high dose group. 8oth the male and female rats of the 0.1 and 1.0 yg/kg groupshad depressed body weight; however, greater relative depression of body weight was observed 1n the high dose females. Other changes such as Increases in bilirubin concentrations, urinary coproporphyrln excretion, and changes 1n relative thymus or Uver-to-body weight ratio occurred 1n the two high-dose female groups, but only In the 1.0 yg/kg male group. Although male rats had significantly decreased hematologic values (packed cell volume, RBC count and hemoglobin) In the two high-dose groups, and these values were normal In all female rats, the authors pointed out that these results may have been an artifact resulting from dehydrationinduced hemoconcentratlon 1n the female rats. No specific data were provided, however, to support this last conclusion. r After necropsy, gross examination revealed subcutaneous edema, a decrease In the size of testes and uteri, and a decrease In the number of corpora lutea. Histologic examination revealed Involution of the thymus, 00110 V-47 08/11/84 decreased number of thymocytes, and focal necroses and pigment accumulation in the liver. These observations were made only In the animals of the high-dose group, with the exception of a slight decrease In the number of thymocytes and mild microscopic distortion of the architecture of the liver1 t, 1n the group fed 0.1 yg/kg. Although histologic evidence from animals killed during the Interim sacrifice was consistent with the Uver and thymus being the primary target organs, in an animal that died during the study there were signs of aortic thrombosis and adrenal hemorrhage, and In a second animal there was severe anemia, suggesting possible Involvement of the hematopoietic system near the time of death. Uver toxicity was the only effect of treatment observed during histo logic examination of rats (Osborne-Hendel) and mice (86C3F^) administered 2,3,7,8-TCDD for 13 weeks In a preliminary subchronic toxicity study designed to define an acceptable dose for a chronic toxicity study (NTP 1980a). The animals 1n groups of 10 males and 10 females were administered the compound 1n corn oil-acetone (9:1) twice a week at doses for rats of 0.0, 0.5, 1, 2, 4 and 8 yg/kg/week, and for mice at doses of 0.0, 1, 2, 5, 10 and 20 yg/kg/week. Deaths occurred at the two high-dose levels In rats, with 4 females In the 8 yg/kg/week and 1 In the 4 yg/kg/week group dying, while only 2 male rats 1n the 4 yg/kg/week group died. Deaths were accompanied by severe toxic hepatitis. Hepatic lesions were observed In all other rats examined 1n groups administered 1-8 yg/kg/week; however, not all animals 1n each group were submitted to necropsy. Normal Uver histology was observed 1n the 2 male rats examined from the low-dose groups and only threshold toxic effects occurred in the low-dose female rats. 00110 V-48 08/11/84 Similar effects of treatment were observed In mice, with a single death occurring In each sex at the high-exposure level, along with reports of hepatic lesions on histologlc examination. In contrast to rats, female mice were less sensitive to the hepatotoxlc effect of 2,3,7,8-TCDD than were the I,, male mice. Hepatic lesions were observed In all dose groups of male mice, while the 1 and 2 pg/kg/week dose groups of female mice had normal livers. Although the group sizes were small, making conclusions tenuous, 1t appeared that sex differences 1n the sensitivity to the toxic effects of 2,3,7,8-TCDD occurred, and that the more sensitive sex may vary with species tested. In a more extensive subchronic study In rats. King and Roesler (1974) followed the development of toxicity by a series of Interim sacrifices during 28 weeks of exposure to 2,3,7,8-TCDD and a 12-week post-treatment recovery period. Groups of 35 male and 35 female Sprague-Dawley rats were Intubated twice weekly with 2,3,7,8-TCDD In corn o1l-acetone (9:1) at cumu lative doses of 0.0, 0.1 and 1 pg/kg/week. No treatment-related deaths occurred; however, 3 animals from each group of each sex were killed after 2, 4, 8 and 16 weeks, and 10 animals of each sex were killed after 28 weeks of treatment. In addition, 3 rats of each sex were killed 4 and 12 weeks after termination of exposure. Animals were mon1toredj for gross changes during the study and were examined for gross and histologic changes at necropsy. Besides a dose-related decrease 1n body weight gain In male rats and a decrease In body weight gain In the high-dose female rats, the only effect of exposure to 2,3,7,8-TCDD was histologic changes In the liver. Liver pathology was normal In all treated groups up through the Interim kill at 16 00110 V-49 08/11/84 weeks. Fatty changes In the liver were considered the most Important obser vation. The fatty changes ranged from single large I1p1d droplets 1n a few centrllobular hepatocytes to I1p1d droplets 1n all centrllobular hepatocytes with extension Into the mldzonal hepatocytes. No clear dose-response pattern was observed In this study; however, 1t did appear that the severity of fatty changes was greater In male rats. During the recovery period, fatty changes progressively decreased 1n severity, but were still present 1n some treated animals 12 weeks after cessation of exposure. Other histologic changes observed 1n the Uver predominantly In the animals killed at 28 weeks Included necrosis. Increased nuclear size, subtle distortion of Uver architecture, and hyperchromatlc nuclei. All of these lesions were consid ered to be slight or mild, and less toxicologically relevant than the fatty changes. The data suggested that the Uver was the most sensitive organ to the toxic effect of 2,3,7,8-TCDD, and although recovery occurred after termination of treatment, the recovery process was slow. The recovery time was also demonstrated to be long in a subchronic study by Goldstein et al. (1982b) of 2,3,7,8-TCDD Induced porphyria. Groups of 8 female Sprague-Dawley rats were given 2,3,7,8-TCDD 1n corn o1l-acetone (7:1) weekly by gavage for 16 weeks at doses of 0.0, 0.01, 0.1 or 10.0 yg/kg/ week and killed 1 week after the last treatment. Additional groups of rats received doses of 0.0 or 1.0 yg/kg/week for 16 weeks and were allowed to recover for 6 months. The high-dose level was lethal to all animals within 12 weeks, while the only other gross sign of toxicity was a decrease 1n body weight gain In the group receiving 1.0 yg/kg/week. After 16 weeks of exposure to 2,3,7,8-TCDD, liver porphyrins were elevated -1000-fold In 7 of 8 animals receiving 1.0 yg/kg/week, but only 1 of 0 animals In the 0.1 00110 V-50 08/11/84 yg/k g / we e k group had e l ev a t ed p orphyrin levels. No effect was observed in the low-dose animals. After a 6-month recovery period the porphyrin level in animals exposed to 1 y g / k g / we e k was still 100-fold higher than values in the control group. A similar pattern was obse r v ed for urinary e xcretion of uroporphyrin. The rate limiting enzy m e in heme synthesis, - a m i n o l e v u linic acid synthetase, was also elevated at both the, time of termination of treatment and at the end of the recovery period; however, other enzymes that were increased after 10 weeks of treatment, cytochrome P-450, AHH and glucuronyl transferase, returned to near normal levels by 6 months. It was clear that a 6-month recovery period from subchronic exposure to 2,3,7,8-TCDD at a dose of 1.0 yg/kg/week was not sufficient for complete reversal of 2,3,7,8-TCDD-lnduced porphyria. D e C a p r i o et al. (1986) fed 2 , 3 , 7 , 8 - T C D D in the diet for 90 days to male and female Hartley guinea pigs and found NOELs of 0.61 and 0.68 ng/kg/day, respectively. Decreased body weight gain, increased relative liver weight, decreased relative thymus weight and hepatocellular cytoplasmic inclusion bodies at 4.90 (males) and 4.86 (females) ng/kg/day and mortality and other toxic effects at 26 (males) and 31 (females) n g / k g / d a y w e r e also observed. In addition to the above laboratory studies, Strik and de Wit (1980) attempted to investigate the toxicologic effect on rabbits of exposure to a natural environment that was contaminated with 2,3,7,8-TCDD. Groups of 20 female rabbits and 1 m a l e rabbit .were housed for 5 mont h s in pens, located in five separate areas, on soil that had been contaminated with 2,3,7,8TCDD. The soil had been cleaned by replacement or cultivation before initi ation of the study. The levels of 2,3,7,8-TCDD before cleaning were from 00110 V -51 07/23/87 0.8-23.2 y g / m 3 ; however, the levels of c o n t a m in a t io n after clea n i ng were not determined. At 1 the end of 5 months liver histology, including the localization of porphyrin, was examined, and the levels of cytochrome P-450 and P-420 were determined along with urinary levels of total porphyrin, creatinine and D-glucaric-acid. All of the parameters examined were consid ered to be within the normal range. Since exposure data were not available, the negative results of this study cannot be compared with the controlled subchronic laboratory studies already described. I n f o r m at i o n on the s u bchronic t o xicity of HxCDD was p r ov i d e d in a p r e liminary range-finding study for a chronic bioassay conducted by NTP (198 0 d ) on a 1-2 mixture of 1,2,3,6,7,8- and 1 ,2,3,7,8,9-HxCDD. Osborne-Mendel rats and B6C3F.J m i c e in grou p s of 10 males and 10 females w e re a d m i n i s t e r e d the HxCDO m i x t u r e in corn o i l - a c e t o n e (9:1) by gavage twice a week for 13 weeks. The total w e e k l y doses g i ven rats w e r e 0.0, 2.5, 5, 10, 50 and 100 yg/kg, w h il e m i ce r e ceived 0.0, 1.25, 2.5, 5, 10 and 50 yg/kg. At week 10 of the study, the body w e ig h t In rats was d e c r e a s e d In a d o s e - r e l a t e d manner to a m a x i m u m of - 2 0 % In the h i g h - d o s e group. In mice, body weig h t was also d e c r e a s e d 1 0 - 2 0 % In the treated animals; however, there a p p e a r e d to be no correlation with dose. At the end of the study the animals were killed and necropsies were performed on selected animals. In both species liver pathology was observed, with threshold to moderate hepatotoxIclty occurring at doses of 5 and 10 yg/kg/week for male and female rats, respectively, and at 10 yg/kg/week for both sexes of mice. At higher exposures, splenic h y pe r p l a s i a and cortical a t r o p h y of the thymus were also d e te c t e d in rats. In rats It was unclear w h e t h e r the l o w- d o se animals w e re free of any p a t h o logic findings or none w e r e s u bj e c te d to necropsy. In m i ce It was stated 00110 V-52 07/22/87 that no changes were observed in males exposed to 2,3,7,8-TCDD at 1.25 yg/k g / we e k or in females expo s e d to 1.25 or 2.5 yg/kg/week. Alth o u gh the data are limited, it appears that the same target organs are sens i t iv e to the toxic effects of both 2,3,7,8-TCDD and this mixture of HxCOD. In addition, a second subchronic range finding study conducted by NIP (1980c) evaluated the dermal toxicity of the above mixture of HxCDD. Groups of 10 male and 10 female Swiss-Webster mice were treated by dermal a p p l i c a tion 3 times/week for 13 weeks. The doses used were from 0.01-50 yg/ a p p l i c a t i o n with the test comp o u nd d i ss o l v e d in acetone. There was 1 0 0% m o r t a l i t y in the 25 and 50 yg/appl Teat ion groups and 8 0% m o r t a l i t y in the 10 yg/appli cati on group. On histologic examination, there were signs of liver dama g e at the lowest dose tested in both sexes; however, the incidence and degree of damage were not well correlated to the dose applied. Chronic T o x i c i t y . In rats and mice the toxic effects of chronic e x p o s u r e to 2 , 3 , 7 , 8 - T C D D are s u mm a r iz e d in T a ble V-5. T h es e studies were predominately designed to assess the carcinogenicity of 2,3,7,8-TCDD and the observations of non-neoplast1c systemic toxicity are, therefore, limited. Van Miller et al. (1977a,b) fed groups of 10 male Sprague-Dawley rats diets c o n t a i n i n g 1, 5, 50, 500, 1000, 5000, 50,000, 500, 0 0 0 or 1 , 00 0 , 0 0 0 ppt 2 , 3 , 7 , 8 - T C D D ( 10" 3 y g/kg diet) for 78 weeks in order to d e te r m i n e the potential toxic and carcinogenic effects of 2,3,7,8-TCDD. The authors estimated that these dietary levels corresponded to doses of 0.0003, 0.001, 0.01, 0.1, 0.4, 2.0, 24, 240 or 500 yg 2,3,7,8-TCDD/kg bw/week, re sp e c tively. All animals that received diets containing >1000 ppt (>0.4 yg/kg 00110 V-5 3 07/22/87 TABLE V-5 Effects of Chrontc Exposure to 2.3,7,0-TCOO In Laboratory Rodents 00110 Specles/Straln Sex/No. Dose Rat/SpragueDawley male/10 male/10 0.0 ppt 1 ppt Treatment Schedule Duration of Study Parameters Monitored NA 95 weeks survival contlnous In diet for 7B weeks 95 weeks survival male/10 5 ppt continuous In diet for 7B weeks 95 weeks survival male/10 iO ppt continuous In diet for 7B weeks 95 weeks survival male/10 500 ppt continuous In diet for 7B weeks 95 weeks survival male/10 1000 and 5000 ppt continuous In diet for 78 weeks 95 weeks survival male/10 50.000. 500,000 and 1,000,000 ppt continuous In diet for 78 weeks 95 weeks survival Effects of Treatment* Reference 40X survived until 95 weeks, the first death occurred at week 68 BOX survived until 95 weeks, the first death occurred at week B6 60X survived until 95 weeks, the first death occurred at week 33 60X survived until 95 weeks, the first death occurred at week 69 50X survived until 95 weeks, the first death occurred at week 17 No animals survived until 95 weeks, the first death occurred at week 31 No animals survived until 95 weeks, the first deaths occurred at weeks 2 and 3 Van Miller et al., 1977a,b V-54 08/11/84 1ABLE V-5 (coni.) 00110 Specles/Stratn Sex/No. Dose Rat/SpragueOawley M and F/ -2193 ppt 50 and 50 (0.1 i>g/kg/day) N and F/ -20B ppt 50 and 50 (0.01 pg/kg/day) r M and Ft -22 ppt (0 .0 0 1 50 and 50 pg/kg/day) Treatment Schedule Duration of Study Parameters Monitored Effects of TreatmHl* Reference continuous In diet for 2 years 2 years continuous In diet for 2 years 2 years continuous In diet for 2 years 2 years extensive hlstopathology, hema tology, urine analyses, and clinical chemistry extensive hlstopathology, hema tology, urine analyses and clinical chemistry extensive htstopathology, urine analyses and clinical chemistry Cumulative mortality, Increased (F); Body weight gain, decreased (.f>; Red blood cell count, decreased (M.F); Packed cell volume, decreased (M.F); Hemoglobin, decreased.(M.F); Reticulocytes, Increased (M.F); White blood cell cotfnt, decreased (F); Serum glutamic pyruvic transaminase. Increased (F); G-Glutamyl transferase. Increased (F); Alkaline phosphatase. Increased (F); Urinary coproporphyrin. Increased (F); Urinary uroporphyrin. Increased (F); Urinary delta-amlnolevullnlc a d d . Increased hepatic dgnrt ton. Increased (M.F) Urinary coproporphyrln. Increased (F); Urinary uroprophyrln. Increased (F); Hepatic degeneration. Increased (M.F) No differences from values obtained from control animals Koclba et al.. 1970a, 1979 V -- 55 09/18/84 TABLE V-5 (cont.) 00110 V-56 Specles/Straln Sex/Ho. Oose Treatment Schedule Duration of Study Parameters Monitored Rat/OsborneHendel H1ce/B6C3F1 Htce/Swlss f H and f/ 75 and 75 H and F/ 50 and 50 0.0 pg/kg/week 0.5 pg/kg/week H and F/ 0.05 pg/kg/week 50 and 50 n and F/ 0.01 pg/kg/week 50 and 50 H and F/ 0.0 pg/kg/week 75 and 75 H and F/ 50 and 50 0.5 pg/kg/week (H); 2.0 pfl/kg/ week (F) H and F/ 50 and 50 H and F/ 50 and 50 H/38 0.05 pg/kg/week (M); 0.2 pg/kg/ week (F) 0.01 pg/kg/week (H); 0.04 pg/kg/ week (F) 0.0 pg/kg/week . H/44 0.007 pg/kg/week H/44 0.7 pg/kg/week H/43 7.0 pg/kg/week NA administered by gavage biweekly for 104 weeks administered by gavage biweekly for 104 weeks administered by gavage biweekly for 104 weeks NA administered by gavage biweekly for 104 weeks administered by gavage biweekly for 104 weeks administered by gavage biweekly for 104 weeks NA administered by gavage weekly for 1 year administered by gavage weekly for 1 year administered by gavage weekly for 1 year 106 weeks 107 weeks 107 weeks 107 weeks 105-106 weeks 107 weeks 107 weeks 107 weeks 588 days 649 days 633 days 424 days extensive hlstopatholdgy extensive hlstopathology extensive hlstopathology extensive hlstopathology extensive hlstopathology extensive hlstopathology extensive hlstopathology extensive hlstopathology histology on all organs histology on all organs histology on all organs histology on all organs Results of statistical analysis were not provided. NA Hot applicable Effects of Treatment1 Toxic hepatitis; 0/74 (ft). 0/75 (f) Toxic hepatitis; 14/50 (N). 32/50 (F) Toxic hepatitis; 0/50 (ft), 1/50 (F) Toxic hepatitis; 1/50 (ft), 0/50 (F) Toxic hepatitis; 1/73 (N). 0/73 (F) Toxic hepatitis; 44/50 (M), 34/47 (F) Toxic hepatitis; 3/49 (H). 2/48 (F) Toxic hepatitis; 5/44 (ft), 1/50 (FI Dermatitis and amyloidosis; 0/38 Dermatitis and amyloidosis; 5/44 Oermatltls and amyloidosis; 10/44 Early mortality, dermatitis and amyloidosis; 17/43 Reference MTP, 1960a NTP. I9B03 Toth et al., 197B. 1979 08/11/84 bw/week) were dead by the end of the study (95 weeks). It a p pe a r ed that diets containing >1000 ppt (>0.4 yg/kg bw/week) 2,3,7,8-TCDD definitely increased mortality. The three highest dietary levels (50,000, 500,000 and 1,000,000 ppt) were acutely toxic, causing death between the second and fourth weeks of the study, with severe liver necrosis, thymic atrophy and cessation of growth. Conclusions could not be drawn concerning the effects of lower doses (lower than 500 ppt) on survival because of the small sample sires and the high m o r t a l i t y in the control g r oup (6 of 10). In the groups r e ce i v in g < 5 00 0 ppt, w e ig h t gain was s i g n i f i c a n t l y d e pr e s se d only in the 5000 ppt group. The o n ly h i s t o p a t h o l o g i c changes reported in these groups were neoplastic changes (see Carcinogenicity Section). Koclba et al. (1878a, 1979} maintained male and female Sprague-Dawley rats (50/sex/dose) on diets containing levels of 2,3,7,8-lCDD that resulted in doses of 0.001, 0.01 or 0.1 yg/kg/day. I ncreased m o r t a l i t y was observed in the h i g h - d o s e females. In the groups r eceiving 0.01 or 0.1 yg/k g / da y , t r e a t m e n t - r e l a t e d changes w e re o b s e r v e d in h e ma t o lo g i c, c l i n i cal chemistry and urinary analysis values. Urinary excretion rates of c o p r o p o r p h y r i n and u r o p o r p h y r i n were increased in these groups. H i stologic e x a m i n a t i o n r e ve a l e d d e ge n e ra t i ve , necrotic and i n fl a m ma t o ry changes in the liver. The NOEL in this study was 0.001 yg/kg bw/day. Toxic hepatitis (lipidosis and hydropic degeneration of the cytoplasm of the hepatocytes) was observed in-both sexes of Osborne-Mendel rats receiving 2 , 3 , 7 , 8 - T C D D by gava g e in corn o i l : a c e t o n e (9:1) (0.25 or 0.025 y g /kg bw), twlce/week for 104 weeks (NTP, 1980a). No other non-neoplastic lesions were observed, even though extensive histological examinations were performed. This study demonstrated a N0AEL of 0.05 yg/kg bw/week for hepatitis. 00110 V-57 07/22/87 In this same study, B6C3F^ mice were treated biweekly For 104 weeks with 2 , 3, 7 , 8- T C DD in corn o i l: a c e t o n e (9:1). Males were given 0.0, 0.01, 0.05 or 0.5 yg/kg bw/week and females were given 0.0, 0.04, 0.2 or 2.0 yg/kg bw/week. Toxic hepatitis was o b served in control and treated groups, but a p pe a r ed to be s i g n i f i c a n t l y elev a t ed only in the high dose groups (NTP, 1980a). In another study, Toth et at. (1978, 1979) Intubated male Swiss mice with 0.0, 0.007, 0.7 or 7.0 yg 2,3,7,8-TCDD/kg bw/week for 1 year. A m y l o i do s i s of the kidney, spleen and liver, and derm a t it i s w e re o b se r v ed in all three treatment groups. This suggests a L0AEL of 0.007 yg/kg bw/week in m ice in this study, but a N0AEL was not established. Some information on the toxicity of chronic dermal exposure can be o b ta i n e d From the NTP (1980b) dermal c a r c i n o g e n i c i t y study in S w is s - We b s te r mice. Thirty males were treated with 0.01 yg 2,3,7,8-TCDD/appl1cation and 30 females were treated with 0.005 yg/applicatlon, 3 times/week for 104 weeks. V e h i c l e - t r e a t e d and u n tr e a te d controls w e re included in the e x p e r i ment. Although no effects on body weight were observed, treated male mice had a significant shortening of lifespan, and treated females showed nonneoplastlc hepatic lesions (hepatic cytomegaly and Inflammatory areas). Target Organ Toxicity Hepatic Effects. As previously described, when administered lethal doses of 2 , 3 , 7 ,8-TCDD, guinea pigs and monkeys have Few or no h i s t o p a t h o logic changes In the liver, whereas rats and mice have extensive h i s t o p a t h o logic effects in this organ. Gupta et al. (1973) and Grieg et al. (1973) 00110 V-58 07/22/87 o b served mark e d d i st o r t i o n of liver a r c h i t e c t u r e in rats given 50 or 100 pg 2 , 3 , 7 ,8-TCOD/kg, with marked necrosis of hepatocytes. The livers of mice given lethal doses of 2 , 3 , 7 , 8 - T C D D also had necrosis (Vos et a 1. , 1974). A c c o r d i n g to Jones and Grieg (1975 ), c e n t r 1lobular necrosis, bile duct p r o l i f e r a t i o n and lipid a c c u m u l a t i o n w e re more severe in mice than In rats. Livers of mice contained excess amounts of porphyrin (McConnell et al., 1978a). Light m i c r o s c o p i c , u l t r a s t r u c t u r a l and h i s t o c he m i ca l changes in the livers of rats given 2,3,7,8-TCDD orally have been described by Fowler et al. (1973), Jones and Butler (1974) and Jones (1975). Fowler et al. (1973) a d mi n i s t e r e d 0.0, 5.0 or 25.0 pg 2 , 3 , 7 ,8-TCDD/kg bw by gavage to groups of 30 m a le rats. The rats w e r e s a c r i f ic e d 1, 3, 6, 9, 16 or 28 days after treatment. Major ul tras t r uc t u ra l changes o c cu r r ed in the cells near the bile c a nallculi in both t reatment groups. A d o s e - r e l a t e d Increase in the smooth and rough e n d o p l as m i c r e t i c u l u m was o b se r v e d s t ar t i n g on day 3, reaching a maximum on days 6-9, and returned to normal by day 28. Jones and Butler (1974) administered 200 pg 2,3,7,8-TCDD/kg bw to groups of 40 male and 40 female Porton rats and sacrificed 4 rats of each sex/week for 10 weeks. The major lesions observed were necrosis and p r o l i f er at i v e changes In the liver. Degene ra t i ng cells were observed near the central vein, progressing to areas of focal necrosis by week 6 and central vein fibrosis with scattered necrosis by week 10. Additionally, hyperplasia of the viable cells, with many multinucleated cells, was prevalent by week 9. Jones (1975) g a ve 23 m a l e Porton rats a single g a v a g e dose of 200 pg 2 , 3 , 7 , 8 - T C D D / k g in arac h i s oil, and 7 control rats r e ce i v e d arachls oil 00110 V - 59 07/22/87 alone. Loss of ATPase activity along the canalicular borders and Increased a c tivity in the sinusoids in the livers of rats were o b se r v ed 3 days f o l l o w ing treatment. The loss of ATPase activity persisted for at least 34 42 days, but returned to normal by 9 months. Peterson et al. (1979a) investigated the effects of lower doses (10 or 25 yg/kg bw, oral) of 2,3,7,8-TCDD on AlPase activity in h e patocyte plasma membranes. Liver surface membranes were Isolated from male Holtzman rats using a m o di f i e d d i s c o n t i n u o u s sucrose g r ad i e nt meth o d on days 2, 10, 20 or 40 p o s t - t r e a t m e n t . Both doses resulted in similar depression of Na/K-ATPase on days 2-40; however, M g f + -ATPase was d e pr e s se d to this extent only in the high-dose groups. In the low-dose group, M g f*-ATPase activity was decreased on day 20, but returned to normal levels by day 40 post-treatment. 2 , 3 , 7 , 8 - T C D D did not inhibit ATPase a c ti v i t y J_n v i t r o , nor did the decrease in ATPase activ i ty c o rre l ate with 2,3,7 , 8- T C DD induced food deprivation. There was, however, a correlation between liver surface membrane activity, bile flow and biliary excretion of ouabain In v i v o . In similar experiments, Hwang (1973) reported that bile flow was stimulated, but Indocyanine green e x cretion was inhibited, in male CD rats following treatment with 5 or 25 yg 2,3,7,8-TCDD/kg bw by gavage. Peterson et al. (1979b) investigated the relationship between AlPase a c tivity and b iliary excr e t io n of ouabain in perfused liver from rats. They discovered that liver surface membrane ATPase activity and ouabain excretion could be segregated by cotreatment with the use of spironolactone, chemicals that increase bile flow, or p r eg n e no l o ne-16-a-carbonitrile, indicating that this ATPase a c tivity was not d i rectly Involved in ouabain transport. 00110 V-60 07/22/87 1 he ability of 2,3,7,8-TCDD to inhibit biliary excretion has been d e m o n strated to correlate with the species sensitivity to the hepatotoxlc effects of 2 , 3 , 7 , 8- T C DD {Seefeld et a l ., 1979, 1980). Yang et al. (1977) reported that the effect of 2 , 3 , 7 , 8 - T C D D on bi li a r y t ransport is c o mpound dependent. In these studies, ouabain excretion, a neutral compound, and bile flow were inhibited, while the excretion of the organic anions phen o l -3,6-dibromophthalein and sulfobromophthalein were essentially unchanged. C u n n i n g h a m and W i ll i a m s (1972) d e te c t ed a d e cr e a s e in j_n v 1vo lipid f ormation in the liver of m a le Wistar rats r e ce i v in g 10 yg 2 , 3 , 7 ,8-lCDD/kg bw, based on [aH]sod1um acetate incorporation. They determined that the synthesis of triglycerides, diglycerides and phospholipids were all depressed. Despite this inhibition of lipid synthesis, Albro et al. (1978) found an increase in total hepatic lipids in rats 13 days f ollowing t r e a t ment with 50 yg 2 , 3 , 7 ,8-TCDD/kg bw. Levels of free fatty acids and cholesterol esters were increased while levels of phospholipids, free cholesterol and triglycerides remained constant. When a sublethal dose, 10 vg/kg bw, was administered, triglycerides and fatty acids were increased and cholesterol esters were decreased. These changes were explained by mobilization of body fat, decreased lysosomal acid lipase, and increased lipid peroxidation. G o l d s t e i n et al. (1978) d e te r m i n e d that 4 w e e k l y doses of 25 yg 2 , 3, 7 , 8 - T C D D / k g bw or higher w ould result in increased hepatic porp h y ri n levels in m a le C57B1 mice. Smith et al. (1981) found that strain d i f f e r ences in s e n s i t iv i t y b e t w e e n C57B1 and D B A/2 m i ce given a single gavage dose of 75 yg 2,3,7,8-TCDD/kg correlated with the ability of 2,3,7,8-TCDD to 00110 V -61 07/22/87 induce increases in hepatic porphyrins; however, there was no c o rr e lation between toxicity and increased porphyrins between sexes within strains. Rats are less s e ns i t iv e to 2 , 3 , 7 , 8- T C DO induced porphyria, with increases in porphyrin levels occurring only after subchronic exposure (>0.01 mg/kg/wcek by gavage for 6 months) (Can ton 1 et a 1., 1981). The major factors involved in 2 , 3 , 7 , 8 - T C D D - i n d u c e d p o r p h y r i a have been i d entified as an increase in ct-aniinolevulinic acid s y nt h e ta s e (Goldstein et a 1. . 1978) and a d e cr e a s e in u r o p o r p h y r i n o g e n d e c a r b o x y l as e activity (Smith et al., 1981; Sweeney and Jones, 1978). - A number of groups investigated the effect of 2,3,7,8-TCDO on hepatic DNA synthesis. Grieg et al. (1974) measured the incorporation of [3H ]thy midine into the hepatic DNA of male and female Porten rats following partial hepatectomy. No effect of 2,3,7,8-TCDD pretreatment (10 or 200 mg/kg bw) was noted. In in vitro studies, however, Conway and Matsumura (1975) and Dickens et al. (1981) demonstrated increased [3H]thymidlne incorporation into DNA in liver slices obtained from rats which had been pretreated with 5 vg 2 , 3 , 7 , 8 - T C D D / k g bw. 2 , 3 , 7 , 8 - T C D D is also a potent inducer of hepatic microsomal enzymes (see Enzyme Induction in Chapter VII). Immunological Effects. The consistent observation of thymic atrophy in early a c ut e t o x i c i t y studies s u gg e s te d that 2 , 3 , 7 , 8 - T C D D may alter the immune r e sp o n s e and p r om p t e d i n v e s t ig a t io n s of i m m u n o t o x i c i t y ,, s u m m a r i z e d in T a bl e V-4. The results suggest -that 2 , 3, 7 , 8 - T C D D interferes with the immu nologic capability of the animals tested. Vos et al. (1973) investigated the effect of orally administered 2,3,7,8-TCDD on the cell-mediated and humoral Immune response of B6D2F^ mice, CD rats and Hartley guinea pigs. 00110 V-62 07/22/87 Pretreatment with 0.04 yg 2,3,7,8-TCDD/kg bw/week reduced the development of skin h y p e r s e n s i t i v i t y to M y c o b a c t e r i u m tubrculos 1s in guinea pigs; no effect on this p arameter was seen in rats. Tetanus a n ti t o xi n levels were reduced in guinea pigs recei v in g 0.2 yg 2 , 3 , 7 , 8 - T C D D / k g bw/week. 2,3,7,8TCOD has also been d e m o n s t r a t e d to inhibit c e l l - m e d i a t e d immunity in "graftversus-host" experiments utilizing spleen cells from mice which had received 4 w e e k l y oral doses of 0, 0.2, 1.0 or 5.0 yg 2 , 3 , 7 , 8 - T C D D / k g bw (Vos et a l ., 1973). Vos and Moore (1974) and Moore and Vos (1974) administered 1 or 5 yg 2 , 3 , 7 , 8 - T C O D / k g bw to female F -344 rats on days 11 and 18 of g e s t a t i o n and days 0, 7 and 14 postpartum. This t reatment resu l t ed in p r ol o n ge d times until graft rejection, decreased spleen cell graft-versus-host activity and decreased binding response to phytohemogglutinln, but an increased humoral immune response to conco n av a l in A in the pups. Thigpen et al. (1975) determined the effect of 2,3,7,8-TCDD on host r e sistance to Injection by Salmonella bern in male SPF C57Bl/6Jfh mice, an effect la rg e l y m e di a t e d by t h y m u s - d e r i v e d lymphocytes. P r e t r e at m e nt with >1 yg/kg bw/w e e k by gava g e resu l t ed in an Increase in m o r t a l i t y and a d e cr e a s e in time until infection. The N0AEL was d e te r m i n e d to be 0.5 yg/kg bw. Pretreatment with 2,3,7,8-TCDD has also been demonstrated to enha n c e the s u s c e p t i b i l i t y of m i ce to E_. col 1 e n do t o x i n (Vos et al., 1978a). Thomas and Hlnsdil! (1979) maintained female Swiss-Webster mice on diets c o n t a i n i n g 1, 2.5, 5, 10 or 20 ppb 2 , 3 , 7 , 8 - T C D D (1, 2.5, 5, 10 or 20 yg/kg diet) from 4 weeks before mating until 3 weeks postpartum. Thymus weight 00110 V-63 07/22/87 I was d ecreased in the pups from groups r e ce i v in g >2.5 ppb, along with a decreased number of pl aq u e -f o r mi n g cells reactive to sheep red blood cells and an increased s u s c e p t i b i l i t y to S. t y p h l m u r i u m . This treatment did not, however, lower the humoral content of anti-RBC antibodies, affect the response to E_. coli LPS or L. m o n o c y t o g e n e s , affect n i t r o g e n - i n d u c e d l y m p h o cyte proliferation, or alter the response of B- and T-cells to conconcanvalin A j_n v i t r o . Similar results have been r e ported in F isher/VJistar rats (Faith and Luster, 1979; Luster et a l ., 1980). Hinsdill et al. (1980) found that feeding Swiss-Webster mice diets c o n taining 100 ppb (100 yg/kg diet) 2,3,7,8-TCDD for 5 weeks increased serum 8-globulins, but suppressed total serum protein, y-globulin and albumin. A d e cr e a s e d immune r e s p o n s e to tetanus, toxoid, sheep red blood cells, S. typhlmur ium and L. m o n o c y t o g e n e s and a lowered c ontact s e n s i t iv i t y to DNFB were noted at dietary concentrations as low as 10 ppb (10 yg/kg diet). Young animals were slightly more susceptible to the Immunosuppressive effects of 2,3,7,8-TCDD than were older animals. Vos and Moore (1974) also reported that 1-month-old mice are more sensitive to 2,3,7,8-TCDD than are 4-month-old mice. M antovani et a l . (1980) a d m i n i s t e r e d single l.p. doses of 1, 2, 6 or 30 yg 2 , 3 , 7 , 8 - T C D D / k g bw to C 5 7B 1 / 6J mice. This treat m en t resulted in a decreased total number of macrophages and splenic natural killer cells, but did not affect the c y to s t a t i c or- cytocldal a c ti v i t y of the r e ma i n i n g cells. McConnell et al. (1978a) suggested that the decrease in peripheral cell counts may be the result of the h y po c e ll u l ar i t y seen in the bone m a rr o w of 2,3,7,8-TCDD-treated mice. 00110 V-64 07/22/87 Other Organ S y s t e m s . One of the characteristic effects of 2,3,7,8- TCDD is the loss of body weight. Because d e c r e a s e d food c o n s u m p t i o n does not totally account for this finding, the potential effects of 2,3,7,8-TCDD on intestinal absorption were of interest. Madge (1977) Investigated the effect of 2 , 3 , 7 , 8 - T C D D on intestinal a b s o r p t i o n in CD-I mice using the everted intestinal sac technique. Treatment of the mice with single gavage doses of 0, 10, 25, 75, 150, 200 or 300 ^g 2 , 3 , 7 ,0-TCDD/kg bw decr e a se d the vitro intestinal absorption of D-glucose, but did not affect a b s o r p tion of D-galactose, L-argenine or L-histldine. These measurements were made 7 days after treatment. In a similar study, Ball and Chhabra (1981) reported that 2,3,7,8-TCDD reduced the absorption of D-glucose and leucine in S p r a g u e - D a w l e y rats. Manis and Kim (1979a,b) found that 22-84 yg 2,3,7,8-TCDD stimulated intestinal trans p or t of iron in male S p r a g u e - D a w l e y rats and in an u n i d e n t i fied strain of mice. Gavage a d mi nistration was more effective than i.p. injection. This s t i m u l a t i o n o c cu r r ed in the duodenum, but the distal segment of the intestine was unaffected. In parallel experiments, calcium transport was decreased and galactose and prollne were unaffected by prior exposure to 2,3,7,8-TCDD. Pegg et al. (1976) and Hook et al. (1977) investigated the effect of 10 or 25 yg 2,3,7,8-TCDD on the Ui vitro function of renal cortical slices obtained from male Sp ra gue- Da wl e y rats 3 or 7 days following intubation. Anion transport, as measured by accumulation of p-aminohippuric acid, was decreased by N-me th yl - n ic o t in a m i de accumulation, at both dose levels. In the jn^ vivo studies, s o d i u m r e a b s o r p t i o n was w i t h i n the normal range and 00110 V -65 07/22/87 ammonlogenesis and gluconeogenesis were unaffected, even when the rats were made acldotic. The authors c o nc l u de d that the o b se r v ed d e crease in kidney function was a result of the generally poor condition of the treated animals and not a direct effect of 2,3,7,8-TCDD. Grieg et al. (1974} found that Intraperltoneal a d m i n i s t r a t i o n of 10 yg 2.3.7.8- TCDD/kg bw Inhibited the ONA synthesis stimulated by folate (250 mg folic acid/kg) or lead acet a t e (40 mg Pb /kg) In the kidneys of m a le and female Ponton strain rats. This inhibition did not involve direct inter action with DNA or the inhibition of protein synthesis, but may Involve the aromatic oxidases of liver microsome. Zinkl et al. (1973) observed a number of hematologic clinical chemistry changes in female CD rats given 30 daily doses of 0.1, 1.0 or 10 yg 2.3.7.8- TCDD/kg bw; however, they concluded that these changes simply reflected damage to other organs, such as the liver, and hemoconcentration. The only other major effect observed was thrombocytopenia, which was also o b se r v ed In mice and female guinea pigs. L y m p h o p e n i a was obse r v ed In guinea pigs that rece i v ed a single d o se of 1, 10 or 50 yg/kg bw. Poll et al. (1980) inves t ig a t ed the induction of h y p e r l i p i d e m i a in male S p ra g u e - D a w l e y rats follo w in g a single l.p. i njection of 2.5, 5, 10 or 20 yg 2 , 3 , 7 , 8 - T C D D / k g bw. T h er e was a d o s e - r e l a t e d increase in total plasma ch ol e s te r o l and high d e n s i t y lipo p r ot e i n c h ol e sterol by 21 days p o s t treatment, but triglycerides and low and very low density lipoproteins were unaffected. In contrast, h y pe r l i p i d e m i a in m a le H a r t l e y guinea pigs g iven a single l.p. injection of 2 yg/kg bw was charac te r i ze d by increases in low and very low density lipoproteins. 00110 V-66 07/22/87 Other E ffects C a r c i n o g e n i c i t y . 2 , 3 , 7 , 8 - T C D D has been tested for c a r e i n o g e n l c i t y In rats and mice by a d m i n i s t e r i n g the comp o u nd in the diet and by gavage. Also, the tumor Incidence In native m i ce inhabiting an area with heavy exposure to the herbicide Agent Orange has been assessed and compared with mice from an uncontaminated habitat. The results of these bioassays are s u m m a r i z e d in T a ble V -6. A l o n g with studies using the oral route, 2,3,7,8TCDD has been tested for tumorIgenicity by dermal application (Table V -7). Using the skin two-stage t u mo r l g e n l c 1ty model, 2,3,7,8-TCDD has been tested for promoting and Initiating activity as well as anticarclnogenic activity. Other model systems have been used to a more limited extent in studies of the effect of 2,3,7,8-TCDD on the carcinogenic potential of chemical care inogens. In a limited study, Van Miller (1977a,b) maintained small groups of male S p r a g u e - D a w l e y rats on diets c o n t a i n i n g 2,3,7 , 8- T C DD . The animals, in, groups of 10, were fed diets containing 0.0, 0.001, 0.005, 0.05, 0.5, 1.0, 5.0, 50, 500 or 1000 ppb (-pg/kg diet) of 2 , 3 , 7 , 8 - T C D D for 78 weeks. As determined from the food consumption of two animals from each group, these exposure levels corresponded to doses of 0.0, 0.0003, 0.001, 0.01, 0.1, 0.4, 2.0, 24, 240 and 500 yg/kg/week, respectively. Animals exposed to >50 ppb died before tumor development could occur. At week 65 of treatment, all surviving animals were examined by laparotomy, and biopsy samples were obtained from any gross tumors., Following termination of treatment, the animals w ere observed for an additional 17 weeks before and until s a c r i f i c ing all surviving animals. At necropsy, performed on animals killed when moribund, found dead, or killed at termination of the study, the animals were examined for both gross and microscopic lesions. 00110 V -67 07/22/87 1ABLE V-6 Carcinogenicity Bioassays of 2,3,7,8-TCDD Administration by the Oral Route oo o Exposure Duration Duration Route Specles/Straln Sex Dose or Exposure of of Study Treatment Vehlcle Tumor Type Tumor Incidence p Value Reference V -68 Gavage rats/ Osborne-Hendel Gavage rats/ Osborne-Hendel r Gavage mtce/B6C3Fi \K O CD CD H 0.0 Mg/kg/week 0.01 |ig/kg/week 0.0S g/kg/week 0.5 ug/kg/week r 0.0 iig/kg/week 0.01 |ig/kg/week 0.0S ig/kg/ueek 0.5 tig/kg/week M 0.0 |ig/kg/week 0.01 |ig/kg/week 0.05 Mg/kg/week 0.5 jig/kg/week 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 104 weeks 105 weeks 107 weeks 107 weeks 107 weeks 105 weeks 107 weeks 107 weeks 107 weeks 105 weeks 107 weeks 107 weeks 107 weeks corn ollacetone (9:1) corn oilacetone (9:1) corn oilacetone (9:1) corn oilacetone (9:1) corn oilacetone (9:1) corn oilacetone (9:1) corn ol1acetone (9:1) corn oilacetone (9:1) corn oilacetone (9:1) corn otlacetone (9:1) corn ollacetone (9:1) corn oilacetone (9:1) follicular-cell adenomas of the thyroid, carcinoma of the thyroid follicular-cell adenomas of the thyroid, carcinoma of the thyroid follicular-cell adenomas of the thyroid, carcinoma of the thyroid folHcular-cell adenomas of the thyroid, carcinoma of the thyroid neoplastic nodule of the liver, hepatocellular carcinoma of the liver neoplastic nodule of the liver, hepatocellular carcinoma of the liver neoplastic nodule of the liver, hepatocellular carcinoma of the liver neoplastic nodule of the liver, hepatocellular carcinoma of the liver hepatocellular carcinoma hepatocellular carcinoma hepatocellular carcinoma hepatocellular carcinoma 1/69 0/69 *0.006 5/48 0/48 *0.042 6/50 2/50 *0.021 10/50 1/50 *0.001 5/75 0/75 <0.001 1/49 0/49 NS 3/50 0/50 NS 12/49 3/49 *0.006 0/73 *0.002 9/49 NS 8/49 NS 11/50 *0.002 NTP, 1980a NTP, 1980a NTP, 1980a TABLE V-6 (cont.) Exposure Duration Duration Route Specles/Straln Sex Dose or Exposure of of Study Treatment Vehlcle Tumor Type Tumor IncIdence p Value Reference Gavage m1ce/B6C3E, F 0.0 pg/kg/week 104 weeks 105 weeks corn oilacetone (9:1) hepatocellular carcinoma. follicular-cell adenomas of the thyroid 0.04 pg/kg/week 104 weeks 107 weeks corn oilacetone (9:1) hepatocellular carcinoma. follicular-cell adenomas of the thyroid 0.2 pg/kg/week 104 weeks 107 weeks corn oilacetone (9:1) hepatocellular carcinoma. follicular-cell adenomas of the thyroid 2.0 pg/kg/week 104 weeks 107 weeks corn oilacetone (9:1) hepatocellular carcinoma. folllcular-cell adenomas of the thyroid Oral ral/ H 0.0 ppb Sprague-Oawley 0.001 ppb 78 weeks 7B weeks 95 weeks 95 weeks In diet In diet all tumors3 all tumors3 0.00S ppb 7B weeks 95 weeks In diet all tumors3 0.05 ppb 78 weeks 95 weeks In diet all tumors3 0.5 ppb 7B weeks 95 weeks In diet all tumors3 1.0 ppb 78 weeks 95 weeks In diet all tumors3 5.0 ppb 78 weeks 95 weeks In diet all tumors3 Oral rat/ H 0.0 pg/kg/day Sprague-Dawley 105 weeks 105 weeks In diet squamous cell carcinoma of the hard palate, squamous cell carcinoma of the longue, adenoma of the adrenal cortex 0.001 pg/kg/day 105 weeks 105 weeks In diet squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue, adenoma of the adrenal cortex 1/73 0/69 *0.006 *0.016 NTH, 1980a 2/50 3/50 NS NS 2/48 l/4r NS NS 6/47 5/46 *0.014 *0.009 0/10 0/10 5/10 3/10 4/10 4/10 7/10 0/85 0/85 0/85 NR NR NR NR NR NR NR NS NS NS Van Hiller et al., 1977a,b KocIba et al., 1978a,b 0/50 1/50 0/50 NS NS NS TABLE V 6 (coni.) 00110 Exposure Duration Duration Route Spectes/Straln Sex Dose or Exposure of of Study Treatment Vehicle Oral rat/ M 0.01 pg/kg/day Sprague-Dawley 10S weeks 105 weeks In diet 0.1 pg/kg/day 10S weeks 105 weeks In diet Oral rat/ r 0.0 pg/kg/day Sprague-Dawley 10S weeks 105 weeks In diet 0.001 pg/kg/day 105 weeks 105 weeks In diet 0.01 pg/kg/day 105 weeks 105 weeks In diet * Oral rat/ Sprague-Dawley F 0.1 pg/kg/day 105 weeks 105 weeks In diet Tumor Type Tumor Incidence p Value Reference squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue, adenoma of the adrenal cortex squamous cell carcinoma of the hard palate, squamous cell carcinoma of the tongue, adenoma of the adrenal cortex hepatocellular carcinoma. squamous cell carcinoma of the tongue, squamous cell carcinoma of the lung hepatocellular carcinoma. squamous cell carcinoma of the tongue, squamous cell carcinoma of the lung hepatocellular carcinoma, squamous cell carcinoma of the tongue, squamous cell carcinoma of the lung hepatocellular carcinoma. squamous cell carcinoma of the tongue, squamous cell carcinoma of the lung 0/50 1/50 2/50 NS NS NS 4/50 <0.05 3/50 . <0.05 5/50 <0.05 0/06 0/86 0/86 0/50 0/50 0/50 2/50 1/50 0/50 11/49 4/49 7/49 NS NS NS NS NS NS NS NS NS <0.05 <0.05 <0.05 Koclba et a1., 197Ba,b Koclba et i . . 1978a,b Koclba el a)., 1978a.b V-70 09/18/84 TA B L E V - 6 ( c o n t .) Exposure Ouratlon Ouratlon Route Specles/Straln Sex Dose or Exposure of of Study Treatment Vehtcle Tumor Type Gavage mice/ Sw1ss/H/R1op Oral mice/ Peromvscus pollonotus h 0.0 pg/kg/week 365 days 5BB days sunflower ol 1 liver tumors** 0.007 pg/kg/week 365 days 649 days sunflower oil liver tumors** 0.7 pg/kg/week 365 days 633 days sunflower oil liver tumors** 7.0 pg/kg/week 365 days 424 days sunflower ol 1 liver tumors** 0.0012 pg/kg/day NA NA contaminated liver soil 0.0 pg/kg/day NA NA contaminated liver soil aNo single target organ for cancer was outstanding. ^Includes hepatomas and hepatocellular carcinomas. NR * Not reported NS Not significant m Tumor IncIdence p Value Reference 7/38 NS 13/44 NS 21/44 <0.01 13/4> NS 0/15 NS 0/15 NS Toth et al.. 1979 Cockerham et al.. 1980 oC--O GO CD 00110 TABLE V-7 Carcinogenicity Bioassays of 2,3,7,B-TCOD Administered by the Dermal Route V-72 Species Sex Doseb Duration of Exposure Target Organ Tumor Type Tumor Incidence Nice N 0.01 pg/appllcatlon 104 weeks Integumentary fibrosarcoma 6/28 system 0.0 iig/appUcatlon (vehicle control) 104 weeks Integumentary system fibrosarcoma 3/42 0.0 pg/app11cation (untreated control) NA Integumentary fibrosarcoma 0/20 system F 0.005 pg/appllcatlon 104 weeks Integumentary fibrosarcoma 8/28 system 0.0 pg/appllcatlon (vehicle control) 104 weeks Integumentary system fibrosarcoma 2/41 0.0 pg/appllcatlon (untreated control) NA Integumentary fibrosarcoma 1/27 system aSource: NTP, 1980b bThc compound was applied 3 times/week In 100 pi of acetone. NA = Not applicable 09/18/84 All animals In groups maintained on diets' containing 1-1000 ppb of 2,3,7,8-TCOO were dead by week 90 of treatment with the first deaths In groups at the 1000 and 1 ppb levels observed at 2 weeks and 31 weeks of treatment, respectively (Van Miller et al., 1977a,b). Animals exposed to (0.001-0.5 ppb of 2,3,7,8-TCOO had similar food consumption (20 g/day) and survival (4/10, 8/10, 6/10, 6/10, 5/10 survived In the 0, 0.001, 0.005, 0.05 and 0.5 ppb treatment groups, respectively) as control animals. All treated and control animals had extensive senile degenerative changes 1n the kidneys. Complete necropsies were done and samples of tissues were taken for microscopic examination from the control groups and each treatment group. Special staining methods were used as an aid 1n the diagnosis of neoplasms. Various benign and malignant tumors were found In each treatment group. No tumors were observed 1n the controls (Table V-8). Statistically significant Increases of squamous cell tumors of the lungs and neoplastic nodules of the Hver were observed 1n rats Ingesting 5 ppb TCD0 (Tables V-9 and V-10). In addition, two animals 1n the 5 ppb dose group and one animal 1n the 1 ppb dose group had liver cholanglocardnomas, which are rare 1n Sprague-Dawley rats. These results provide evidence of a carcinogenic effect. The observation of no tumors of any kind In the controls 1s unusual for Sprague-Dawley rats. In addition, the reporting of the study was not exten sive. These factors may tend to lessen the reliance which can be placed on the positive results of this study. However, this study Is suggestive of a carcinogenic response upon exposure to TCDD In rats. 00110 V-73 09/18/84 I TABLE V-8 2,3,7,8-TCOD Intake and Mortality In Male Sprague-Dawley Ratsd Doseb (ppb) Weekly Dose/Rat (vig/kg bw) Week of First Death Number of Rats Dead at 95th Week 0.0 0.001 0.005 0.05 0.5 1 5 0.0003 0.001 0.01 0.1 0.4 2.0 68 6/10 (60%) 86 2/10 (20%) 33 4/10 (40%) 69 4/10 (40%) 17 5/10 (50%) 31 10/10 (100%) 31 10/10 (100%) aSource: Adpated from Van Miller et al., 1977a,b bRats at 50, 500 and 1000 ppb dose levels were all dead within 4 weeks. 00110 V-74 09/18/84 TABLE V-9 Benign and Malignant Tumors In Rats Ingesting 2,3,7,8-TC00a Doseb t Benign Malignant Number of Tumors Number of Rats With Tumors 0 1 ppt 5 ppt 50 ppt 500 ppt 0 0 1 2 2 0 0 5 1 2 0 0/10 (0%)c 0 0/10 (0%) 6d 5/10 (50%}e 3f 3/10 (30%) 49 4/10 (40%)^ 1 ppb 0 4 51 4/10 (40%) 5 ppb B 2 i o J 7/10 (70%) 3Source: Adapted from Van Miller et al., 1977a,b bRats at dose levels of 50, 500 and 1000 ppb were all dead within 4 weeks. C4G male rats used as controls for another study, received at the same time and kept under Identical conditions, did not have neoplasms when kl 1led at 18 months. ^1 rat had ear duct carcinoma and lymphocytic leukemia 1 adenocarcinoma (kidney) 1 malignant histiocytoma (retroperitoneal) 1 angiosarcoma (skin) 1 Leydlg cell adenoma (testis) 3 rats died with aplastic anemia ^1 fibrosarcoma (muscle) 1 squamous cell tumor (skin) 1 astrocytoma (brain) Si fibroma (striated muscle) 1 carcinoma (skin) 1 adenocarcinoma (kidney) 1 sclerosing seminoma (testis) ^1 rat had a severe Uver infarction ^1 rat cholanglocarclnoma and malignant histiocytomas (retroperitoneal) 1 angiosarcoma (skin) 1 glioblastoma (brain) 1 malignant histiocytoma (retroperitoneal) Jl rat had squamous cell tumor (lung) and neoplastic nodule (liver) 2 cholanglocardnomas and neoplastic nodules (Uver) 3 squamous cell tumors (lung) 1 neoplastic nodule 00110 V-75 09/18/84  TABLE V-10 Liver Tumors 1n Rats Ingesting 2,3,7,,8-TCODa Oose (ppb) Neoplastic Nodules Cholanglocardnomas Squamous Cell Tumors of the Lungs 0 0/10 (OX) 0/10 (OX) 1 0/10 (0%) 1/10 (10X) 5 4/10 (40X) 2/10 (2QX)b p=0.043 0/10 0/10 4/10 (40X) p=0.043 ^Source: Adapted from Van Miller et al., 1977a,b bThe two animals had both neoplastic nodules of the liver and cholanglocarclnomas. 00110 V-76 09/18/84 The second study employing oral administration to rats, performed by Kodba et al. (1978a,b), was more extensive. Groups of 100 Sprague-Oawley rats (50 males and 50 females) were exposed for 2 years to diets containing -22, 208 and 2193 ppt of 99% pure 2,3,7,8-TCDD. The dietary levels were Adjusted according to body weight 1n order to maintain 2,3,7,8-TCDO dose levels of 0.001, 0.01 or 0.1 yg/kg/day. The control group consisted of 172 rats (86 males and 86 females) maintained on unadulterated diets. Dur ing the study, the animals were palpated periodically for gross tumors and clinical chemistry analyses were performed on the blood and urine. Gross and histopathologic examinations of major organs were conducted In animals removed from the study before week 105, while extensive histopathologic examinations of a large number of organs and tissues were conducted on animals killed at the termination of the study. Gross signs of 2,3,7,8-TCDD toxicity Included significantly Increased cumulative mortality In the high dose female rats, and lower body weights In the high dose male and female and medium dose female rats (p values not given). Hematologic values Including packed cell volume and hemoglobin concentration were decreased (p<0.05) In both sexes at the 0.1 ug/kg/day dose, and urinary coproporphyrln and uroporphryln were Increased (p<0.05) 1n females receiving 0.01 and 0.1 yg/kg/day. Statistically significant Increases In tumors at several sites occurred In both male and female rats 1n the high dose group and liver lesions, Including hepatocellular neoplas tic nodules and lung lesions Including focal alveolar hyperplasia in mlddosed females. Tumors of the hard palate, tongue and adrenal cortex were Increased In high-dosed males; tumors of the liver, tongue and lung were increased 1n high-dosed females. The most sensitive target organ appeared 01330 V-77 09/18/84 to be the liver of female rats with hepatocellular carcinoma Incidences of 0/86, 0/50, 2/50 and 11/49 for the control, low, medium and high dose groups, respectively. There was no Increase in the hepatocellular carcinoma Incidence In male animals. Other tumors commonly observed In male and female rats were significantly <p<0.05) decreased In the treated animals (Table V-ll). There was no Increase In the neoplastic lesions In low-dosed males and females. In a study to determine the carcinogenic potential of the herbicide 2,4,5-trIchlorophenoxyethanol (2,4,5-TCPE), Toth et al. (1978, 1979) tested both this compound containing the known contaminant 2,3,7,8-TCOO and 2,3,7,8-TCDD alone by gavage 1n Sw1ss-H/R1op mice. Groups of 45 male mice were administered 2,3,7,8-TCDD dissolved 1n sunflower oil at dose levels of 0.007, 0.7 or 7.0 yg/kg bw once a week for 1 year. Other groups consist ing of 100 male and 100 female mice were administered 2,4,5-TCPE contami nated with measured amounts of 2,3,7,8-TCDD suspended In 0.5% carboxymethy1cellulose once a week for 1 year. Control groups administered each vehicle alone were Included 1n the study for comparison. Following the treatment period, the animals were observed until moribund or until spontaneous death. At autopsy, animals were examined for tumors, and organs were obtained for histopathologic examination. The only tumors that were observed to occur at a significantly greater Incidence in either the 2,3,7,8-TCDD or the 2,4,5-TCPE/2,3,7,8-TCDD treat ment groups compared with the appropriate vehicle control were tumors of the liver. These tumors consisted of both benign hepatomas and hepatocellular carcinomas. In the 2,3,7,8-TCDD treated mice, the Incidence of liver tumors 01330 V-78 09/18/84 01330 TABLE V-ll Tumors That Were Significantly Decreased In Rats Following Exposure to 2,3,7,8-TCDDa V-79 Sex Tumor Type 0.0 tig/kg/day Tumor Incidence 0.1 pg/kg/day 0.01 pg/kg/day 0.001 pg/kg/day M Acinar adenoma of the pancreas H Pheochromo'cytoma of the adrenal H Subcutaneous flbroadenoma/fIbroma/lipoma F Benign tumors of the uterus F Benign tumors of the mammary gland F Carcinoma of the mammary gland F Pituitary adenoma 14/85 28/85 10/85 28/86 73/86 8/86 43/86 2/50b 4/50b 6/50 7/49b 24/49b 0/49b 12/49b 5/50 10/50 5/50 11/50 36/50 4/50 13/50 7/50 6/50 l/50b 12/50 35/50 4/50 18/50 aSource: Koclba et al., 1978a bThe tumor Incidence was significantly (p<0.05) different from the Incidence In this control group. 09/18/84 was 7/38, 13/44, 21/44 and 13/43 for groups administered 0.0, 0.007, 0.7 and 7.0 yg/kg bw/week. The incidences of benign and malignant tumors were not enumerated separately. Only the 0.7 yg/kg/week group had a statistically significant (p<l%) increased Incidence of tumors as compared with the c o n trol animals. The high dose group had a shorter lifespan, 424 days as c o m pared with 588 days in the control group, and this may have affected tumor yield. The tumor incidence for mice receiving 2,4,5-TCPE containing 2 . 3 . 7 . 8 - TCDD is p r es e n te d in T a ble V-12. The auth o r s c o n c l u d e d that both 2,4,5-TCPE and 2,3,7,8-TCDD were carcinogenic since an increased incidence of liver tumors was o b se r v e d in groups r e ce i v in g 2,4,5-TCPE even though the 2 . 3 . 7 . 8 - TCDD level in one group was 0.007 yg/kg/day, w hich was shown to be nontumorigenic when 2,3,7,8-TCDD was administered alone. An exact quantita tive comparison between the groups receiving 2,3,7,8-TCDD and the groups r e ce i v i n g the m i x t u r e is diff i c ul t to assess as a result of the use of two vehicles (sunflower oil and carboxymethylcellulose) with drastically differ ent physical properties. Under the National T o xi c o lo g y Program, 2 , 3,7,8-TCDD has been tested for c a rc i n og e n ic i t y by oral a d mi n i s t r a t i o n in Osborne-Mendel rats and B6C3F^ mice (NTP, 1982a) and by dermal application to Swiss-Webster mice (NTP, 1982b). For both oral and dermal studies, the compound was custom synthe sized and shown by analysis using gas chromatography to be 99.0-99.4% pure. The two major impurities tentatively identified by gas chromatography were trichlorodibenzo-p-dioxin and PeCDD, with HxCDD detected at levels of 0.1-0.2% by gas chromatography mass spectrometry. The chemical was protect ed from light and kept at room temperature until used to make stock solution at 3-month intervals. In both studies, the approximate maximal tolerated dose was determined by a preliminary subchronic toxicity study. 01330 V-80 04/08/88 01330 TABLE V -- 12 Tumor Incidence In Nice Treated with 2,4,5-TCPE Contaminated with 2,3,7,8-TCDDa V-81 Group TCPEb (mg/kg) Treatment Effective TCDD Veh1clec Sex Number of (wg/kg| (mg/kg) Nice 1 67.0 2 70.0 3 0.112 (1.6 ppm) 0.007 (0.1 ppm) control 4 7.0 . 0.07 (10 ppm) 5 7.0 0.0007 (0.1 ppm) * G.7 0.00007 (0.1 ppm) 7_ 8 control 9 10 11 12 "" 7.0 0.7 0.007 -- 50 50 50 50 50 50 10 10 10 10 N F N F M f N F N F H F N F N F N .M N N 88 83 98 96 93 84 93 96 94 93 97 94 96 84 96 91 43 44 44 38 aSource: Toth et al., 1979 **TCPE Trtchlorophenoxy ethanol cCarboxymethyl cellulose In groups 1-8, sunflower oil In groups 9-12. dp<l* ep<0-lX Number of Tumor BearIng Nice 69 61 78 59 63 57 79 60 77 71 78 64 74 55 78 57 27 36 39 27 Liver (X) Number of Animals with Tumors of: Lung Lymphomas Other Organs Average Lifespan 42d (18) 7 (B) 57 (SB) 9 (9) 24 (26) 4 |5) 25 (27) 10 (10) 23 (24) 8 (9) 24 5 ((52)5) 32 4 ((53)3) 32 (33) 4 (4) 13 (30) 21 (48) 13 (29) 7 (18) 50 52 18 39 44 41 38 38 50 42 51 38 44 38 38 31 11 18 27 15 7 16 15 25 11 16 15 - 23 8 17 23 13 IB 22 19 19 23 17 36 21 20 17 22 21 14 22 18 17 22 15 24 19 67 12 4 10 6 67 595 652 571 5B2 577 639 641 589 660 590 643 S66 615 565 651 549 424 633 649 568 CD CD 09/1 In the oral study, (NTP, 1982a) groups of 50 male and 50 female rats received 2 , 3, 7 ,8-TCDD by gavage in corn o i l: a c et o n e (9:1) 2 d a ys/week for 104 weeks. The weekly dose of 2,3,7,8-TCDD was 0.01, 0.05 or 0.5 yg/kg bw. The male mice r e ce i v ed identical oral doses of test c o mp o u n d on the above schedule, while female mice received weekly doses of 0.04, 0.2 and 2.0 wg/kg bw for 104 weeks. In both species an additional 3-week observation period followed treatment. Control groups for both rats and mice consisted of 75 untreated animals and 75 vehicle-treated animals of each sex. At death or termination of the study, gross and histopathologic examinations were performed on all major organs and gross lesions. In this c hronic study the only overt sign of toxicity in rats was a s l ig h t ly lower body w e ig h t in the high dose m a le and female a nimals after 55 and 45 weeks of treatment, respectively. Survival among control and treated groups was not significantly different. Histologic examination revealed that toxic h epatitis was comm o n in the high dosed animals, with the liver developing lipidosis and hydropic degeneration of the hepatocytes, and pro liferation and fibrosis of the peripheral bile ducts. Histologic examina tion also revealed increased Incidences of neoplastic lesions in both liver and thyroid. The Incidence of hepatic neoplastic nodules or hepatocellular carcinomas was 0/74, 0/50, 0/50 and 3/50 (all neoplastic nodules) for males, and 5/75, 1/49, 3/50 and 15/49 for females in the control, low, m e d i u m and high dose groups, respectively. The incidence of hepatic neoplastic nodules in females was 5/75, 1/49, 3/50 and 12/49, respectively. In both males and females, the incidence of these combined lesions had a positive dose-related trend by the Cochran-Armitage test (p=0.005 and p=0.001 for males and females, respectively); however, the Incidence of these tumors was 01330 V-82 04/08/88 significantly (p=0.001} higher than controls only 1n female rats In the high dose group. The Incidence of hepatic neoplastic nodules In high-dose females was statistically significantly elevated (p=0.006) as compared to controls. In male rats, the Incidence of thyroid follicular cell adenomas or carcinomas was 1/69, 5/48, 8/50 and 11/50 In the control, low, medium and high dose groups, respectively, and these tumor Incidences showed a significant dose-related trend along with the Incidences In the two higher doses being significantly (p=0.021 and 0.001, respectively) Increased over controls. Incidence of other tumors, Including thyroid fol Hcular-cell adenomas, adrenal adenomas or carcinomas and subcutaneous adenomas 1n females, subcutaneous fibromas and cortical adenomas showed a dose-related trend or a significant Increase over controls at the low or medium dose level, but did not meet the requirements for overall significance of 0.5 using the Bonferronl inequality criteria. The only tumors considered by the NTP to be related to exposure to 2,3,7,8-TCOO were the thyroid tumors in male rats and the liver tumors 1n female rats. As observed In the rats, administration of 2,3,7,B-TCDD by gavage produced no overt signs of toxicity 1n mice with the only non-neoplast1c histopathologic effect being toxic hepatitis. Neoplastic lesions that demonstrated both a significant dose-related trend and a greater Incidence 1n the high dose animals Included hepatocellular adenomas or carcinomas in both male and female mice and thyroid follicular adenomas 1n female mice. The Incidence of liver tumors was 8/73, 9/49, 8/49 and 17/50 1n males and 1/73, 2/50, 2/48 and 6/47 In females, while the Incidence of thyroid tumors 1n females was 0/69, 3/50, 1/47 and 5/46 for the controls, low, medium and high dose groups, respectively. The Incidence of histiocytic lymphomas and 01330 V-83 09/18/84 s u bcutaneous fibromas in female mice and al v e o l a r / b r o n c h i o i a r adenomas or carcinomas of the lung in male mice were significant by either the Cochran- Armitage test or the Fisher exact test but not both, and did not meet the Bonferroni inequality criteria for overall significance. Under test c o n d i tions it was conc l u de d by the NTP (1982a) that 2 , 3 , 7 , 8 - T C D O was carci n og e n ic to both male and female B6C3F^ mice. . Cockerham et al. (1980) performed a field study on beach mice, Peromyscus polionotus. that inhabited an area which was heavily treated with the herbicide 2,4,5-T of which 2,3,7,8-TCDD was a contaminant. Analysis of the soil in the contaminated area revealed average 2,3,7,8-TCDO levels of 150 ppt at the surface. Me as u r ed levels of 2 , 3,7,8-TCDD in the liver of beach mice from the c o nt a m in a t ed area was determined to be 1300 ppt in males and 960 ppt in females. D e tec t ion of 2 , 3,7,8-TCDD in the liver Indicates that the compound was absorbed; however, since seeds in the area did not contain 2 , 3 , 7 , 8 - T C D D it was b e li e v e d that the an im a l s Ingested the c o mp o u n d from contaminated dust while grooming. In the 10 male and 5 female animals c a p tured in the c o n t a m i n a t e d area, there w e re no h i s t o p a t h o l o g i c d i fferences, Including neoplastic lesions, observed In the liver as compared with 9 male and 6 female beach mice captured In a noncontaminated area. The only obse r v ed d i ff e r en c e in the two groups of mice was a s t a t i s t i c a l l y s i g n i f i cant (95% confidence) Increase In liver to body weight ratios. The authors back-calculated from the 2,3,7,8-TCDD levels of the U v e r and estimated a daily 2,3,7,8-TCDD dose of 0.0012 pg/kg bw. It was noted that this e x p o sure was much lower than the exposure used In laboratory studies to produce tumors. 01330 V-84 04/08/88 2,3,7,8-TCDD (NTP, 1982b) has been tested in mice for tumorigenic poten tial by dermal a p plication. This study was c o nd u c te d under the NTP and the d e s c r i p t i o n of the chemicals used was the same as p r e v i o u s l y p r es e n t e d in the discussion of NTP (1982a). Groups of 30 male and 30 female Swiss-Webster mice were treated with 100 yi of a solu t i on of the test comp o u nd in a c e t o n e 3 t i mes/week for 104 weeks. Groups of 45 animals were employed as vehicle controls and 2 groups of 15 animals were used as untreated controls. The concentration of 2 , 3 , 7 , 8 - T C D D used r e su l t ed in a dose of 0.01 y g / a p p l l c a t l o n in male and 0.005 y g / a p p l i c a t i o n in female mice. Subchronic toxicity studies used to define the dose levels for the chronic bioassay Indicated that all the doses used resu l t ed in some liver dama g e but no Increase in m o rt a l it y . In the chronic study, animals were killed when moribund at the termination of the study and examined for gross tumors. Microscopic examinations of all major organs were also made. In mice exposed dermally to 2,3,7,8-TCDD (NTP, 1982b), there was no t r e a t m e n t - r e l a t e d d i f f e r e n c e in body weig h t of either sex b e t w e e n exposed animals and control groups; however, male mice treated with 2,3,7,8-TCDD had a s i gn i f i c a n t s h o r t e n i n g of life s p an (l.e., 10/30, 7/45 and 3/30 survived to the end of the study In the untreated control, vehicle control and 2,3,7,8TCDD-treated groups, respectively). NontumorIgenlc hepatic lesions were observed 1n treated female mice, but not In treated male mice. The only tumors that were treatment related were Integumentary system fibrosarcomas, with tumors developing on or near the site of application. The Incidence of 01330 V -85 04/08/88 these tumors in male mice was 3/42 and 6/28, and 1n female mice the inci dences were 2/41 and 8/27, respectively, for the vehicle control groups and the treated animals. Only the tumor incidence 1n female mice was statisti cally (p=0.Q07) greater than control values; however, life table analyses Indicated that the time to tumor was shorter 1n both the male and female treated mice. The Incidence of tumors In untreated and vehicle control groups was slmllar. Using the mouse skin two-stage tumorlgenesis model, 2,3,7,8-TCOO has been tested for Initatlng and promoting activity. In this model, an Initia tor 1s a chemical that 1s applied to the skin for a very limited time undera dose schedule which will not result In tumor formation during the-course of the study. Treatment with the Initiator, however, Is considered to have caused some Irreversible change 1n the treated cells since subsequent repeated exposures to a promoting agent (a compound that does not produce tumors at the concentration tested after chronic exposure) results 1n the development of tumors. The results of these studies and similar studies assessing promotion of hepatocarclnogenesls are summarized In Table V-13. Also, in additional Investigations, the effect of previous or simultaneous exposure to 2,3,7,8-TCDD on the tumor yield of known chemical carcinogens has been studied. OIGIovannl et al. (1977) tested 2,3,7,8-TCDD, which was 98.656 pure, for Initatlng activity on the skin of female CD-I mice. Groups of 30 animals received an Initial single dermal dose of 2,3,7,8-TCOO of 2 yg/mouse followed by twice weekly promotion with 5 yg of the known promoter 12-otetra-decanocyl-phorbol-13-acetate (TPA) for 32 weeks. The level of 01330 V-86 09/ 18/84 TABLF V 13 Assessment of th Inltlatlon and Promotion Activity of 2,3,7,,8-TC0D In Laboratory An Ima 1s o ccooo Number of Specles/Straln Sex An Ima Is/Group initlator/Dose Promoter/Dose Tumor Type % or IncIdence of Animals With Tumors Reference Nlce/CD-1 f 30 2.3.7,8-TCDD/ TPA/5 pg/applIcatlon, dermal papilloma 14 2 |ig/mouse 2 ttmes/week for 32 weeks F 30 DNBA/ TPA/5 pg/applIcatIon, dermal papilloma 63 2.56 pg/mouse 2 tlmes/week for 32 weeks DIGIovannl et al.. 1977 Nice/ H 5 none Swlss-Uebster none dermal papilloma 3/42 NTP, 1982b N 30 none 2.3,7.8-TCDD/0.001 Mg/ application, 3 tlmes/week for 104 weeks dermal papilloma 6/28 N 30 DNBA/50 pg 2,3,7,8-TCDD/O.OOl pg/ dermal papilloma 5/30 (single application. 3 tlmes/week appiIcatlon) for 104 weeks I OD Nice/ F 45 none Swlss-Webster none dermal papilloma 2/41 F 30 none 2,3,7,8-TCDD/O.005 Mg/ application, 3 tlmes/week for 104 weeks dermal papilloma 8/27 F 30 DNBA/50 pg 2.3.7.8-TCDD/0.005 pg/ dermal papilloma 8/29 (single application, 3 tlmes/week appiIcatlon) for 104 weeks Hlce/CD-1 F 30 none 2.3.7,8-TCD0/0.1 Mg/ application, 2 tlmes/week for 30 weeks dermal papilloma o.ox Berry et al., 1978, 1979 F 30 DNBA/200 nmol 2.3.7.8-TCDD/0.1 pg/ dermal papilloma o.ox (single application, 2 tlmes/week appiIcatlon) for 30 weeks F 30 DNBA/200 nmol TPA/2 pg/applIcatIon, dermal papilloma 92% o (single 2 tlmes/week for 30 weeks oc\o appiIcatlon) co co TABLE -13 (cont.) 01330 V-88 Number of Spectes/Stratn Sex Anlmals/Group Initiator/Dose Proooter/Dose Mlce/HRS/J f 20 DHBA/0.2 tdBol none (hr/*) (single application) f 20 none 2,3,7,8-TCOO/btweekly application of SO ng/ application for 0 weeks followed by 20 ng/appltcatlon for U weeks F 20 DMBA/0.2 nmol 2,3,7,8-TCDD/blweekly (single application of SO ng/ application) application for B weeks followed by 20 ng/ application for 17 weeks. r 20 DHBA/0.2 nmol TPA/blweekly application (single of 2 nQ/mouse application) Mlce/HRS/J F 20 DMBA/0.2 nmol none (hr/hr) (single application) F 20 none 2,3,7,8-TCDD/blweekly application of SO ng/ application for 8 weeks .. followed by 20 ng/appltcatlon for 17 weeks F 20 DMBA/0.2 nmol 2,3,7,8-TCOD/blweekly (single application of SO ng/ application application for 8 weeks followed by 20 ng/appll- catlon for 17 weeks F 20 DMBA/0.2 nmol TPA/b1weekly application (single of 2 n9 /mouse application) Rats/ Charles River F 4 DFN/10 mg/kg none F 4 none 2.3,7,8-TCDD/O.14 Mg/kg twice a week for 28 weeks* Tumor Type * or Ipcldence of Animals With Tumors Reference dermal papilloma 0/20 Poland et at.. 1982 dermal papilloma 0/20 - dermal papilloma 0/20 dermal papilloma dermal papilloma dermal papilloma 20/20 1/20 0/20 dermal papilloma 15/19 dermal papilloma hepatocellular carcinoma hepatocellular carcinoma -70* 0/4 0/4 Pitot et al., 1980 09/18/84 TABLE V-13 (cont.) 088 LO Number of Specles/Stratn Sex An Ima Is/Group Intttator/Dose Promoter/Oose Tumor Type Rats/ Charles Rtver F F F 5 none 2,3,7,8-TCDD/l.4 ,,g/kg twice a week for 28 weeks* S DEN/10 mg/kg 2.3.7.8-TC0D/0.14 wg/kg twice a week for 28 weeks* 7 DEN/10 mg/kg 2.3.7.B-TC0D/1.4 wg/kg twice a week for 28 weeks* *The 2,3,7,8-TCDD was administered by subcutaneous Injection. hepatocellular carcinoma hepatocellular carcinoma hepatocellular carcinoma % or Incidence of Animals Ulth Tumors Reference 0/5 Pitot et al., 19B0 0/5 5/7 68-A t8/81/60 2.3.7.8- lCDD exposure was chosen from the E D ^ for induction of ary] h y dr o c a r b o n h y droxylase activity, and this level resulted in the death of 1/3 of the. a nimals before the end of the study. In the group initiated with 2.3.7.8- TCDD and promoted with TPA, there was a skin papilloma incidence of 14% with an average of 0.1 papi 1l o m a s / m o u s e . This was in comparison with animals initiated with d i m e t h y l b e n z a t h r a c e n e {D M B A ), a pote n t tumor initiator, and promoted with TPA where the papilloma incidence was 63% with 2.2 papillomas/ mouse. The authors concluded that 2,3,7,8-TCDD was a weak tumor initiator; however, no vehicle control groups or TPA-treated-only control groups were Included in the study for comparison. The tumor promoting activity of 2,3,7,8-TCDO was investigated, along with the c o mplete c a rc i n o g e n i c i t y in the NTP (1982b) bloassay. In the tumor promotion study, groups of 30 mice of each sex were Initiated by a single dermal application of 50 yg of the tumor initiator (DMBA), A week after the initiation dose, the male mice received applications of 0.001 yg 3 times weekly and the female received 0.005 yg of 2,3,7,8-TCDD for 104 weeks. The skin tumor incidence in both male and female m ice exposed to DMBA prior to 2,3,7,8-TCDD application or the same level of 2,3,7,8-TCDD alone w e re near l y Identical with r e s p e c t i v e tumor inci d e nc e in males of 5/30 and 6/28, and incidences in females of 8/29 and 8/27. This NTP (1982b) dermal b l oa s s a y found no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e in the incidence of tumors between the groups of animals treated with 2,3,7,8-TCDD alone and those treated with' DMBA prior to 2,3,7,8-TCDD application. Furthermore, the i n cidence of h e m a n g i o s a r c o m a was higher in male mice in 2 . 3 . 7 . 8- TCDD treated group than in the animals treated with DMBA prior to 2.3.7.8-TCDD. 30 female CD-I mice were initiated with a single 200 nmol application of DMBA followed by twice weekly applications of 0.1 yg of 01330 V-90 04/12/88 2 . 3 . 7 . 8 - TCDD for 30 weeks In a bioassay by Berry et al. (1978, 1979 ). At. the t e rm ination of promotion, there were no dermal p a pillomas in either the DMBA plus 2 , 3 , 7 , 8-TCDO group or the 2,3,7,8-TCDD group. In an additional group of mice initiated with DMBA followed by promotion twice a week with 2 yg of the known promoter (TPA) there was, however, a 92% incidence of skin papillomas. The average number of tumors/mouse was 8.1. In the m ous e skin two-stage tumorigenesis model, 2,3,7,8-TCDD did not demonstrate tumor promoting activity. Slaga and Nesnow (1985) observed that 2,3,7,8-TCDD either had no p r o m o t i n g a c t i v i t y or very weak p r om o t i n g a c ti v i t y in Senear mice skin. Poland et al. (1982) described studies which indicate that genetic d i f f e r en c e s In mice affect the tumor p r o m o t i n g c a p a c i t y of 2 , 3 , 7 , 8 - T C D D in the skin two-stage tumorlgenesls model. Both 2,3,7,8-TCDD and TPA were compared for tumor prom o t in g a c tivity in D B A-initiated HRS/J mice that were either heterozygous (hr/+) or homozygous (hr/hr) for the recessive "hair less" trait. Promotion with biweekly applications of 2 ug of TPA for 25 weeks resulted In p a pil l oma incidences of 100 and 70% 1n (h r /+} and (hr/hr) mice, respectively. Promotion of 0M B A - 1n 11 1a ted (hr/f) mice with 2,3,7,8TCDD (50 ng/application for 8 weeks followed by 20 ng/applIcation) did not result in the f ormation of tumors, while promotion of (hr/hr) mice resulted in both the same I n ci d e nc e and m u l t i p l i c i t y of tumors as o b se r v e d in TPAp r om o t e d mice. W i t h either DMBA or N - n i t r o s o g u a n i d i n e ( M N N G ) - i n i t 1a ted (hr/hr) mice, the effe c t iv e doser of 2,3, 7 , 8- T C DD was ~100 - f ol d less than TPA on molar basis. Histologic examination of the skin showed that TPA produced both acute inflammation and hyperplasia In {h r /+) and (hr/hr) mice, while 2.3. 7 . 8- TCDD produced hyperplasia and hyperkeratosis only In (hr/hr) mice 01330 V-91 04/ 12/88 with no inflammatory response. The lack of a 2 , 3 , 7 ,8-TCDD-induced inflamma tory response suggested to the authors that 2 , 3 , 7 ,8-TCDD-promoted skin p a pi l l om a s in (hr/hr) m i ce by a m e c h a n i s m d i ff e r e n t from TPA. Pitot et al. (1980) investigated the ability of 2,3,7,8-TCDD to act as a promoter for the hepatocarcinogen diethylnitrosamine (DEN). Female C h a r l e s River rats were given DEN at a single intragastric dose of 10 mg/kg during the time of liver regeneration after a 70% partial hepatectomy. Following this Initiation with DEN, rats received twice weekly subcutaneous injections of either 0.14 or 1.4 yg of 2 , 3 , 7 ,8-TCDD/kg bu for 28 weeks. Additional groups of partially hepatectomized animals received only the initiation t reatment or the p r o m o t i o n treatment. No tumors w e re o b s e r v e d in the rats given DEN or 2,3,7,8-TCDD alone, or in the animals exposed to DEN plus the low dose of 2,3,7,8-TCDD. In the rats exposed to DEN plus the high dose of 2.3.7.8- TCDD, however, the hepatocellular carcinoma incidence was 5/7. Similar high incidences, 8/10, of h e p a t o c e l l u l a r c a r c i n o m a s were o b s e r v e d in rats exposed to DEN followed by dietary a d m i n i st r a ti o n of p h e n o b a r b l t a l , a promoter of liver carcinogenesis. Foci of cells with altered enzyme p a t terns indicative of preneoplastlc lesions were identified in rats initiated with DEN and promoted with either dose level of 2,3,7,8-TCDD or phenobarbitol. A l t h o u g h this study is limi t e d by the small size oF the exper i me n t al groups, the authors concluded that 2,3,7,8-TCDD was a promoter for DENinitiated hepatocarcinogenes 1s in rats. Abernathy et al. (1985) investigated the promotion effects of 2 .3.7.8- TCDD in vitro using C3H/10T1/2 cells initiated with N-methylN 1- n i t r o - N - n i t r o s o g u a n i d i n e . Maximal e n h a n c e m e n t of focus f ormation 01330 V-92 04/12/88 occurred at 40 pM 2,3,7,8-TCOQ, a concentration 10,000-fold lower than the optimal concentration of 12-o-tetradecanoylphorbol-13-acetate. Inve s t ig a t io n s have a l so been c o nd u c te d on the effects of prior or simultaneous treatment with 2,3, 7 , 8- T C DD on the subsequent development of skin tumors by chemical carcinogens. When 2,3,7,8-TCDD (0.1 y g ) was administered simultaneously with DMBA (200 nmol) to the backs of CD-I mice in a single i n itiation dose, the skin papi l l om a incidence foll o w in g p r o m o tion with TPA was nearly the same as when DMBA alone was used as the initia tor (DIGiovanni et al., 1977). Although simultaneous exposure to 2,3,7,8TCDD and DMBA did not appreciably affect tumor yield, Berry et a l . (1979) d e monstrated a marked 9 3 % decrease in the incidence of DMBA initiated tumors when CD-I mice were pretreated 3days before DMBA Initiation with 1 yg/ m ouse of 2 , 3, 7 ,8-TCDD. The time of treatment with 2 , 3 , 7 , 8 - T C D D in rela t i on to initiation was shown to be critical in the anti t u mo r i ge n i c effect of 2,3,7,8-TCDD (Berry et al., 1979; Diglovanni et al., 1979b, 1980). Maximum tumor inhibition of between 86 and 95% occurred when pretreatment was between 1 and 5 days before initiation. If pretreatment was 10 days before DMBA initiation, the tumor yield was decreased by 78%, while 2,3,7,8-TCDD treatment 5 minutes before or 1 day after DMBA initiation had no effect on tumor yield. There was some indication of an inverse relationship between the pretreatment (3 days before DMBA initiation) dose of 2,3,7,8-TCDD and the incidence of tumors. Doses of 2,3,7,8-TCDD of 0.0, 0.01, 0.1 and 2 yg/mouse resulted in d e creases in tumor yields, respectively, of 0, 83, 92 and 96% (DIGiovanni et al., 1979b). Also under similar experimental condi tions Cohen et al. (1979) observed a 75% decrease in the incidence of skin tumors in Senear mice p r etr e at e d with 1 yg of 2,3,7 , 8- T C DD 3 days before initiation by DMBA. Kouri et al. (1978) reported that i.p. injection of 01330 V-93 04/12/88 1-100 mg 2 , 3 , 7 , 8 - TC D D / k g p r ec e d ed by MCA but not t r io ctanoin raised the c a r c i n o g e n i c index in B6 but not D2 mice. The term " c ar c i no g e ni c index" was defined by Kouri et al. (1978) as percentage of tumor incidence 8 months after treatment divided by the average latency in days mult i p li e d by 100. DiGiovanni et al. (1980) investigated the anti tumorigenic effect of 2 . 3 . 7 . 8 - TCDD in CD-I mice w i th chemical c a rc i n o g e n s other than DMBA. As observed with DMBA, exposure to 2,3,7,8-TCDD 3 days before initiation with either b e n z o ( a ) p y r e n e (BaP) or 3-MC resulted in a d e cr e a se in tumor yield as c o mp a r e d with a c et o n e p r e t r e a t e d animals, w h i l e 'p r e t r e a t m e n t with 2 , 3, 7 , 8 TCDD 5 m i n u t e s before or 1 day after i n it i ations was i n ef f ective in c h anging the tumor yield. The m a x i m u m d e c r e a s e in tumor p r o d u c t i o n was 86 and 57%, respectively, for BaP and 3-MC initiated mice. A different temporal r ela tionship was observed in the ability of 2,3,7,8-TCDD to inhibit tumor f o r m a tion by B a P - d 1o 1 -epoxide as compared with the previously studied p o l y a r o m a tic hydrocarbons (PAH). When 2,3,7,8-TCDD was applied 3 days or 5 minutes before, or 1 day after initiation with B a P - d i o l - e p o x i d e , decreases in tumor yield w e r e 81.5, 49 and 39%, r e sp e ctively. E x a m i n a t i o n of PAH m e t a b o l i s m in the skin of mice treated with 2,3,7 , 8- T C DD showed a 21-fold Increase in aryl hydrocarbon hydroxylase (AHH) activity 72 hours after treatment (DiGiovanni et al., 1980). The jn. v i tro m e t a b o l i s m of DMBA by dermal h o mo g e n a t e s from 2.3.7.8- TCDD-treated mice indicated both qualitative and quantitative changes in m e t a b o l i s m (i.e., changes in both rates of m e t a b o l i s m and r e l a tive types of metabolites formed) (Cohen et al., 1979; DiGiovanni et al., 1979b; Berry et al., 1979). The s i milarity in the time frame of AHH i n d u c tion and the antitumorigenic effect of pretreatment with 2,3,7,8-TCDD sug gested that the antitumorigenic properties of 2,3,7,8-TCDD resulted from 01330 V-94 04/08/88 2,3,7,8-1COD- induced alteration in the m e t a b o l i s m of the initiating c h e m i cal. Although metabolic change was a possible m e c h a n is m for the inhibition of D M B A , 3-MC and BaP initiation, the ability of 2,3,7 , 8- T C DD to inhibit tumor yield when administered 1 day after initiation with BaP-diol-epoxide (which does not require metabolic activation) Indicated to DiGiovanni et al. (1880) that more than one m e c h a n i s m may participate in the anti c a rc i n og e n ic effect of 2,3,7,8-TCDD. M u t a g e n i c i t y . S h o r t - t e r m Ui vitro test systems have been developed to assess the biologic, toxic and genotoxic effects of chemicals. These assays have proven to be useful indicators of potential activity of diverse indus trial chemicals, a broad range of drugs and xenobiotics, carcinogens and crude environmental extracts. The most widely used short-term test system, the Ames test for bacterial mutagenesis, employs several strains of Salmo nella typhimur1um that are highly susceptible to the effects of mutagenic chemicals. Despite the obvious utility of the Ames test and related short term assays, their predictive capabilities (i.e., the correlation between bacterial mutagenicity and carcinogenicity) have not been fully assessed (Bartsch et a l ., 1982). M u ta g e ni c i ty assays in m i cr o o r g a n i s m s have been used to assess the g e n o toxic effects of 2,3,7,8-TCDD; however, the results of most of these assays have indicated little potential for mutagenic effects (Table V-14). Huss a i n et al. (1972) expo s e d S. t y p h i m u r 1urn h i s t i d i n e - d e p e n d e n t strains T A 1 5 3 0 and T A 1 5 3 2 in liquid s u sp e n si o n to 2 , 3 , 7 , 8 - T C D D f o ll o w ed by plating into s e le c t iv e m e d i u m to o b s e r v e r e ve r s io n to prototypes. No increase in the reversion rate was observed with strain TA1530 at exposure levels of 1 01330 V-95 04/08/88 01330 TABLE V-14 The Results of Mutagenicity Assays for 2,3,7,8-TCDO In Salmonella typhlmurlum V-96 Type of Assay Strains of Salmonella typhlmurlum S-9 TA98 TA1530 TA1535 TA1537 TA1S38 TA1532 TA1950 TA1975 TA1978 G4G TA100 TA1S31 TA1534 Reference Spot test /- NT NT 00 0 0 NT NT NT NT NT NT NT McCann, 1978 Plate */- NT NT 00 0 0 NT NT NT NT NT NT NT McCann, 1978 incorporation Plate Incorporation* 00 0 0 0 0 0 0 0 0 0 NT NT Gilbert et al., 1980 Fluctuation test 00 0 0 0 0 0 0 0 0 0 NT NT 61 lbert et al.. 1980 Spot test - NT 0 NT NT NT NT NT NT 0 NT QR QR Seller, 1973 Plate 0 NT 00 0 NT NT NT NT NT 0 NT NT Geiger and Neal, 1981 Incorporation Plate - NT NT NT 0 Incorporation NT NT NT NT NT NT NT NT NT Geiger and Neal, 1981 Suspension assay - NT 0 NT NT NT NT NT NT NT NT NT NT Hussain et al., 1972 Suspension assay /- 0 NT 00 NT ,, NT NT NT NT NT 0 NT NT Zelger, 1983 `The assay was performed under both aerobic and anaerobic conditions. NT - Not tested; QR - Questionable response; 0 - Negative response; - Positive response 09/18/84 and 10 yg/mi. These exposures resulted in cell survivals of 90 and <1%, respectively, In strain TA1532, increased reversion frequency was not obse r v ed at 2 , 3 , 7 , 8- T C DD c o n c e n t r a t i o n s of 2-3 yg/mi, w h ich resulted in a 0 - 5 0 % d e crease in survival; however, at 2 , 3 , 7 , 8 - T C D D levels that resulted in a 99% d e cr e a se in survival, there was an increased number of revertant colonies/surviving cells. The dose levels were not specified. The source of the 2 , 3 , 7 , 8 - T C D D sample s tudied in this paper was the Food and Drug Administration, and Its reported purity was 99%. Also, Seiler (1973) observed a positive mutagenic response in a spot test of 2,3,7,8-TCDD p e r formed in the absence of a metabolic acti v a ti o n system. However, the purity of the sample studied was not provided. In tester strains 046 and TA1530, the ratio of r e v e r t a n t s / 1 0 8 cells in the treated plates di vi d e d by s p o n taneous revertants/100 cells was <1. In strains TA1531 and TA1534, the ratio was b etween 1 and 2, w h i c h was c o n s i d e r e d a "doubtful" m u ta g e n i c response, while in strain TA1532, the ratio was >10. There was no mention of the 2,3,7,8-TCDD levels tested in this assay. The positive controls, d i ethylsulfate, 2-ami nopu r i ne and 2- am i n of l u or e n e, p roduced ratios of 2 to 5, <1 and 5 to TO, resp e c ti v e ly , in strain TA1532. In both the study by Hussain et al. (1972) and the study by Seiler (1973), 2,3,7,8-TCDD produced a p o si t i v e m u ta g e n i c resp o n se only in the S. t y p h i m u r l u m strain T A T 532, w h ic h is s e ns i t iv e to f r ameshift mutagens. Hussain et al. (1972) also performed a mutagenicity test of 2,3,7,8-TCDD in two other m i cr o b ia l test systems. A p o si t i v e resp o n se was o b se r v e d in Escherichia coli Sd-4 as indicated by a reversion to streptomycin Independ ence. In this assay, cells w e r e treated in s u sp e n si o n for 1 hour with 2,3,7,8-TCDD at 0.5-4 yg/mi. The greatest mutation frequency (256 mutants x 10_ e , as compared with the control frequency of 2.2 mutants x 01330 V-97 04/08/88 TO"8} occurred at a dose level of 2 yg/ml. The absolute number of c o lo n i e s / p l a t e was 7 for the control and 4-6 for the treated plate. The dose of 2 ng/mi caused an 89% decrease in cell survival. In the second test system, the ab il i t y of 2 , 3 , 7 , 8 - T C D D to increase p r op h a ge Induction in E_. coli K -39 cells was examined. The vehicle control, DMSO, inhibited prophage induction as comp a r ed with the untreated controls, w h ile the most effective dose level of 2 , 3 , 7 , 8 - T C D D (0.5 ^g/ms.) r e sulted in an increased p r op h a ge induction as compared with the vehicle control but not as compared with the untreated controls. Hussain et al. (1972) concluded that 2,3,7,8-TCDD was capa b l e of c a u s i n g increases in the reverse m u ta t i o n rate in coll Sd-4 and that 2,3,7,8-TCDD had a weak ability to induce prophage in E. coli K -39 cells. . The studies that followed these two early reports of Hussain et al. (1972) and Seiler (1973) failed to detect mutagenic activity of 2,3,7,8-TCDD in S. typhimuriurn. W a s s o m et al. (1978) cited a personal c o m m u n i c a t i o n from McCann (1978), which reported that 2 , 3,7,8-TCDD was Inactive in both the spot test and p late i n co r p o r a t i o n a ssay w i th S. t y p h i m u r i u m strains 1 Al 532 , TA1535, TAl537 and TA1538. Doses and other experimental protocols were not mentioned except that the tests were performed both with and without metabo lic activation. Gilbert et al. (1980) reported that 2,3,7,8-TCDD gave "substantially negative results" with S. t y p h i m u r 1um strains TA98, TA100, TAl530, TAT 535, TA1537 , TA1538, G46, TA1532, TA1950, TA1975 and TA1978. Both the standard plate incorporation assay and the bacterial fluctuation test were used, and both were performed with and without S-9 prepared from the livers of Aroclor 1254 pretreated rats. In the plate Incorporation assay, the test compound was tested at 1-2000 pg/plate under both aerobic and anaerobic conditions. Details were not provided for the fluctuation 01330 V-98 04/08/88 assay. It is difficult to assess p o ss i b le reasons for the c o n f l i ct i n g results between the earlier studies and these later mutagenicity assays, since information on experi me n t al cond i t io n s was limited in the n e gative studies. . In an attempt to resolve the conflicting results and observe a mutagenic response, Geiger and Neal (1981) tested 2 , 3 , 7 , 8 - T C D D in the standard plate incorporation assay using S-9 prepared from different sources. In order to maximize the amount of compound tested, dloxane, a better solvent for 2.3.7.8- TCDD than the commonly employed DMSO, was used. Even with the use of dioxane, the limited solubility of 2,3,7,8-TCDD allowed only 20 yg/ plate to be tested, a dose that was shown to be non-toxic to the cells. The S-9 used in these assays was prep a r ed from the livers of Aroclor 1254 p r e treated male Sprague-Dawley rats and male Golden Syrian hamsters, and from 2.3.7.8- TCDD induced ma le hamsters. In all assays at 2,3,7,8-TCDD c o n c e n trations of 0.2, 2, 5 or 20 y g/plate, and rega r d le s s of the source of the S-9, there was no observed m u ta g e ni c response. In further attempts to duplicate the previous positive results, Geiger and Neal (1981) tested the same concentrations of 2,3,7,8-TCDD in strain TA1537, a more sensitive dire c t d e s c e n d e n t of strain TA1532, for m u t a g e n i c a c t i v i t y In the a b s e n c e of S-9. Again, no increase in the number of revertants was observed. In assays either with or without S-9, positive controls had predictable Increases in the number of revertant colonies. The authors concluded that 2.3.7.8- TCDD was not active under the conditions of this assay; however, testing at higher concentrations may elicit a positive response. It was also noted that many other polychlorinated aromatic compounds are not muta genic in the Ames test, even though there is p o s i t i v e e v id e n c e of carcinogenicity. 01330 V-99 04/08/88 M u ta g e ni c effects of 2 , 3 , 7 , 8 - l C D D in yeast were observed by Bronzetti et al. (1983). Positive results for reversion and gene conversion were o b ta i n ed j_n vitro and in the h o s t - m e d i a t e d assay. The _i_n v itro e x pe r iments yielded small d o s e - r e l a t e d increases in t r p f c o n v e r ta n t s and ilv*" revertants. An S I 0 metabolic activation system was required. Exposure of the yeast to 2 , 3 , 7 , 8 - T C D D at the highest level tested (10 yg/mi.) r e s u l t ed in 16% survival and yiel d e d 4-fold Increases in r eversion and gene conversion. In the host-med i a te d assay, male mice were exposed to 25 yg of 2 , 3 , 7 ,8-TCDD/kg (Bronzetti et al., 1983). After 5, 10, 20 or 30 days, 0.2 ma, of a yeast c u l t u r e (4 x 1 0 s cells) was instilled r e troorbital 1y . Four hours later, the liver and kidneys were removed and the yeast cells in these organs were assayed for mutagenic responses. Increases (4- to 6-fold) in reversion and gene c o nv e r si o n were o b served in yeast cells obtained from the livers and kidneys. The toxic r e sp o n se of the animals to an e x po s u re of 25 yg/kg was not descr i bed in this report. The p o sitive results described in this paper suggest that 2 , 3 , 7 , 8 - T C D D Is m u t a g e n i c in yeast, but more definitive studies are needed before a firm conclusion can be drawn. Hay (1982) has found that 2 , 3 , 7 , 8 - T C D D d i s s o l v e d in DMS0 tran s f or m e d baby hamster kidney cells (BHK) in v i t r o . The dioxin isomers 2,8-dichloroand 1 ,3,7-trichlorod1benzo--d1oxin also transformed BHK cells, but the response was weak. The unchlorinated dibenzo--d1oxin and the fully chlori n a te d o c t a c h l o r o d i b e n z o - E - d l o x i n w e re both n e ga t i v e in the BHK a s sa y (l.e., there was no cell transformation). More recently, Rogers et al. (1982) 01330 V-100 04/08/88 reported that 2,3,7,8-TCDD induced mutations in the excess thymidine, thioguanine and methotrexate selective systems in L5178Y mouse lymphoma cells in culture. The National Toxicology Program (NTP) {Zeiger, 1983) provided data on 2 , 3 , 7 , 8 - T C D D from four a ssay systems: the S. t y p h i m u r i u m (strains TA98, TA100, TA1535 and TA1537) histidine reversion assay, the sex-linked reces sive lethal test in D r o s o p h i l a . and c y togenetic studies (sister chromatid e x change and c h r o m o s o m e a b e r r a t i o n s ) in C hinese hamster o vary cells. N e g a tive results were obtained in all of these assays. These studies cannot be evaluated, however, because the procedures used to obtain the data were not described. In vitro reactions of 2,3,7,8-TCDD with bacteriophage QB RNA were e v al uated by Kondorosi et al. (1973). Active RNA was purified from QB phage followed by incubation for 1 hour at 37C with 0.0, 0.2, 2.0 or 4.0 yg/ma. of 2 ,3,7,8-TCDD. At all c o ncentrations tested, 2 , 3,7,8-TCDD had no effect on the transfectivity of QB RNA. Other compounds tested Included the .alkylating agents methyl, ethyl and isopropyl methane-sulfonate, and diethyl pyrocarbonate, all of which inactivated QB RNA under the same exper imental conditions. The a uthors sugg e s te d that 2 , 3 , 7 , 8 - T C D D inactivity in this assay indicated that 2,3,7,8-TCDD was an intercalating agent, and hence w o uld require d o ub l e s t ra n d ed DNA in order to interact. The data p r es e n t e d in this study, however, were insufficient to support this conjecture. In vivo binding of radiolabeled 2,3,7,8-TCDD to liver macromolecules was s tudied in S p r a g u e - D a w l e y rats by Poland and Glover (1979). Both m a le and female animals were administered [ 1 ,6-3H]2,3,7,8-TCDD i.p. at a dose of 01330 V-101 04/08/88 7.5 pg/kg. This dose corresponded to a tritium level of 0.87 mCi/kg. 1he animals were killed 12, 48 and 168 hours after treatment, or 24 hours after treatment when the animals were pretreated with the enzyme inducers phno barbital or unlabeled 2 , 3 , 7 ,8-TCDD. Following sacrifice, isolation of macromolecules, and removal of free labeled 2 , 3 , 7 ,8-TCDD, the amount of label bound to protein, RNA and DNA was determined. The greatest n o n extractable binding of labeled 2,3,7,8-TCDD occurred to protein; however, the amount of label bound was small and only amounted to 0 . 0 3 - 0 .1 % of the total radioactivity administered. The total amount of label associated with RNA and DNA was, respectively, only 50 and 4 cpm above background. Time after exposure, sex or prior enzyme induction had no significant effect on 2.3.7.8- TCDD binding. As a result of the extremely low levels of radio a c t i v i t y a s s o c i a t e d with RNA and DNA, it is uncer t ai n whether 2 , 3 , 7 , 8- T C DD truly binds c o v a l e n t l y to these m a c r o m o l e c u l e s and, if so, whether there is any biological significance to this low level of apparent binding. The effects of 2,3,7,8-TCDD exposure on the extent of chromosomal a b er rations in the bone m a r r o w of male rats were r e po r t ed in an abstract by Green and M o r e l a n d (1975). In the initial e x pe r iment, no increase in chromosomal aberration was observed after five dally gavage treatments at a 2.3.7.8- TCDD dose of 10 yg/kg. In the second portion of this study, rats were ex po s e d by a single intraper i t on e a l Injection of 2 , 3 , 7 , 8 - T C D D at 5, 10 or 15 yg/kg or a single gavage treatment at 20 yg/kg. The animals at the two highest exposure levels were killed 24 hours post-treatment, while the remaining animals were killed 29 days post-treatment. Again, no increase in chromosomal aberr a tions was observed, except in the positive control group exposed to triethylenemelamine. 01330 V-102 04/08/88 In a later report, a small but significant increase in chromosomal a b e r r a ti o n s was obse r v ed in the bone m a r r o w cells of m a le and female Osborne-Mendel rats (Green et al., 1977). Bone marrow cells for cytogenetic analysis were o b ta i n e d f r om Osbo r n e- M e nd e l rats used in a r a n g e - f i n d i n g study preliminary to a chronic bioassay (Green et al., 1977). The animals in groups of 8 males and 8 females rece i v ed twice w e e k l y intubations of 2.3.7.8-TCDD at respective doses of 0.25, 1.0, 2.0 and 4.0, or 0.25, 0.5, 2.0 and 4.0 yg/kg for 13 weeks. Because it was not required for the range-finding study, a control group was not included. Bone marrow cells w e re a n al y z e d for a b n o r m a l i t i e s and cells in mitosis in the animals that s u rvived to the end of the study (4-8 a n i m a l s / g r o u p ). The only significant increases in c h r o m o so m a l a b er r a t i o n s in c o m p a r i s o n w i th the low dose group were in males at 2 and 4 yg/kg and females at 4 yg/kg. The greatest incidence o b se r v ed was 4 . 6 5 % of the cells with ch ro m o so m a l breaks in the high-dose males, and this was considered only weakly positive. The weak response, as well as the lack of data from control animals and the reported difficulty of obtaining cells from the high-dose animals as a result of 2.3.7.8-TCDD toxicity, makes the conclusion from this study that 2,3,7,8TCDD produced chromosomal breaks tenuous. A similar weak r e sp o n s e was o b se r v ed by L o pr i e no et al. (1982) in male and female CD-I mice which received an l.p. injection of 2,3,7,8-TCDD at a dose of 10 yg/kg. At 96 hours post-treatment, there was a significant (p<0.01) increase in bone marrow cells with gaps and chromatid aberrations. When chromosomal aberrations were analyzed at 24 hours post-treatment, there was no significant change in the incidence of cells with aberrant c h r o m o somes. The study was continued with a more extensive experiment using CD-C0BS female rats. The rats were treated weekly by gavage (vehicle 01330 V -10 3 04/08/88 a c e t o n e - c o r n oil 1:6) at doses of 0, 0.01, 0.10 or 1.00 ^g/kg for 45 weeks. Analysis of bone ma rr o w cells for chromosomal aberrations 24 hours after the last treatment failed to detect any significant increases. A limited number of initial studies on the m u tagenicity of 2,3,7,8-TCDD in bact e r ia reported p o si t i v e results in S. t y p h i m u r i u m strain TAT 532 in the abse n c e of a m a m m a l i a n m e ta b o li c a c ti v a t i o n system (Hussain et a 1. , 1972; Seiler, 1973). More recent attempts to repeat these results with strain TA1532 or related strains have failed (Geiger and Neal, 1981; Nebert et al., 1976; Gilbert et al., 1980; McCann, 1978). These authors have also reported no increase in m u t a t i o n rate when 2 , 3 , 7 , 8 - T C D D was tested in the p r es e n c e of a m a m m a l i a n m e ta b o l i c a c t i v a t i o n system. In other j_n v itro assays, 2,3,7,8TCDD has p r oduced a p o s i t i v e resp o n se in r e ve r s io n to s t r e p t o m y c i n i n d e p e n d ence in i . coli Sd-4 cells and q u e s t i o n a b l e p o si t i v e resp o n se with p r op h a ge induction in E_. col i K -39 cells (Hussain et al., 1972). Also, 2 , 3 , 7 , 8 - T C D D has been reported to be m u t a g e n i c in the y e ast S. c e r e v l s i a e in both the 1n vitro a s s a y with S-10 and the h o s t - m e d i a t e d a s say (Bronzetti et a l ., 1983). Rogers et al. (1982) h a ve also reported p o s i t i v e m u t a g e n i c i t y results in the mouse lymphoma assay system. In the . col 1 studies, the poor survival of the cells or the interference of the vehicle solvent, DMS0, with the assay m a kes the e v a l u a t i o n of the studies difficult. With the data a v ailable, it is not p o s s i b l e to r e s o l v e the c o n f l i c t i n g reports on the m u t a g e n i c p o t e n tial of 2,3,7,8-TCDD. Overall, the data indicate little potential for the interaction of 2,3,7,8-TCDD with nucleic acids or the ability of 2,3,7,8-TCDD to produce chromosomal aberrations. Kondorosi et al. (1973) demonstrated that 2,3,7,8- 01330 V -104 04/14/88 TCDD did not react with RNA j_n vitro in the absence of a metabolic a c t i v a tion system. I_n vivo studies using r a d i o l ab e l ed 2 , 3 , 7 , 8 - T C D D indicated some association of non-extractable label with RNA and DNA (Poland and Glover, 1979); however, the level of bound label was very low. Similar marginal data were a v ai l a b l e on the cl as t o ge n i c effect of 2,3,7,8-TCDD. Alth o u gh two in vivo studies in rats (Green and Moreland, 1975; L o p r i e n o et at., 1982) failed to demonstrate any treatment-related chromosomal aberration, a second study by the same authors (Green et al., 1977 ) using a longer exposure period reported a small increase in the number of a b er r a ti o n s. A similar small increase was observed by Loprieno et al. (1982) following a single i.p. Injection of 2,3,7 , 8- T C DD in mice. In humans exposed to 2,3,7,8- TCDD during the manufacture of 2,4,5-TCPE and Buminol, Czeizel and Kiraly (1976) r e po r t ed an increase in the number of c h ro m osomal a b er r a ti o n s, w h i l e no increase was d e te c t ed in i n di v iduals expo s e d to 2 , 3 , 7 , 8 - T C D D f ollowing an industrial acci d e nt in Seveso, Italy (Reggiani, 1980; Mott u r a et al., 1981). The studies of the clastogenic effect of 2,3,7,8-TCDD were presented with little or no e x pe r i me n t al detail to assist in e v a l u a t i n g the merits of the reports. The data available are too limited to indicate whether 2,3,7,8TCDD can interact with nucleic acids or produce chromosomal aberrations. Conflicting evidence exists on the mutagenic and genotoxlc effects of 2 . 3 . 7 . 8 - TCDD, and a l t h o u g h there is e v id e n ce for the genotoxlc effect of 2.3. 7 . 8- TCDD additional testing will be required to demonstrate this activity with certainly (Giri, 1986). The differences among the results reported could be due to several factors, such as treatment protocols, solubility problems, purity of the samples tested and the high toxicity of 2,3,7,8-TCDD. This chemical may be G1330 V-105 04/14/88 a weak mutagen, but because it is very toxic, the dose range for detecting a posi t i ve genetic effect may be very narrow. Therefore, a d ditional e x p e r i m e n t a t i o n is n e ce s s ar y before any c o n c l u si v e d e t e r m i n a t i o n can be made. Suggested further testing Includes the ability of 2,3,7,8-TCDO to induce forward m u ta t i on s in m a m m a l i a n cells in culture, additional yeast and bacterial studies and the s e x-linked rece s s iv e lethal test in D r o s o p h i l a . Other m u t a g e n i c i t y studies involving humans are found in the M u t a g e n i c i t y Section of Chapter VI. Teratogenicity and Reproductive T o x i c i t y . A number of investigators have studied the role of 2 , 3 , 7 , 8 - T C D D c o n t a m i n a t i o n in 2 , 4 , 5 - T - 1 n d u c e d teratogenicity (Table V-15). Neubert and Dillmann (1972) investigated the teratogenicity of purified 2,3,7,8-TCDD, purified 2,4,5-T and samples of 2,4,5-T containing 0.05+0.02 ppm (0.05 mg/kg sample) or an undetermined amount of 2,3,7,8-TCDD. The E D 5Q for the production of cleft palate by 2,3,7,8-TCDD was determined to be 4.6 ug/kg bw. Lamb et al. (1980) studied the effect of herbicides containing 2,3,7,8TCDD on the reproductive behavior of male C57B1/6 mice. 2,3,7,8-TCDD was mixed with 2,4-D and 2,4,5-T to simulate Agent Orange and added to the diet. At doses that resul t ed in hepatic and thymic toxicity, no significant changes w e re noted in m a ting frequency, a verage fertility, percentage implantation, number of resorption sites, percentage of fetal malformations, survival of offspring or neonatal development. Rats are apparently less sensitive than mice to the teratogenic effects of 2,3,7,8-TCDD contaminated 2,4,5-T. Only skeletal anomalies (wavy ribs, fused sternum) indicative of fetal toxicity have been reported (Khera et 01330 V-106 04/14/88 o uCJ TABLE V--15 o Studies on the Potential Teratogenic.Effects of 2.3.7.B-TC00 Contaminated 2,4.5-T ---------------------- :----------------------------------------------------------------------- J------------------- Species/ Strain Vehicle Form of 2,4.5-T TCDD Level Oally Oose Treatment Observation Days Day Maternal Response fetal Response Reference Hice/ NHRI Rape-seed oil acid <0.02 ppm (Sample A) a. 15. 30. 45. 60. 90, and 120 mg/kg 6-15 Rape-seed oil acid 0.05*0.02 ppm - (Sample B) 30, 60 and 90 mg/kg Rape-seed oil acid NR 90 mg/kg (Sample C) 6-15 6t.15 Mice/ .NHRI Rape-seed oil butyl ester NR acid NR 0.05*0.02 ppm 12 and IT mg/kg i r 20. 35. 60. 90 and 130 mg/kg 6-15 6-15 Mice/ CD-I Corn oil: acetone (9:1) acid <0.05 ppm 115 mg/kg 10-15 IB No toxic effects; Significant Increases In Neubert and Dlllmann. decreased maternal the Incidence of cleft 1972 weight at doses of palates at doses above 90 mg/kg and 30 mg/kg (see text for greater additional details). Significantly decreased (p<0.005) fetal weight at all dose levels. 18 No toxic effects; Increases In the Incidence decreased maternal of cleft palate at 60 and weight at 90 mg/kg 90 mg/kg; significant decrease In fetal weight (p<0.005) at all dose levels 18 No toxic effects Increase In the Incidence but decreased of cleft palate; significant maternal weight (p<0.005) decrease In fetal weight IB No toxic effects Significant decrease In fetal weight but no effect on mortality; Increase In the frequency of cleft palate similar to that seen with acid (see text) NR Toxic effects observed at 90 and 130 mg/kg Increases In the percent age of resorptions and/or dead fetuses at 90 and 130 mg/kg; Increases In the Incidence of cleft palate and retardation of skeletal development at 35 mg/kg and above Roll. 1971 (Taken from EPA, 1979a) 18 No significant No effect on fetal mortal Courtney, 1977 effect on weight ity or fetal weight but gain or llver-to- an Increase In the Inci body weight ratios dence of cleft palate 04/14/88  co TABLE V-15 (coni.) oco Species/ Strain Vehicle Form of 2.4,5-T TCOD Level Dally Dose Treatment Observation Days Day Naternal Response Fetal Respoqse Reference Nice/ CS7B/6 or Nice/ AKR Honey:water (1:1) or 0NS0 acid 30 ppm 21.5, 46.4 and 113 mg/kg 6-14 Nice/ AKR Honey-.water acid (1:1) 30 ppm 113 mg/kg 6-15 Rats/ SpragueOawley {groups of 25 rats) Gavage/ hydroxypropylmethyl- cellulose acid 0.5 ppm 1. 3. 6. 12 or 24 mg/kg/ day 6-15 Rats/ Ulslar r Gavage/ aqueous gelatin or corn oil add <0.5 mg/kg i 25. 50. 100 ' or ISO mg/kg/ day 6-15 * Gavage/ aqueous gelatin or corn oil butyl ester <0.5 mg/kg 50 or 150 mg/kg/day 6-15 10 NR Significant (p<0.01) Increases In the Incidence of cleft palate In the high dose group and cystic kidney In both dose groups; Increased fetal mortality also observed In the high dose group Cour tney et al., 1970a,b 19 Increase In 11ver-to-body weight ratio Significant (p<0.05) Increases In the Incidence of cleft palate and fetal mortality Courtney et al.. 1970a.b 20 No effect on body A slight but statistically Emerson et a l ., weight and no significant (p<0.05) 1970, 1971 N.B. observable signs decrease In Implantations This appears to of toxicity and litter size In lowest be a full publi dose group only; no frank cation of the teratogenic effects based abstract summary on a detailed examination by Thompson of the control and 24 mg/kg et al., 197) dose group; the only effect noted was an Increase In the Incidence of 5th par tially ossified stemebrae 22 Some maternal mor At 100 or 150 mg/kg. Khera and HcKInley, tality and decreased fetal weight, 1972; Khera et al.. decreased body Increased fetal mortality 1971 weight gain at and an Increase In the 150 mg/kg; no Incidence of skeletal signs of toxicity anomalies; no significant at 100 mg/kg or effect at the two lower below dose levels 22 NR No significant effect on fetal mortality, fetal weight, or the Incidence of anomalies Khera and HcKInley, 1972; Khera et al., 1971 04/14/88 O OCCJO TABLE V-15 (cont.) Species/ Strain Vehicle Form of 2.4.5-T TC00 Level Dally Oose Treatment Observation Days Day Maternal Response Fetal Response Reference Rats/ Ho Itm a n Gavage/ 1:1 solu tion of honey and water add 30 ppm 4.6. 10.0. and 46.4 mg/kg/day 10-15 Rats/CO Gavage/ 15X sucrose solution acid 0.5 ppm 10.0, 21.5, 46.4 and 80.0 mg/kg/ day i OvD Rats/ Strain not speci fied Gavage/ methocel Gavage/ methocel f acid 0.5 ppm 50 mg/kg acid 0.5 ppm 100 mg/kg Syrian hamsters/ Hesocrlcetus euratus Gavage/ acetone, corn oil, and carboxymethyl cellulose In ratio of 1:5.8: 10 acid <0.1-4.5 ppm 20. 40. 80 and 100 mg/kg 6-15 6-15 6-10 6-10 20 NR Significant (p<0.01) Increases In fetal mor tality at the 2 higher dose levels; dose-related Increases In the percentage of abnormal fetuses per litter; a high Incidence of cystic kidneys In treated groups Courtney et al., 1910a.b 20 Reduced maternal Increase In the Incidence Courtney and Moore. weight gain at the of kidney anomalies but 2 higher dose no Increase In cleft 1971 levels (p<0.05) palate and Increased ltver-to-body weight ratio at the highest dose level (p<0.05) NS No effect on mor No significant effect on Sparschu et a l .. tality or body fetal mortality or fetal 1971b weight gain weight; a significant (p<0.05) Increase In the Incidence of delayed ossification NS Increased mor tality and decreased body weight gain Increase In the Incidence of delayed ossification and poorly ossified or malallgned sternabrae (p<0.05) Sparschu et al., 1971b 14 NS Dose-related Increases In fetal mortality, gastro intestinal hemorrhages, and fetal abnormalities; Collins et al., 1971 NS b Not specified; NR ^ Not reported 04/14/88 a 1. , 1971; Khera and McKinley, 1972). At doses <100 mg/kg bw/day of c o n t a m inated 2,4,5-T, the only skeletal effect appeared to be delayed ossification {Emerson et al., 1970, 1971; Sparschu et al., 1971b). The t e r a t o ge n i ci t y of p u rified samples of 2 , 3 , 7 , 8 - T C O D is pres e n te d in Table V-16, Courtney and Moore (1971) administered subcutaneous injections of 1 or 3 yg of purified 2 , 3 , 7 ,8-TCDD/kg bw/day to CD-I, DBA/2J and C57B1/6J mice on days 6-15 of gestation. This treatment had no effect on fetal mortality; however, cleft palate and unspecified kidney anomalies were found at all dose levels in all strains, with C 57B1/6J being the most s e n s i tive strain. M o ore et al. (1973) a d m i n i s t e r e d oral doses of 2 , 3 , 7 , 8 - T C D D (1 or 3 yg/kg/day) to C57B1/6 mice by gavage on days 10-13 of gestation. Cleft palate and h y dronephrosis were observed in both dose groups. These kidney lesions were apparently reversible, since very few litters (1/14) that were c r os s - n u r s e d on control mothers had pups with kidney abnormalities. In contrast, kidney lesions were found in 4 of 14 control litters that were nursed on 2,3,7,8-TCDD treated mice. Neubert and Dillman (1972) and Neubert et al. (1973) administered daily oral doses of 0.3, 3.0, 4.5 or 9.0 yg/kg bw to NMRI mice on days 6-15 of gestation. E x tensive resorption (6 of 9 litters completely resorbed) o c c u r red in the h i gh - d o s e group. C.left palate, but not kidney a b no r m al i t ie s , were observed at a dose of 3.0 yg/kg bw/day or higher, with 0.3 yg/kg bw/day being the N0AEL in this study. A single dose of 45 yg/kg bw could pr od u c e cleft pala t e if g i ven befo r e day 13 of g e station, with m a x i m u m incidences occurring when the dose was given on day 8 or 11. 01330 V-110 04/14/88 04/14/88 O co Oco Specles/Straln Vehicle* Mouse/C0-1 mouse/0BA/2J mouse/C57Bl/6J Mouse/C57BI/6 Mouse/CD-1 OHSOb Acetone:* corn oil (1:9) 0NS0b or corn oil House/CF-1 Mouse/NMRI corn o l 1*: acetone (98:2) rape-seed* oil Rat/CD * DHSOb Rat/SpragueOawley corn oil*/ acetone TABLE V-16 Studies on the Potential Teratogenic and Reproductive Effects of 2,3,7,8-TCOD Compound Bally Oose Treatment Observation Days Day Maternal Response ----------- 1---------Fetal Response Reference 2,3.7.8-TCDD 1. 3 pg/kg 6-15 17c or 18 Increased liver/ body weight ratio cleft palate, kid Courtney and Moore, ney anomal les*1 1971 2.3.7.B-TC00 1. 3 pg/kg 10-13 or 10 18c none reported cleft palate, kid Moore et al., 1973 ney anomalies'* 2.3.7.B-TCDD 25. 50. 100, 200. 400 pg/kg 7-16 18e 2,3,7,8-TCDD 2.3.7.B-TCD0 0.001, 0.01, 0.1. 1.0. 3.0 pg/kg 0.3, 3.0, 4.5. 9.0 pg/kg 6-15 6-15 18c 18 2.3.7.B-TC00 0. 0.5. 2.0 pg/kg 2,3,7,8-TCOO 0. 0.03. 0.125, 0.5, 2.0 and 8.0 pg/kg , 6-15. 9 and 10, or 13 and 14 6-15 20c 20c Increased liver/ body weight ratio none reported no effect observed none reported cleft palate, hydronephrotlc kidneys, hydrocephalus, open eyes, edema, petechlae cleft palate, dilated renal pelvis fetocldal at the high dose, cleft palate at doses at or above 3 pg/kg kidney malforma tions at both dose levels Courtney, 1976 Smith et al.. 1976 Neubert and Dlllmann, 1972 Courtney and Moore, 1971 vaginal hemorrhage at 2.0 and 8.0 pg/kg Intestinal hemorrhage at 0.125 and 0.5 pg/kg. fetal death of higher doses, subcutaneous edema Sparschu et al., 1971a o is Oi s TABLE V-16 (coni.) Specles/Straln Vehicle3 Compound Dally Dose Treatment Observation Days Day Maternal Response Fetal Response Reference Rat/Ulstar corn oil*/ anlsole 2.3,7.8-TCDD 0.0, 0.125, 0.25, 0.5, T. 2. . 8, 16 ug/kg Rat/SpragueDawley corn oil3/ acetone (9:1) 2,3,7,8-TCDD 0.0, 0.125, 0.5, 2.0 ug/kg Rat/SpragueDawley diet 2,3,7,8-TCDD 0.001, 0.01 and 0,1 *>g/kgf Rabbit/ New Zealand rHonkey/rhesus corn oil3/ acetone (9:1) diet 2,3,7.8-TCOD 2.3.7,8,TC00 0.0, 0.1, 0.25, 0.5 and 1 ug/kg 8.6 pg/kg/day Monkey/rhesus diet 2,3,7,8-TCOO 55.7 pg/kg/day Administration was by gavage. Administration was by subcutaneous Injection. cf1rst day of gestation designated day lero. ^Kidney anomalies were not specifically defined. ef1rst day of gestation designated day one. M c r n n I 1 n i f p * 6-15 1-3 throughout gestation 6-15 7 months before and during gestation 7 months before and during ges tat Ion 22 21 postparturltlon 28 at term at term maternal toxicity observed at or above 1 ug/kg Increased fetal death observed at or above 1 ug/kg. subcutaneous edema and hemorrhages In the 0.25-2.0 ug/kg groups Khera and Ruddlck, 1973 decrease In body weight gain In the high dose group decreased fetal weight In the 0.5 and 2 ufl/kg group, cystic kidneys and dilated renal pel vis occured In the 2 ug/kg group Gtavlnt et al., 1982a low fertility at 0.01 and 0.1 ug/kg. decreased body weight at 0.01 and 0.1 ug/kg. dilated renal pelvis low survival at 0.01 and 0.1 ug/kg, decreased body weight at 0.01, slight dilated renal pelvis at 0.001 ug/kg Hurray et at., 1979 maternal toxicity at doses of 0.25 ug/kg and above 6/8 conceived, nor mal serum estradiol and progesterone Increases In extra Glavlnl et al.. 1982b ribs and total soft tissue anomalies 3/8 normal births Allen et al.. 1979 3/8 conceived, de creased serum estra diol and progester one 1/B normal births Allen et al.. 1979 >. in.j f o r M l Hv In adults. 04/14/88 Courtney (1976) compared the effectiveness of oral and subcutaneous a d m i n i s t r a t i o n in CD-I mice. S u bc u t an e o us a d m i n i s t r a t i o n was found to p r o duce a greater teratogenic response at a lower dose than did oral administration. Smith et al. (1976) determined the minimum effective oral dose (MED) for p r od u c in g teratogenic effects in C F -1 m i ce to be 1 yg 2 , 3 , 7 , 8 - T C D D / k g bw/day. The NOAEL in this study was 0.1 y g/kg bw/day. Courtney and Moore (1971) determined the teratogenic potential of s u b c u t a n e o u s l y Injected 2 , 3 , 7 , 8 - T C D D (0.5 or 2 yg/kg bw/day) in CD rats. The c o mpound was a d m i n i s t e r e d in d i m e t h y l s u l f o x i d e on days 6-15, 9-10 or 13-14 of gestation. The only developmental anomalies observed were kidney malfor ma t i o n s in all dose groups. Six hemorrhagic gastrointestinal tracts were found; however, this was considered a primary fetotoxic effect and not a malformation. Sparschu et al. (1971a) admin i st e r ed 0.03, 0.125, 0.5, 2.0 or 8.0 yg 2,3,7,8-TCDD/kg/day by gavage to Sprague-Dawley rats on days 6-15 of g e s t a tion. A number of dose-related fetotoxic effects were found, including edema, increased numbers of resorptions, numbers of dead fetuses and Intes tinal hemorrhage. No teratogenic effects were reported in the surviving fetuses. Khera and Ruddick (1973) intubated groups of 7-15 pregnant Wistar rats with 0.125, 0.25, 0.5, 1, 2, 4, 8 or 16 yg 2 , 3 , 7 , 8 - T C D D / k g bw/d a y on days 6-15 of gestation. Severe fetotoxic effects were observed at doses of 1 yg/kg bw/day or higher, with no live fetuses found in the groups exposed 01330 V-113 04/14/88 to 4, 8 or 16 yg/kg bw/day. No anomalies were observed in the group receiving 0.125 yg/kg' bw/day. In the intermediate dose groups, 0.25-2.0 yg/kg bw/day, anumber ofanomalies, including s u bcutaneous edema of the head and neck and hemo r r ha g e s in the intestine, brain, and subcutaneous tissue, were observed. When the dams were allowed to litter and wean the pups, none of the pups in groups receiving >1 yg/kg bw/day survived until weaning. Fostering pups from dams exposed to 1 yg/kg bw/day to control dams did not appreciably increase survival (36/42 died). Giavini et al. (1982a, 1983) a d m i n i s t e r e d 0, 0.125, 0.5 or 2.0 yg 2 , 3 , 7 ,8-TCDO/kg/day by gavage, to Sprague-Oawley rats on days 1-3 days of gestation or to female CRCD rats daily for 2 weeks before mating. In the groups dosed on days 1-3 of gestation, fetal weight was significantly decreased in the 0.5 and 2.0 yg/kg bw/day groups, but no statistica lly si gn i f ic a n t Increases in m a l f o r m a t i o n s were noted. When adult female rats were treated for 2 weeks before mating, an increased number of cystic k i d neys and dilated renal pelvis were o b se r v e d in the pups in the high dose group. In these studies, 0.125 yg/kg bw/day was the NOEL for both m a t e r nal toxicity and adverse effects on the fetus. The r e p r o d u c t i v e e ffects of 2 , 3 , 7 , 8 - T C O D were also studied in a 3 - g e n e r ation study using Sprague-Oawley rats (Murray et al., 1979). Throughout the study, animals were c o n t i n u o u s l y m a i n t a i n e d on diets p r o v i d i n g doses of 0, 0.001, 0.01 or 0.1 y g 2 , 3 , 7 ,8-TCDO/kg/day. The parental g r oup (f ) was m a in t a in e d for 90 days on the test diets prior to mating. The f rats were mated twice, producing the filial generations ( f ^ and f^g). Selected f,D and f_ rats were mated at -130 days of age to produce the 2f and f^ litters, respecti v e ly . In, later g enerations, the high dose 01330 V-114 04/14/88 group (0,1 yg 2 , 3 , 7 , 8 -TCQD/kg/day) was discontinued because few offspring were p r oduced in this group. At the inter m ed i a te dose (0.01 yg/kg/day), 2 . 3 . 7 . 8 - TCDD caus e d lower body w e ig h t in exposed rats of both sexes (f^ and fp). At the low dose, no toxic effects were discerned. Fertility was gr ea t l y reduced in the f g e n e r a t i o n exposed to 0.1 yg 2.3.7.8- TCDD/kg/day. At 0.01 yg 2,3,7,8-TCDD/kg/day, fertility was s i g n i f ic a n tl y {P < 0 .05) redu c e d in the f^ and f^ rats. F e r t i l i t y in rats (of any generation) exposed to 0.001 yg 2 , 3 , 7 , 8- T C QQ / k g/ d a y was not d i ff e r en t from that of control rats. Decreases in litter size w e re noted in the f g r o u p exposed to 0.1 yg/kg/day and the f^ and f^ litters e xposed at 0.01 yg/kg/day. S t a t i s t i c a l l y s i gn i ficant decr e a se s in fetal survival throughout gest a t io n were noted in f^ and f^ litters of the 0.01 yg 2,3,7,8-TCDD/kg/day exposed dams. At 0.001 yg 2,3,7,8-TCDD/kg/ day, a decreased gestational survival was reported for the f2 litters, but not for other generations. Decreased neonatal survival was noted among f1A and f^ pups exposed to 0.01 yg 2,3,7,8-TCDD/kg/day, but not among f^g or fg pups. Postnatal body weights of the f^ and f^ litters at 0.01 yg 2,3,7,8-TCDD/kg/day were significantly depressed. At the low dose (0.001 yg 2,3,7,8-TCDD/kg/day), necropsy of 21-day-old pups revealed a s t a t i s t i c a l l y s i g n i f ic a n t (P<0.05) Increase in dila t e d renal pelvis in the f^ generation. S u bs e q u e n t ge ne r a ti o n s at this dose level or any at the intermediate dose (0.01 yg 2,3,7,8-TCDD/kg/day) did not have a significant increase in this a bnormality. .Significantly decr e a se d thymus weig h t and increased liver w e ig h t were repo r t ed in the f^ g e n e r a ti o n , but not in the f1 generation (f2 generation data not obtained) of the intermediate dose group. Murray et al. (1979) concluded that 2,3,7,8-TCDD ingested at 0.01 or 01330 V-115 04/14/88 0.1 yg/kg/day impaired reproduction among rats, and NQAELs were associated with 0.001 y g 2 , 3, 7 ,8-TCDD/kg/day. Nisbet and Paxton (1982) reevaluated the primary data of Murray et al. (1979) using d ifferent statistical methods. From this r v a l ua t i on it was concluded that 2,3,7,8-TCDD significantly reduced the gestational index, decreased fetal weight, and increased liver to body weight ratios and the incidence of dilated renal pelvis in both lower dose groups. Nisbet and Paxton (1982) concluded that the dose of 0.001 yg/kg/day was not a NQAEL in this study. The FIFRA Scientific Advisory Panel has also reviewed the data from this three generation study and concluded that the effects observed at the 0.001 yg/kg dose were not consistent enough between the different generations to consider them treatment-related (U.S. ERA, 1979b). Although the panel considered the data suggestive of an embryotoxic effect, they concluded that 0.001 yg/kg represented a NOEL. Subsequently, EPA did further evaluation of Murray et al. (1979) data and arrived at a conclusion that 0.001 yg/kg represents a L0AEL (U.S. EPA, 1984a), It has been demonstated that both genetic susceptibility and concomitant expo s u re to other compounds affect the d evelopmental toxicity of 2,3,7,8-TCDD. Poland and Glover (1980) and Dencker and Pratt (1981) demonstated genetic differences in the susceptibility of mice; only responsive C57B1/6J mice developed the characteristic cleft palate and hydroneophrotic kidneys after treatment. This indicates that developmental toxicity, as many other toxicologic endpoints of 2,3,7,8-TCDD, segregates with the Ah locus. Additionally, it was demon s tr a t ed that simultaneous exposure to 2,3,7,8-TCDD and specific polychlorinated biphenyls (Birnbaum et al., 1985), or the hormones h y d r o c o r t i s o n e ( Birnbaum et al., 1986) or 01330 V-116 04/14/88 thyroxine and t r i i o d o t h y r o x i n e (Lamb et a 1. , 1986) increases the s e ns i tivity of mice to the d evelopmental effects of 2 , 3 , 7 , 8 - T C D D , and an addi t i ve effect was observed by Weber et al. (1985) for simultaneous exposure to 2,3,7,8-TCDD and 2,3,7,8-tetrachlorodibenzofurans (TCDF). Berry et al. (1976, 1977 ) treated S p r a g u e - D a w l e y rats on day 17 of gest a t io n with 0.2, 0.5, 2.5 or 6 ^g 2,3,7 ,8-TCDD/kg bw intraper i tonea 11 y . Maximal induction of fetal hepatic aryl hydrocarbon hydroxylase (A H H ) a c t i vity (measured by fluorometric methods) and N-hydroxylation of FAA (measured by autoradiography) was observed at a dose of 2.5 pg/kg bw. At lower doses these effects were not observed in either the fetus or the dams. Electron microscopic examination revealed cellular necrosis, increased glyc o g en and rough e n doplasmic r e t i c u l u m and swollen m i t o c h o n d r i a in the liver at dose levels that Induced AHH activity. Induction of epoxide hydratase was also o b ser v ed in the lungs (ratio 2 , 3 , 7 , 8 - T C 0 D / c o n t r o l = 2 . 8 5 ) , kidney (ratio 2,3,7,8-TCDD/control = l .06) and skin (ratio 2,3,7,8-TCDD/ c on t r o l ^ . 62). Lucier and McDaniel (1979 ) intubated CD rats with 0 or 3 yg 2,3,7,8T C D D / k g on days 5, 10 or 16 of gestation. They m e a s u r e d the a c t i v i t y of fetal and newborn hepatic microsomal benzo[a]pyrene hydroxylase and p-nitrophenol glucuronide formation on gestation day 21, postnatal day 8 and p o st natal day 21. The BaP hydroxylase activity of controls on gestation day 21, treated rats on g e s t a t i o n day 21, cont r o ls on postnatal day 8, treated rats on postnatal day 8, c o nt r o ls on post n a ta l day 21 and treated rats on p o s t natal day 21 was 0.003, 0 .032 (p<0.01), 0.214, 0.719 (p<0.01), 0.224 and 0.863 (p<0.01) nmol/mln/mg, respectively. The rate of p-nitrophenyl glu- 01330 V -11 7 04/14/88 curonide formation at the above intervals was 13.4, 14.4, 38.4, 99.5 (p<0.01), 23.1 and 164.2 (p < 0 .01} nmol/min/mg, respectively. Giavini et at. (1982b) administered 0.1, 0.25, 0.5 or 1 yg 2,3,7,8TCDD/kg bw/day to groups of 10-15 New Zealand rabbits by gavage on days 6-15 of gestation. Maternal toxicity was observed at doses of 0.25, 0.5 or 1.0 yg/kg bw/day. There were increases in abortions and resorptions at doses of 0.25 or 0.5 yg/kg bw/day, with no live fetuses found in the 1.0 yg/kg bw/day dose groups. Extra ribs were found in all dose groups. H y d r o n e p h r o sis was a common finding in all groups, but the increase in treated groups over control values was not statistically significant. 2,3,7,8-TCOD also induced fetal liver m i cr o s om a l enzymes in New Zeal a n d rabbits (Norman et a 1., 1978). The BaP hydrox yl a se activity of the liver microsomal fraction of adult males and newborn pups was determined following subcutaneous injec tion of 30 nmol/kg into adult animals. Dams received treatment on the 24th day of gestation. The benzo[a]pyrene hydroxylase activity of newborn c o n trols, adult controls, treated newborns and treated adults was 0.3, 1.8, 1.6 and 3.7 nmol/mg protein, respectively. Allen et al. (1979) fed adult female rhesus monkeys on diets containing 50 or 500 ppt (50 or 500 ng/kg diet) 2,3,7,8-TCDD for 7 months. The a n i mals consumed a total dose of 1.8 and 11.7 yg 2,3,7,8-TCDD, respectively. The anim a l s w e r e bred at 7 m o nt h s of treatment r e su l t i n g in p r eg n a n c y in 6 of 8 females in the l o w-dose g r ou p and 3 of 8 in the h i gh - d o s e group. They w ere c o nt i n ue d on treatment duri n g pregnancy. In both groups, two-thirds of the pregnancies ended in spontaneous abortions. There were no reported m a l f o r m a t i o n s in the three s u rv i v in g Infants. All of the cont r o ls (one group of 8 and another of unspecified size) conceived and gave birth to 01330 V - 118 04/14/88 normal offpsring. M c N u l t y (1978) reported a d o s e - r e l a t e d increase in spontaneous abor t i on s in rhesus monkeys given oral doses of 0.0, 0.2, 1 or 5 vig/kg bw 3 times/week for 3 weeks starting ~20 days p r econception. The group sizes (2-4 animals) were too small for adequate statistical analysis. Summary There are wide variations in the species sensitivity to the acute t o x i city of 2 , 3 , 7 ,8-TCDD. L D ^ s ran9e from 0-6 yg/kg bw for the male guinea pig to >5000 yg/kg bw for the male hamster (Schwetz et al., 1973; Olson et a 1., 1980b; Henck et al., 1981). The toxic mani f e st a t io n s seem to be the same whether the c o mp o u nd is given as a single oral dose or as a limited number of multiple treatments, with death occurring from 5-45 days p o s t treatment. Lethal exposures result in weight loss, often described as "wasting away," and thymic atrophy. In some species, particularly rats and mice, extensive liver damage is observed (Gupta et al., 1973). In general, no specific cause of death has been identified, although extensive hemor rhaging has been implicated in mice (Vos et al., 1974). In rats and mice, single high doses produce liver necrosis (Jones and Butler, 1974), while lower doses produce fatty changes and proliferation of the e n do p lasmic r e t i c u l u m (Fowler et al., 1973). Other effects seen in some species Include induction of microsomal enzymes, degeneration of plasma membranes with loss of ATPase activity, a decreased ability to excrete some xenobiotics in the bile, porphyria, altered g a s t r o i n t e s t i n a l absorption of some nutrients and decreased blood cellularlty. 2 , 3 , 7 , 8 - T C D D is an immunotoxin, p r e d o m i n a t e l y a f f e c t i n g cel 1 -mediated immunity. Hypersensitivity, adverse effects on the thymus and increased 01330 V-119 h 04/14/88 sensitivity to antigens have demonstrated the immunotoxic potential of 2,3,7 ,8-TCDD. In rats and mice, the liver appears to be the most sensitive organ following chronic or subchronic exposure. H e patotoxicity develops following a long induction period and the changes persist for long periods following the termination of exposure (King and Roesler, 1974; Goldstein et al., 1982b). In the subchronic studies reviewed in this report, the NOAEL of 0.01 yg/kg bw/day (Kociba et al., 1976 ) and 0.07 yg/kg bw/day (N T P , 1980a) have been reported for rats. A NOAEL of 0.29 yg/kg bw/day was evident for female m i ce and a L0EL of 0.14 yg/kg bw/day for m a le mice in the NTP (1980a) study. A NOAEL of 0.001 yg/kg bw/day, a NOAEL of 0.05 yg/kg bw/day, and a frank effect level (FEL) of 0.1 yg/kg bw/day have been reported for rats following chronic exposure (Kociba et al., 1978a,b, 1979; NTP, 1980a). Toth et al. (1978, 1979) o b se r v ed toxic effects in mice at doses as low as 0.007 yg/kg bw/week. In a p r e l i m i n a r y study by Van Miller (1977a,b), 2 , 3 , 7 , 8-TCDD was tested for c a r c i n o g e n i c i t y f o ll o w in g oral a d m i n i s t r a t i o n to rats. Increases in the incidence of total tumors was o b se r v e d in some groups; however, the group sizes, -10 animals/group, were too small for an assessment of a treatmentrelated response. In a second, t more extensive study by Kociba et al. (1978a) a positive carcinogenic response was detected. In this study the estimated Intake of 2,3,7,8-TCDD from the diet was 0.0, 0.001, 0.01 and 0.1 01330 V-120 04/14/88 vig/kg/day. In the high dose group, both m a l e , a n d female animals had s i g nifi c a nt increases in site specific tumors. The target organs and tumor types in male animals were squamous cell carcinomas of the tongue and hard palate, and a d en o m as of the adrenal cortex; in female animals the target organs and tumor types were hepatocellular carcinomas, squamous cell car cinomas of the tongue and of the lung. The data demonstrate that dietary exposure to 2,3,7,8-TCDD at levels that produce a daily dose of 0.1 yg/kg pr od u c e increased tumor incidences in both male and female rats. Though the increase was not significant, these tumor types were also found in lower dose groups. Under the National Toxicology Program, 2,3,7,8-TCDD was tested for c a r c i n o g e n i c i t y in rats f o llowing ad mi n i st r a ti o n by gavage (NTP, 1982a). Both male and female animals were exposed to weekly doses of 0.0, 0.01, 0.05 and 5 yg/kg bw. The only tumors that appeared to be treatment-related were follicular cell a d en o m a s or c a rcinomas of the thyroid in male animals, and neop l a st i c nodules or h e p a t o c e l l u l a r c a r c i n om a s of the liver in female animals. The incidence of these tumors was significantly greater than control in the high dose groups and the i ncidence of both tumors showed a positive dose-related trend. Under the conditions of this assay, 2,3,7,8TCDD was concluded to be c a rcinogenic in both m ale and female rats. Further studies in mice exposed by gavage have provided support for the carcinogenicity of 2,3,7,8-TCDD. Toth et aT. (1979) exposed male mice to 2,3,7,8-TCDD at doses of 0.0, 0.007, 0.7 and 7.0 yg/kg week in a study to determine whether 2,4,5-TCPE, its contaminant 2,3,7,8-TCDD, or both were carcinogens. At the 0.7 yg/kg/week level there was a significantly Increased incidence of liver tumors. Liver tumors were not significantly 01330 V-121 04/14/88 increased in the high dose group; however, early m o rt a l it y in this group from high doses may have p r ec l u de d o b se r v i n g late d e ve l o p i n g tumors. Simi larly increased incidences of liver tumors were o b se r v ed in the NTP (1980a) study in the high dose male mice exposed to 0.5 y g / k g / we e k and in the high dose female mice exposed to 2 yg/kg/week of 2,3,7,8-TCDO by gavage. Female m i ce also had an Increased incidence of foll i c ul a r -c e l l a d enomas of the thyroid. In both studies, 2,3,7,8-TCDO was carcinogenic to mice with effective doses ranging between 0.5 and 2 yg/kg/day depending on sex and the individual study. ` The m ouse skin two-stage tumor 1gen i d t y model has also been used to test the carcinogenic potential of 2,3,7,8-TCDO. Following long-term dermal application 3 times/week of 2,3,7,8-TCDO at levels of 0.01 and 0.005 yg/ application to male and female mice, respectively, there was an increased incidence of skin tumors only in female mice (NTP, 1982b). Along with the indication that 2 , 3,7,8-TCDD was a complete carcinogen in this system, OiGiovanni et al. (1977) reported that 2,3,7,8-TCDO was also a tumor initia tor in m ouse skin. The a b i l i t y of 2 , 3 , 7 , 8 - T C D D to initiate, however, has yet to be confirmed since appropriate vehicle and promotion-only control groups w e re not included. Attempts, to demonstrate tumor p r omoting a c tivity with 2 , 3 , 7 , 8 - T C D D on m o us e skin have p r od u c e d n e ga t i ve results in some assays (NTP, 1982b; Berry et al., 1978, 1979; Slaga and Nesnow, 1985); however, Poland et al. (1982) reported that 2,3,7,8-TCDD was a tumor promoter when tested on the skin, of mice homo z y go u s for the "hairless" trait but not in mice h e t e r o zyg o us for this recessive trait. Pitot et al. (1980) also reported that 2,3,7,8-TCDD was a promoter for DEN-initiated hepatocarc i nogenes i s in rats following parenteral a d m i n i st r a ti o n of the compounds. On mouse skin, 2,3,7,8-TCDD was a complete carcinogen and possibly a tumor 01330 V-122 04/14/88 initiator, while no tumor promoting activity could be attributed to 2 , 3, 7 , 8- T C DD in the assays. In rat liver Initiated with DEN, 2,3,7 , 8- T C DD was a tumor promoter. In studies of- the inte r a ct i o n of 2 , 3 , 7 , 8 - T C D D with other chemical c a r c i nogens, Kouri et al. (1978) reported that 2,3,7,8-TCDD was a cocarcinogen with 3-MC when administered by subcutaneous injection. In the mouse skin bioassay, initiation with simultaneous administration of 2,3,7,8-TCDD and OMBA, however, did not affect tumor yield (DiGiovanni et al., 1977). S i m i larly, no effect was observed when 2,3,7,8-TCDD was administered either i m mediately b e fore (5 minutes) or 1 day after DMBA Initiation (Berry et al., 1979; DiGiovanni et al., 1977, 1979a; Cohen et al., 1979). When treatment with 2,3,7,8-TCDD occurred 1-10 days before DMBA initiation, 2,3,7,8-TCDD demonstrated a potent anticarcinogenic action. Although 1-5 days prior exposure to 2,3,7,8-TCDD Inhibited tumor Initiation by BaP, 3-MC and BaPdiol-epoxide, the tumor initiating ability of the latter compound was also inhibited when 2,3,7,8-TCDD exposure occurred either 5 minutes before or 1 day after initiation (DiGiovanni et al., 1980). The increased AHH activity resulting from 2,3,7,8-TCDD exposure may account for the anticarcinogenic activity by altering the metabolism of the initiating compound; however, DiGiovanni et al. (1980) suggest that the inhibition of the initiating activity of BaP-diol-epoxide 1 day after initiation indicates that more than one m e c h a n i s m p a r t i c ip a t es in the a n t i c a r c i n o g e n i c a c ti v i ty of 2,3,7,8-TCDD. Early reports i ndicated that 2 , 3 , 7 , 8 - T C D D was m u t a g e n i c in S. t y p h i m u r lum strain TA1532 (Hussain et al., 1972; Seiler, 1973); however, later attempts to confirm these results have been unsuccessful (Geiger and Neal, 1981; Nebert et al., 1976; Gilbert et al., 1980; McCann, 1978). 2,3,7,8- 01330 V-123 04/14/88 TCOD has been reported to be m u ta g e ni c to E_. col i in vitro (Hussain et a 1. , 197?) and to S. cerevi siae j_n v i t r o , and in the h o s t - m e d i a t e d assay (Bronzetti et a 1., 1980). Covalent interactions with nucleic acids are minimal if they occur at all (Kondorosi et a l . f 1973; Poland and Glover, 1979). Only marginal effects have been observed on the incidence of c h r o m o somal a b e r r a ti o n s j_n vivo (Green and M oreland, 1 975 ; Green et al., 1977 ). 2 , 3 , 7 ,8-TCDD has been d e m o n s t r a t e d to be teratogenic in all strains of mice tested. The most common malformations observed are cleft palate and kidney anomalies; however, other malformations have been observed occasion ally. W i t h a MED of 1 jjg/kg/day, 2 , 3 , 7 , 8 - T C D D is the most potent t e r a t o gen known. At higher doses, 2 , 3 , 7 , 8 - T C D D has a marked fetotoxic effect, as measured by decreased fetal weight and increased fetal toxicity. Hemor rhagic GI tract has been a s sociated with 2,3,7 , 8- T C DD fetal toxicity. In rats, it has a l so been c o n s i s t e n t l y o b se r v e d that 2 , 3 , 7 , 8- T C DD produced te ra t o ge n i c and f etotoxic r esponses in all strains tested. In this species, the most common fetal anomalies observed were edema, hemorrhage and malformation of the kidney with effects observed at doses of >0.01 pg/kg/day. In addi t i on , there Is some e v id e n ce that 2 , 3 , 7 , 8 - T C D D can Induce m i cr o s om a l enzymes in the fetus e x p o s e d _1n u t e r o , and this induction is a c c o m p a n i e d by d a m a g e to the fine s t ru c t u r e of the liver cell; however, other reports Indicate that enzyme induction occurs only after birth follow ing exposure to 2,3,7,8-TCDD through the mother's milk. As in mice, h e m o r rhagic GI tracts have been observed in rat fetuses exposed in utero to 2,3,7,8-TCDD. 01330 V-124 04/14/88 Rabbits and monkeys are also susceptible to the fetotoxic effects of 2,3,7,8-TCDD; however, the studies of these species have been too limited to clearly demonstrate a teratogenic response or define a threshold dose for fetotoxldty. 01330 V-12 5 04/14/88 VI. HEALTH EFFECTS IV HUMANS Clinical Case Studies Acute exposure to 2,3,7,8-TCDO results 1n nausea and vomiting, headache, and Irritation of the eyes, skin and respiratory tract. The Initial skin ^reaction 1s a cutaneous reaction rese m b li n g a chemical burn, followed several days to weeks after by chloracne (Taylor, 1979). Chloracne, the typical human dermal reaction to 2,3,7,8-TCDD, 1s a cutaneous eruption of comedones, cysts, and In severe cases, pustules. These usually occur on the face and shoulders as a result of squamous metaplasia of the dermal glands (Crow, 1978; Passl et al., 1981). Host of the d o cumented acute exposures to 2,3,7,8-TCDO have been the result of chemical Industry accidents Involving 2 , 4, 5 - t r l c h l o r o p h e n o x y a c e t l c acid, which 1s c o nt a m in a t ed with 2 , 3, 7 , 8 - T C D D . A c co r d i n g to H o lm s t ed t (1980), the first cases of chloracne associated with exposure to dioxins occurred following an explosion 1n a chemical plant producing 2,4,5-T 1n 1949. Zack and Susklnd (1980) recorded nausea, head aches, fatigue, muscular aches and pains, and chloracne as the frequent com plaints among the 228 workers exposed. Chemical tests revealed elevated lipid levels and prolonged prothrombin times. Residual chloracne, pe ripheral neuropathy, fatigue and severe aches and pains persisted for up to 2 years. Holmstedt (1980) and May (1973) reviewed reports of three other Indus trial explosions: a BASF factory In" L u d w l g s h a f en, Germany 1n 1953; the Coalite and Chemicals plant 1n England In 1968; and a 2,4,5-T producing factory In Amsterdam In 1963. Severe chloracne was the most common symptom among the exposed workers. Nervous system and unspecified Internal organ 01340 VI-1 08/10/84 damage were reported 1n the German workers. Clinical examinations w e re p e r formed on 14 of the -90 workers In the Coalite and Chemicals plant at the time of the explosion. Eleven of the 14 had altered liver function, altered hematological parameters or glucosurla, fc An accident at the ICHESA plant at Seveso, Italy, 1n 1976, resulted In the exposure of at least 8655 workers and nearby residents when a reactor vessel used 1n m a nuf a cturing 2,4,5-T exploded (Garattlnl, 1982). From this population, there were a total of 447 reported cases of chloracne, along with complaints of nausea, vomiting, headache, diarrhea, hyperhldrosls and Irritation of the eyes (Taylor, 1979; Glanottl, 1977; Crow, 1981). Serious cases of chloracne occurred 1n children within several weeks of the exposure. The Lombardy Regional Authority has compiled extensive data regarding the health effects of 2,3,7,8-TCDO on children and adults at Seveso (Pocchlarl et a 1., 1979; Boerl, 1978; Chla p p ln o et al., 1978; Slrchla, 1978). Reduced peripheral nerve conduction velocities occurred In both adults and children, with a correlation between the Incidence and the dis tance from the plant. Total serum complement activity, lymphocyte blasto- genlc response and peripheral blood lymphocytes were elevated In children exposed to the accident (Tognonl and Bonaccorsl, 1982). The limited number of studies regarding the Immunological effects of 2,3,7,8-TCDD In adults have not revealed any reduction In Immunocapablllty (Regglanl, 1980; May, 1982). . Caramaschl et al. (1981) reported an Increase In the frequency of h e a d aches, eye Irritation, gastrointestinal tract symptoms and abnormal y-GT, 01340 VI-2 09/18/84 serum GPT and aminolevulinic acid levels In children, living 1n the Seveso area, who developed chloracne. Increased urinary glucarlc acid levels, Indicative of Increased microsomal enzyme activity, were found In children 3 years after the accident (Ideo et a!., 1982). t, Six children dermally exposed to contaminated soil (30 ppm, 30 mg/kg soil) 1n horse arenas 1n Eastern Missouri developed headaches, skin lesions and p o ly a r th r a lg l a (Kimbrough et al., 1977). In the most severe case, eplstaxls and lethargy were reported. Numbness of the extremities, skin rashes and Irritation, U v e r dy sf u n c tion, weakness, loss of sex drive and psychological changes have been asso ciated with exposure to 2,3,7,8-TCDD and other dioxins, which occur a s .c o n taminants 1n Agent Orange, 1n veterans and residents of Vietnam. The r e la tionship between exposure to 2,3,7,8-TCDD and the development of these symp toms 1s, as yet, unknown (Holden, 1979; Bogen, 1979). Stevens (1981) estimated the cumulative m i ni m u m toxic dose of 2,3,7,8TCDD 1n man to be 0.1 yg/kg, based on analogy to 2 ,3,7,8-tetrach1orod1benzo-^-furan. Given the conditions prevailing 1n Vietnam, Stevens esti mated that 5 years of exposure to Agent Orange would be required to reach this dose level. Cytogenetic studies of lymphocytes from Individuals exposed to 2,3,7,8TCDD, usually by exposure to conta m in a t ed 2,4,5-T, have been performed. In two of the studies Involving exposure during the Seveso Incident, no Increase In the Incidence of chromosomal a b er r ations was observed as corn- 01340 VI-3 08/10/84 pared with nonexposed controls (Regg1an1, 1980; M ottura et al., 1981). In a third such study, changes suggestive of a muta t i on 1n functional nucleolar organizing regions of lymphocyte chromosomes were observed (QILernla et al., 1982), but this assay has yet to be validated. Workers exposed to the fc herbicides 2,4,5-trlchlofophenoxy-ethanol (2,4,5-TCPE) and Bumlnol, contami nated w i th 2,3,7,8-TCDD, were found to have an Increased Incidence (p<0.001) of chromatid-type and unstable chromosomal aberrations In peripheral lympho cytes {Czelzel and Klraly, 1976). No Increased Incidences of chromosomal aberrations were observed 1n soldiers exposed 10 years previously to Agent Orange, as compared with unexposed subjects (Mulcahy, 1980). With the exception of the Mulcahy (1980) study, the studies described In this para graph were performed within a few weeks of exposure. All studies Involved exposure to other chemicals and exposure levels were not characterized. Thirty-five cases of various types of cancer were reported In 570 V i e t n a m veterans. The cancers (Including 1 lung, 3 kidney and 2-3 1n testes) were attr i b ut e d to 2 , 3, 7 , 8- T C DD exposure during "Agent Orange" (a mixture of 2,4-D, 2 , 4, 5-T and 30-50 ppm 2,3,7,8-TCDD) sprayings 1n Vietnam (Holden, 1979). These data are of little value because the 570 veterans were not selected at random; Instead, they were selected because they com plained of symptoms related to Agent Orange exposure. According to the study, there was no reference group with which to compare these statistics. A few occurrences of soft tissue sarcoma have been reported among chemi cal Industry workers 1n the United States who were exposed to varying levels of 2,4,5-T, 2,4,5-TP, chlorophenols and 2,3,7,8-TCDD contaminants (Cook et al., 1980; Moses and Sellkoff, 1981). Honchar and Halperln (1981) reported 01340 VI-4 08/10/84 that 3 of 105 deaths among phenoxy acid workers reported by two chemical companies were from soft tissue sarcoma. This represents a 2.9% mortality rate due to this cancer while only 0.07% of the deaths among males in the United States were due to soft tissue sarcoma. Cook (1981) reported a I fourth case of malignant fibrous histiocytoma in a phenoxy acid chemical worker. For a similar industrial setting, except that 2,3,7,8-TCDD levels were <1 ppm, Ott et al. (1980) reported no increase in the cases of cancer among 204 chemical workers. This study reported on 22 deaths among the workers, however, and >75% of the men worked for <12 months in a Job Involv ing some 2,3,7,8-TCDD exposure. Epidemiological Studies Poland et al. (1971) reported the results of a health survey conducted among 73 workers who had been exposed to 2,4,5-T, d1- and trichlorophenols, dioxin contaminants and 2,4-D. Thirteen of the workers developed moderate to severe cases of chloracne. Other complaints included minimal active acne, eye irritation, hyperpigmentation, hirsutism and gastrointestinal symptoms. These effects could not be attributed to exposure to any specific compound. In a study by Walker and Martin (1979), chloracne and elevated y -GT, triglyceride and cholesterol levels have been associated with occu pational exposure to 2,3,7,8-TCDD. Other studies have reported a correla tion between the extent of occupational exposure to 2,3,7,8-TCDD and the development of chloracne (Ott et al., 1980; Cook et al., 1980). Neuropath ies have been reported in workers involved In the production of 2,4,5-sodium trichlorophenoxyacetate and trIchlorophenoxyacetate butylester (PazderovaVejlupkova et al., 1981) and phenoxy acid herbicide (Singer et al., 1982). Dioxin contaminants were suspected to be the causative agents in these cases. 01340 VI-5 09/18/84 A positive association between 2,4,5-T exposures and Increases 1n birth defects or abortions has been reported 1n human populations 1n Oregon (U.S. EPA, 1979a), New Zealand (Hanlfy et al., 1981) and Australia (Field and Kerr, 1979 ). A lack of any such association has been reported 1n human fc , populations 1n Arkansas (Nelson et al., 1979), Hungary (Thomas, 1980), New Zealand (Dept, of Health, New Zealand, 1980; McQueen et al.( 1977) and Australia (Aldred, 1978). Almost all of the reports are geographic correla tion studies, and because of the uncertainties Inherent 1n this type of epidemiologic Investigation, as well as the difficulties 1n distinguishing the effects of 2,4,5-T from those of 2,3,7,8-TCDD contamination, none of the reportedly positive associations unequivocally Identify either 2,4,5-T or 2,3,7,8-TCDO as the causative agent. Similarly, the reportedly negative associations do not rule out 2,4,5-T or 2,3,7,8-TCDD as potential teratogens or abortlfadents In humans. Based on a report of a high Incidence of abortions 1n a small group of women living around Alsea, Oregon, who may have been exposed to the herbi cide 2,4,5-T from aerial spraying (Smith, 1979), the U.S. EPA (1979a) Ini tiated a study, often referred to as the "Alsea II study," to determine 1f spontaneous abortion rates differed between the exposed and unexposed popu lation, If spontaneous abortion rates evidenced seasonal variation In these two groups, and 1f such seasonal variations were associated with 2,4,5-T spray application. . Spontaneous Abortion Rate Index, as defined by the U.S. EPA, 1s "basic ally the ratio of the number of hospitalized spontaneous abortions to the number of births corresponding to the spontaneous abortions, based on the 01340 VI-6 09/18/84 residence zip code of the women contributing to each event.* Upon comple tion of the study, the U.S. EPA concluded that (1) the 1972-1977 Spontaneous Abortion Rate index for the study area was significantly higher than In the Rural Control Area or the Urban area; (2) there was a statistically slgnlfl- I cnt seasonal cycle In the abortion Index In each of the areas with a period of ~4 months. In particular there was an outstanding peak In the study area 1n June; and (3) there was a statistically significant correlation between the Spontaneous Abortion Rate Index and spray patterns In the study area when a lag-time of 2 or 3 months was Included. The U.S. EPA concluded how ever, "This analysis Is a correlational analysis, and correlation does not necessarily mean causation.11 H1lby et al. (I960), citing three critiques of the Alsea II study that were not published 1n the open literature, state that the statistical method and basic design of the Alsea II study were sufficiently flawed to make this study of no use In human risk assessment. The Alsea II study has also been reviewed by a panel of epidemiologists who, In a published report of their meeting, also concluded that the basic design of the study was inadequate to demonstrate either an effect or absence of an effect of exposure to 2,4,5-T (Coulston and Olajos, 1980). The major Inadequacies of the study were that the data collection methods were biased and would likely result In the underestimation of abortions, particularly 1n the urban area (the Incidence of abortions 1n all three groups was within the expected background rate of 8-15%); only a small portion, of the area from which the exposed subjects were selected was actually sprayed with 2,4,5-T; and the study was not con trolled for other factors such as age, smoking habits and alcohol consump tion, which may affect the spontaneous abortion rate. Based on a report 01340 VI-7 09/18/84 by Smith (1979 ), the II.S. EPA is attempting or has attempted to correlate 2,3,7,8-TCDD levels in the affected areas with the observed rate of abor tion. No published reports have been encountered on the outcome of this eIffor t. In the only other report encountered on a population in the United States, Nelson et al. (1979) noted a general increase in the reported Inci dence of facial cleft in both high and low exposure groups in Arkansas from 1948 to 1974. In this study, exposure estimates were based on average rice production in different areas of Arkansas, and the incidence of cleft palate was determined by screening birth certificates and checking records of the Crippled Children's Services. No consistent exposure/effeet correlations were noted, and the general increase with time 1n the Incidence of facial clefts was attributed to better reporting procedures; however, there does not have to be a direct correspondence of malformations 1n human beings and experimental animals. Of the four reports available from New Zealand (Dept, of Health, New Zealand, 1980; McQueen et al., 1977; Hanlfy et al., 1981; Smith et al., 1982a), the report by the Department of Health Is essentially anecdotal, Involving two women who gave birth to malformed children (one with an atrial septal defect and a malformation of the tricuspid valve of the heart and the other with biliary atresia). In both cases, exposure to 2,4,5-T could not be ruled out. Based on an analysis of spraying records, the time course of the pregnancies and plant damage near the women's homes, however, the Department of Health, New Zealand (1980) concluded that there was insuffl- 01340 VI-8 08/10/84 dent evidence to Implicate 2,4,5-T spraying as a causative factor. Even if the spraying had been implicated, a lack of information on 2,3,7,8-TCDQ levels In the spray and the absence of any monitoring data on 2,4,5-T or 2,3,7,8-TCDD would limit the usefulness of this report, fe , The study by McQueen et al. (1977) is not published in the open litera ture but 1s summarized by Mil by et al. (1980). According to the summary, McQueen et al. (1977) "...examined the epidemiology of neural-tube defects In three areas in New Zealand and concluded 'there 1s no evidence to Impli cate 2,4,5-T as a causal factor in human birth defects.'* No additional details are provided. Hanlfy et al. (1981) performed an epidemiologic study in Northland, New Zealand, in areas where spraying of 2,4,5-T was conducted by various com panies for a number of years. The rate of birth defects was obtained from an examination of hospital records 1n seven mutually exclusive areas on a monthly basis over a period extending from 1959-1977. The rate of birth defects from 1959-1965 represented the rate for a nonexposed population since this occurred before the use of 2,4,5-T, while the Incidence of birth defects from 1972-1976 represented the rate for the exposed population. Ouring the time of the survey there were 37,751 births, 436 stillbirths, 264 deaths shortly after birth and 510 congenital anomalies. Three categories of birth defects, heart abnormalities, hypospadias and epispadias, and talipes, had elevated rate ratios of >1 (p=0.05) In comparisons between the exposed (1972-1976) and control (1959-1965) populations. Exposure estimates were made for the seven areas and for different years using company records of aerial spraying and a model that factored In assumed fractional removal 01340 VI-9 08/10/84 rates/month (this factor was assumed to be either 1.0 or 0.25). Comparisons of the rate of specific malformations with exposure demonstrated a statis tically significant association between the occurrence of talipes and expo sure when the fractional removal rate was assumed to be 0.25. There was, t however, no statistically significant association where 1.0 was used as the fractional removal rate. Smith et al. (1982a) Investigated the outcome of pregnancy In families of professional 2,4,5-T applicators and agricultural contractors In New Zealand. Agricultural contractors were chosen as the control population since both sprayers and contractors were of the same economic group with similar outdoor occupations. The survey was conducted by mall with 8914 of the chemical applicators responding and 83% of the agricultural contractors responding to questions asking whether they used 2,4,5-T and Its temporal relationship to reproductive histories regarding birth, miscarriages, still births and congenital defects. The relative risks of congenital defects and miscarriages were 1.19 (0.58-2.45% confidence limits) and 0.89 (0.51-1.30% confidence limits) for the wives of chemical sprayers as compared with the wives of agricultural contractors. These data Indicate that exposure of fathers and mothers (e.g., while cleaning clothes) had no effect on the out come of pregnancy. Biases that may have affected the results, such as the age of the mother at childbirth, smoking habits and birth to Maori parents were Investigated and eliminated as possible confounders. t The two reports from Australia (Aldred, 1978; Field and Kerr, 1979) also present apparently conflicting results. The report by Aldred (1978) is not published In the open literature, but the following summary 1s taken from 01340 VI-10 08/12/84 M11by et al. (1980): 'The report concluded that birth defects In a group of babies born in the [Yarram] district 1n 1974 and 1976 could not be attrib uted to exposure to 2,4,5-T or 2,4-0.'. Additional details that might be useful 1n assessing the rationale for this statement are not provided In the summary. The report by Field and Kerr (1979) plotted the Incidence of neural-tube defects (anencephaly and meningomyelocele) 1n New South Wales, Australia, over the years 1965-1975, and the previous years usage of 2,4,5-T 1n all of Australia. The authors noted a decrease 1n the incidence of neural-tube defects expected on the basis of the plotted line 1n 1975 and 1976, when Australia Instituted monitoring of 2,4,5-T to ensure a 2,3,7,8TCDO level <0.1 ppm. The data were not tested for significance, although Field and Kerr (1979) indicate that they consider the epidemiological data on neural-tube defects to be 'relatively complete'; however, they do not comment on the Increasing Incidence of neural-tube defects with time and whether or not an Increase 1n the thoroughness of reporting neural-tube defects could have contributed to the apparent correlation of 2,4,5-T exposure with these defects. A visual replotting of the data suggests that the Incidence of cleft palate correlates better with 2,4,5-T usage than with time. Nonetheless, the appropriateness of correlating 2,4,5-T usage In all of Australia with the Incidence of defects In one area of Australia Is questionable. Thomas (1980) used an approach similar to that of Field and Kerr (1979) on data from Hungary. One major difference, however, is that Thomas (1980) compared the Incidence of stillbirths, cleft Up, cleft palate, spina bifida, anencephalus and cystic kidney disease 1n all of Hungary betweer 1976 and 1980 with 2,4,5-T use 1n 1975 In all of Hungary. Because Hungary requires compulsory notification of malformations diagnosed from birth to 01340 VI-11 08/10/84 age 1 year, because a relatively large percentage (5576) of the Hungarian population lives 1n rural areas where 2,4,5-T exposure may be expected to be greatest, and because annual use of 2,4,5-T In Hungary had risen from 46,000 k^ 1n 1969 to 1,200,000 kg 1n 1975, Thomas (1980) considered Hungary to be ...probably the best country In which to examine possible health effects of this herbicide.* In any event, all Indices of birth defect rates decreased or remained stable over the period of study. In addition to contamination of 2,4,5-T being a potential source of 2,3,7,8-TCDD exposure, 2,3,7,8-TCDD Is also an Inadvertent contaminant of 2,4,5-trlchlorophenol (TCP). Chronic exposure to 2,3,7,8-TCDD may occur during the manufacture of TCP and high level acute exposure to 2,3,7,8-TCDD has occurred after an accident In July 1976, at the ICHESA TCP chemical factory In Seveso, Italy (Bonaccorsl et al., 1978). In this accident, the reaction used to produce TCP became uncontrolled, producing conditions favorable for 2,3,7,8-TCOO formation before venting the contents of the chemical reactor Into the atmosphere. The resulting cloud of chemicals settled over a heavily populated area. Although the amount of 2,3,7,8-TCDD released was not known, the reported cases of chloracne, a symptom of acute exposure to 2,3,7,8-TCOD, Indicated that exposure to 2,3,7,8-TCDD had occurred. Some preliminary results are available from epidemiologic studies of reproductive events 1n the Inhabitants of Seveso, and recently a study has become available on the reproductive history of men employed In the chemical manufacturing Industry with possible chronic exposure to 2,3,7,8- TCDD (Townsend et al., 1982). 01340 VI-12 08/10/84 Epidemiologic studies to determine the reproductive effects In Individ uals exposed to 2,3,7,8-TCDD and TCP following the accidental contamination of a populated area around Seveso, Italy, are not completed. The Incidence of spontaneous abortions occurring between March 1976 and January 1978 have 1,, been reported for Inhabitants 1n the area around Seveso by Bonaccorsl et al. (1978), Regglanl (1980) and B1sant1 et al. (1980). The spontaneous abortion rate In the contaminated area for the three trimesters following the acci dent was 13.1, 11.0 and 13.05%, which was similar to the worldwide 15-20% frequency of spontaneous abortion. Subdividing the contaminated area Into highly, moderately, and least contaminated, and examining the rates for each area individually, also failed to demonstrate any change 1n the spontaneous abortion rate. The Incidence rates of malformations were also examined; however, the numbers were too small for meaningful assessment. There are several reasons why these studies would not Indicate that the effect of 2,3,7,8-TCOD exposure In this accident had no effect on human reproduction. The authors note that there are many difficulties In Interpreting these data. The Incidence rates of spontaneous abortions and birth defects were not adequately available for the region before the accident as a result of suspected under-reporting. The Inadequate reporting even after the accident was due to political turmoil with regard to the management of health services. Also, an unknown number of pregnancies were surgically aborted for fear of 2,3,7,8-TCDD-lnduced birth defects. In a recent review of the progress of epidemiologic Investigations of the Seveso accident, Tognonl and Bonaccorsl (1982) Indicated that the data on spontaneous abortions and mal formation rates still needed verification and that these data were too pre liminary to allow for conclusions. 01340 VI-13 08/10/84 Townsend et al. (1982) Investigated the reproductive history of wives of employees potentially exposed to 2,3,7,8-TCDO during chlorophenol produc tion. A total of 930 'potential ly exposed males were Identified who had worked for >1 month between January 1939 and December 1975 In a Job with t potential 2,3,7,8-TCDD exposure. Exposure estimates of low, moderate and high were made by an Industrial hygienist primarily from Job description and surface contamination data; however, the high potential exposure group was reserved for process workers during 1963-1964 when changes In operations resulted 1n a number of cases of chloracne. The control population was an equal number of male employees not Involved In any process that might cause exposure to 2,3,7,8-TCDD and matched for date of hire. In these groups, 586 wives were Identified and 370 agreed to participate as the exposed group, while 345 wives In the control group agreed to participate. After Identifi cation of the participants, a personal Interview was conducted with the wives to determine pregnancy outcome. Of the total of 737 conceptions In the exposed category and 1785 conceptions 1n the control category (concep tion that occurred In the exposed group before availability of work records Indicating potential exposure to 2,3,7,8-TCDD were placed In the control group), there was no statistically significant Increase 1n spontaneous abortions, stillbirths. Infant deaths or selected congenital malformations. Sample sizes were too small to provide meaningful data If the populations were subdivided by extent of exposure. It was suggested that many confounding factors could account for these negative results, such as the Inappropriate selection of 'the populations, unidentified covarlables and Insufficient power; however, the authors maintained that these results were consistent with animal data, which report that paternal exposure to 2,3,7,8-TCDD does not affect the conceptus. 01340 VI-14 08/10/84 Poole (1983), In testimony before the House Committee on Science and Technology, described a reanalysis of the primary data used by Townsend et al. (1982). In this reanalysis, the rate of cleft palate and cleft U p were reported to be elevated by 1.9 (90* confidence Intervals of 1.0-3.6) In the I, years 1971-1974 for both the control and exposed groups (the comparison population was not described). At the same House Committee hearing, Houk (1983) presented data from the Birth Defect Monitoring Program of the Center for Disease Control on the yearly rate of cleft palate alone or cleft U p with or without cleft palate for births In Midland County, Michigan (the site of a chlorophenol production facility) during the years 1970-1981. The data Indicated an Increased rate for these defects of between 50 and 100% in the years 1971-1975, with the rate returning to expected from 1976-1981. The observed Increase was only statistically significant 1f the rates for cleft palate alone and cleft lip with or without cleft palate were combined; however, 1t was the opinion of Houk (1983) that these defects should not be combined since the causal mechanism may be different. The Michigan Depart ment of Public Health (1983a) also reported these results and, In addition, demonstrated that the same results occurred If the comparison was made with other counties 1n Michigan as well as with the general population of the United States. It was noted In this report that "runs* of Increases In oral cleft for successive years have occurred 1n six other counties with no obvious potential for chemical exposure described. The Michigan Department of Public Health (1983a) Interpreted the data to Indicate that a more detailed case control study was necessary to determine If any common factors may exist, such as exposure to chemicals contaminated with 2,3,7,8-TCDO. 01340 VI-15 08/10/84 Several Investigators have suggested that 2,3,7,8-TCDD 1s the causative agent of excess Hver carcinoma and soft-tissue sarcoma associated with occupational or accidental exposure to phenoxyacetlc acid herbicides. Direct evidence 1s limited since the dioxins usually occur 1n conjunction fc with other chemicals and quantitative exposure data are not available. Observations of an unusual occurrence of relatively rare soft-tissue sarcomas were first made by Harden (1977). Of some 87 patients seen from 1970-1976 at the Department of Oncology, University Hospital, Umea, Sweden, seven individuals with soft-tissue sarcomas were Identified. All seven had had occupational exposure to phenoxy acids 10-20 years earlier. The tumors were 2 leiomyosarcomas, 1 llposarcoma, 1 rhabdomyosarcoma, 1 myxofibrosar coma and 2 additional sarcomas of which the hlstopathology was uncertain, but one was probably a neurofibrosarcoma and the other a rhabdomyosarcoma. The clustering of this rare tumor type among these patients prompted the author to suggest that epidemiological studies be done to determine 1f exposure to phenoxy acids and the Impurities they contain are related to the occurrence of soft-tissue sarcomas. Zack and Susklnd (1980) reported a soft-tissue sarcoma death In a cohort study of workers exposed to 2,3,7,8-TCDD 1n a trIchlorophenol process acci dent 1n N1tro, West Virginia. This tumor, a fibrous histiocytoma, was noted by the author as a rare event. This study, referred to as the Nltro study, 1s discussed later. - Cook et al. (1980) 1n a cohort mortality study of 61 male employees of a trlchlorophenol manufacturing area, who exhibited chloracne following a 1964 01340 VI-16 08/10/84 exposure incident, noted four deaths by the end of his study period, one of which was due to a fibrosarcoma. The authors did not seem to attribute any special significance to this finding at the time. Ott et al. (1980) in a cohort m o r t a l i t y study of 204 e mployees ex po s e d to 2,4,5-T during its manufacture from 1950 to 1971, found no soft-tissue sarcomas a mong 11 deaths that had occurred by 1976. One of these 11 deaths was due to a malignant neoplasm. In a review of the studies of Zack and Suskind (1980), Cook et al. (1980), an unpublished study by Zack (in which a liposarcoma was found), and a study by Ott et al. (1980), Honchar and Halperin (1981) noted 3 (2.9%) s o f t - t is s u e sarcomas in a total of 105 deaths, c o mp a r ed r oughly to 0 . 0 7 % deaths due to soft tissue sarcoma e x pected In United States males 20-84 years old, (ICO 171, 8th Revision, 1975)* indicating an unusual excess of such tumors. The researchers underestimated the results because of the possiblity that some soft- t is s u e sarcomas may have been coded to categories other than ICO 171. Individually, none of these case studies reported a significant excess of soft-tissue sarcomas. Cook (1981) found an ad di tional malignant fibrous histiocytoma after a later review of the medical records from his earlier cohort study. Cook, who was familiar with the three earlier cases, noted that frank c h l o r a c n e o c cu r r ed p r e v i o u s l y in two cases of the four having a diagnosis of malignant fibrous histiocytoma. A third person d i a g n o s e d as havi n g a f i br o s ar c o ma w o rk e d in a trichlor o p he n o l `Department of Health, Education, and Welfare. U.S. Public Health Service. National Center for Health Statistics of the United States, 1974. Vol. II. M o rtality, Part A. 01340 VI-17 04/14/88 (TCP) process area cont a m in a t ed with 2,3,7,8-TCDD. This individual e x h i b i t ed facial dermatitis but no diagnosis of chloracne. The fourth case (diag nosed as a liposarcoma) was an individual who had been empl o y ed earlier in a plant producing 2,4,5-T. Cook (1981) noted that although chloracne was not reported, it could not be discounted. He also noted that all four were smokers and suggested that c i ga r e tt e smokers with c h lo r a cn e caused by 2 , 3 , 7 ,8-TCDD exposure may be subject to an increased risk of fibrous softtissue sarcomas. H a r d e n and Eriksson (1981) discounted this hypothesis by citing that only one of Hardell's seven cases exhibited chloracne before the appearance of the soft-tissue sarcomas, and that in their subsequent case control study, they found no d i f f e r e n c e in smoking habits b etween his cases and controls. Moses and S e likoff (1981) r e po r t ed a fifth s o f t - t is s u e sarcoma in a worker employed at the Monsanto Chemical Company at a time when trichlorophenol and 2,4,5-T were being produced. He died of a retroperitoneal neuro genic sarcoma (malignant schwanoma) in 1980 at the age of 58. The employee, prior to his death, in a deta i l ed occup a ti o n al hist o r y said that he believed he was exposed to these chemicals while he was a truck driver, hauler and m a i n t e n a n c e worker, but that he did not work in the production of either chemical. He was a nonsmoker and did not have a history of chloracne. Johnson et al. (1981) treated a father and son with soft-tissue sarcomas (the 33-year-old son was diagnosed as having a fibrosarcomatous m e s o t h e lioma, while the 53-year-old father had a liposarcoma). Both were exposed 01340 VI-18 04/14/88 to halogenated phenol derivatives. The author noted that 2,4-dlchlorophenal can be a precursor of 2,4-D and 2,4,5-T. The father had had prolonged expo sure before the diagnosis. The son supposedly had a shorter latency, according to the author. In neither case was the follow-up time given, t. Sarma and Jacops (1981) reported three cases of thoracic soft-tissue sarcoma In Individuals who were presumably exposed to Agent Orange while serving 1n Vietnam. The diagnoses were fibrous histiocytoma, mediastinal fibrosarcoma, and a pleural/dlaphragmatlc leiomyosarcoma. All three served In areas where defoliants were used at the time. One was drenched with the material 1n a single spraying. Bishop and Jones (1981) found two cases of non-Hodgkin's lymphomas of the scalp 1n a related clinical study of 158 employees of a pentaehlorophenol manufacturing plant In Wales. Homologues of 2,3,7,8-TCDD occurred as contaminants at up to 300 ppm at Intermediate manufacturing stages and 5 ppm 1n the final products. Mild, moderate and severe cases of chloracne were seen 1n many employees, Including the two men who subsequently developed lymphomas. Both men worked In processes where exposure to other chemicals occurred. Including exposure to aromatic hydrocarbons. The authors reported that only 0.28 tumors of this type could be expected to occur In a group of 158 workers (ICO 200 and 202), although the basis for the computation of expected numbers Is not stated. p Olsson and Brandt (1981) noted that of 123 male patients seen at their clinic In Sweden with a recent diagnosis of non-Hodgkin's lymphoma (NHL), 5 had cutaneous lesions as the only clinically detectable manifestation of NHL. four of them were reported to have had repeatedly sprayed large areas 01340 VI-19 08/10/84 with phenoxy acid herbicides. In the remaining 118 NHL patients, only seven had a similar occupational exposure to phenoxy acids. The authors reported this to be significant at p<0.001. Olsson and Brandt (1981) suggested that a relationship exists between cutaneous presentation of NHL and occupational t exposure to phenoxy acids, and believed their observations were similar to those of Bishop and Jones (1981). The total number of workers with these Illnesses who were exposed to phenoxy acids or chlorophenols or both Is small, but considering the rarity of this cancer, It 1s unusual that so many cases of soft-tissue sarcomas have occurred. A Lancet editorial (Anonymous, 1982) calls this phenomenon "disturbing.* Soft-tissue sarcomas (STS) constitute a collection of heterologous lesions that Include both malignant and nonmallgnant tumors. Not all of them have their origin In primordial mesenchymal cells. Some exceptions are tumors of peripheral nerves, and neuroectodermal tumors that are classified as STS, but are derived from nonmesenchymal cells. Classification, grading and staging of STSs 1s difficult because of the capacity of such cells to differentiate Into many different tissues. Fairly precise hlstogenetlc classification of such tumors Is accomplished through consideration of growth patterns and cell morphology, and evaluation of Intracellular and extracellular products of tumor cells. There are a dozen distinctly dif ferent classes of mesenchymal cells that develop Into the following six well-defined tissue complexes: fibrous tissue, tendosynovlal tissue, adipose tissue, muscle, vessels and bone. STSs can be Induced In any of these tissue types (Hajdu, 1983). The classification of STSs for cause of death coding 1n the ninth and latest revision of the International Classlfl- 01340 VI-20 08/10/84 cation of Diseases (ICO, 1975) places STSs Into one of several categories. But chiefly, they fall Into 'malignant neoplasms of connective and other soft-tissue' (ICO 171). Lymphosarcomas, retroperitoneal sarcomas and extra skelt etal STSs of the bone are coded elsewhere. In some Instances, If site Is mentioned, It Is coded to the site, e.g., leiomyosarcoma of the stomach (ICO 151.9), neurofibroma of the chest wall (215.4). Questions have been raised concerning the appropriateness of lumping together malignant tumors of different sites and tumor types 1n order to derive risk estimates. It may not be scientifically appropriate to do so because an elevated risk cannot readily be ascribed to a particular site or type as 1s usual with most carcinogenic chemicals and substances. Unfortu nately, with respect to STSs, tallies of deaths caused by STSs of particular sites and types are not maintained separately by the vital statistics offices because of their rarity, and therefore, 1t Is Impossible to derive risk estimates for particular types at given sites. Altogether, -2000 deaths/year can be attributed to STSs In the United States, most of which are coded to ICO category 171 for purposes of developing Incidence and mor tality rates for this composite cause. Within ICO 171, Individual types that may be correlated with exposure cannot be Identified. A separate problem that potentially could arise from assigning STSs to multiple IC.D codes 1s that Incidence and death rates from STSs may be underestimated. Furthermore, risk estimates derived from dividing observed cases (or deaths) by expected cases (or deaths) could be biased upward. This could happen when observed STSs classified to ICO codes other than ICO 01340 VI-21 08/10/84 171 are lumped together while expected STSs are based upon ICD 171 only. Thus, action of this sort, especially with respect to cohort studies of Individuals exposed to dioxin-containing herbicides or chlorophenols or both, could Vead to risk estimates that may be biased upward by the Inclusion of STSs 1n the observed category for risk estimation that should be coded to categories other than 171. Prompted by clinical observations over a 7-year period of malignant sar comas 1n seven men with previous occupational exposure to phenoxyacetlc acid herbicides (Harden, 1977), researchers at the Department of Oncology, University Hospital, Umea, Sweden, Initiated case-control epidemiologic studies (Cole, 1979) to test the hypothesis of an etlologlc association (Hardell and Sandstrom, 1979). Cases were defined as male patients with sarcomas of soft connective tissue, such as smooth muscle (leiomyosarcoma) and fat (llposarcoma). The distribution of tumor types In the two studies Is shown In Table VI-1. Sarcomas of tissues, such as bone and cartilage, were excluded as cases. According to the authors, these tumors may have a different etiology and there occurred a different age-d1str1but1on In patients with these tumors as compared with that of STS (Harden, 1983). Two case-control studies were conducted, the first In northern Sweden (referred to as Study A), and the second in the southern part of the country (Study B). The exposures to the substances of primary Interest are shown in Table VI-2. In the north (Study A), occupational exposure to phenoxyacetlc acids took place In both forestry and agricultural work. In the south (Study B), these exposures were predominantly agricultural. The phenoxy- 01340 VI-22 08/10/84 TABLE VI-1 Distribution of Tumor Types 1n Two Case-Controls Studies of Soft-Tissue Sarcoma Diagnosis Tissue of Origin Percent of Cases Study Aa (n=52) Study Bb (n=l10) Leiomyosarcoma Fibrous histiocytoma * Llposarcoma Neurogenic sarcoma Angiosarcoma Myxosarcoma Fibrosarcoma Other sarcomas Total Smooth muscle Subcutaneous connective tissue Fat tissue Nerve tissue Blood vessels Primitive connective tissue Fibrous tissue 30 17 14 10 8 6 4 11 100 23 25 6 4 2 8 8 24 100 aUnpubl1shed Information supplied by Harden to EPA (Harden and Sandstrom, 1979) bEr1ksson et al., 1979, 1981 01340 VI-23 08/10/84 I TABLE VI-2 Exposure Frequencies In Two Case-Control Studies of Soft-Tissue Sarcoma Substance(s) Percent Exposed Study A Study B Cases (n52) Controls (n=2Q6 ) Cases (n=l10) Controls (n=219) Phenoxyacetlc acids only Chlorophenols only Both Total 23.1 11.5 1.9 36.5 6.3 12.7 2.3 2.4 10.0 3.6 0.5 0 0 9.2 22.7 5.9 Sources: Study A, Harden and Sandstrom, 1979; Study B, Eriksson et al., 1979, 1981 01340 VI-24 08/10/84 acetic acids to which exposure occurred consisted p r ed o m inantly of 2,4,5-T and 2,4-D in both studies. E x po s u re to 2 ,4,5-T in the ab se n c e of 2,4-D was rarely reported in either study. E x po s u re to c h lo r o ph e n ol s , which contain c h l o r i n a t e d d i b e n z o d i o x i n impurities (Levin et al., 1976) occurred most l y in sawmill work and paper pulp production. Very few persons reported exposure both to phenoxyacetic acid and chlorophenols in these studies. Of the two p r e d o m in a n t p h e n o x ya c e ti c acids, only 2,4,5-T is known to be c o n t a m i n a t e d with 2 , 3, 7 ,8-TCDD. In S t udy B, a rela t i ve risk of 4.9 (9Q% c o n f i d en c e intervals 1.6-11.1) was found in relation to e x po s u re to p h e n o x ya c e ti c acid herbicide other than 2,4,5-T (2,4-D, MCPA, mecoprop, dichloroprop). R e la t i ve risks in r e la t i on to the three m ajor cate g o ri e s of e x po s u re are shown in Table VI-3.* Studies A and B indicate a risk of d e v e l o p i n g STSs among work e r s exposed to phenoxya c e ti c acids only, chlo r o ph e n ol s only, or phenoxyacetic acids and/or chlorophenols several times higher than among persons not exposed to these chemicals. In each comparison, the relative risk is high {p < 0 .005) and u n li k e ly to have r e sulted by chance alone. Since little is k nown of the e t io l o g y of STSs, the c o n s i d e r a t i o n of c o n founding factors in these studies was largely a hypothetical matter. The authors prevented the effects of age, sex, and place of residence as possible *In the analyses considering phenoxyacetic acids only and chlorophenols only, persons exposed to the other categories of substances were excluded. In Study A, the three persons exposed to both c h l o r o ph e n ol s and p h e n o x y acetic acids were included in all comparisons. 01340 VI-25 04/14/88 01340 TABLE VI-3 Relative Risks of Soft-Tissue Sarcoma 1n Relation to Exposure to Phenoxyacetlc Acids and Chlorophenols 1n Two Case-Control Studies3 VI-26 Phenoxyacetlc Acids Only Study A Study B Chlorophenols Only Study A Study B Phenoxyacetlc Acids and/or Chlorophenols Study A Study B Relative risk*1 90% Confidence Interval Significance level1* 5.3 2.7-10.2 <0.001 6.8 3.1-14.9 <0.001 6.6 2.8-15.6 <0.001 3.3 1.6-7.0 <0.005 5.7 3.2-10.2 <0.001 4.7 2.7-8.3 <0.001 aSource: Study A, Hardell and Sandstrom, 1979; Study B, Eriksson et al., 1979, 1981 ^Unmatched odds ratio cTest-based method of Mlettlnen, 1976 *Ch1 square statistic, no continuity correction, one-ta1led test 04/05/84 confounding factors in the selection of controls.* Because of the high cor relation between exposure to the substances of interest and employment in agriculture and forestry, a possible alternative hypothesis could be developed that some other unknown factor present in these occupations was i responsible for the elevated relative risks. To test this hypothesis, 1t 1s possible to calculate the relative risk 1n relation to the phenoxyacetic acid exposure 1n Study 8, restricting the analysis to workers within agriculture and forestry. The result 1s a rela tive risk of 6.1 (90% confidence Interval 2.4-15.4). This finding strongly suggests that some confounding risk factor for STS distributed throughout agriculture and forestry work was not responsible for the overall Increase in risk found In relation to phenoxyacetic acid exposure. 8ecause exposure histories were obtained by means of questionnaires and interviews, the major potential source of bias 1n these studies stems from the need to rely upon the personal recollection of cases and controls for exposure histories. The published papers Indicate that the researchers paid a great deal of attention to this potential problem and specific efforts were made to avoid 1t during the conduct of the study. In addition, the relative risk calculated by considering the agriculture and forestry workers who did not report exposure to phenoxyacetic acids or Controls were matched individually to cases on the basis of these factors. Unmatched analyses are presented 1n Table VI-3 for the sake of simplicity. The matched-method relative risks for exposure to phenoxyacetic acids and/or chlorophenols were 6.2 (p<0.001) in Study A and 5.1 (p<0.001) in Study B. 01340 VI-27 08/10/84 chlorophenols and comparing them with unexposed persons 1n other occupations was 0.9 (90% confidence Interval 0.3-2.4) In Study 8, This suggests that little recall bias was present (Axelson. 1980). *In an update of their earlier study, Eriksson et al. (1981) obtained Information on the effects of phenoxy acids 1n the absence of the Impurlt1es--polychlor1nated d1benzod1ox1ns and dlbenzofurans. The risk ratio given exposure to phenoxy acids free of polychlorinated dlbenzodloxlns and dlbenzofurans equaled 4.2 based upon 7 out of 14 respondents who Indicated exposure to phenoxy acid herbicides. When consideration was given to persons exposed to only phenoxy acids that contain such Impurities, the relative risk was 17.0. A description of the basis for the determination of exposure or nonexposure to dioxins Is not well presented In this study. The authors concluded that exposure to phenoxy a d d s and chlorophenols might constitute a risk factor 1n the development of soft-tissue sarcomas." This risk relates not only to 2,4,5-trlchlorophenoxy acids containing dioxin Impurities, but to other phenoxy ad d s as well. Some doubt was raised con cerning the possible mlsclasslfIcatlons of Individuals who were exposed to phenoxy adds free of polychlorinated dlbenzodloxlns [1.e.t 1n particular, "dlchoroprop* 1n the Eriksson et al. (1981) study]. In a recent communica tion from Harden (1983), Eriksson recalculated risk estimates after reclassifying dlchoroprop-exposed cases and controls Into the category of probable exposure to phenoxy a d d s contaminated with polychlorinated dlben zodloxlns and removing them from the nonexposed category. The new estimates were 4.0 based upon 5 out of 8 respondents who were exposed to phenoxy adds free of contamination and 10.9 for those exposed to contaminated phenoxy 01340 VI-28 09/18/84 acid. The first estimate was of only borderline significance utilizing the Mletlnen test based statistic, thus, weakening any finding that the risk of STS extends to phenoxy acids free of dioxin. * In a cohort mortality study Cook et al. (1980), studied 61 males Involved In a 1964 exposure Incident who had chloracne caused by absorption of 2,3,7,8-TCDO. The skin lesions characterizing chloracne ranged from a few comedones on the back of one employee (predating his entry Into the process area where exposure could occur) to severe cysts and comedones over the faces, scalps, ears, necks and backs of the remaining employees of the group. Since the main route of exposure was not through the respiratory tract, no measurements of dioxin 1n the air were provided by the author. On the other hand, the author did divide the cohort of 61 males Into potent ially high' vs. "low* exposure by place of work based upon dermal exposure, although not stated. Vital status was traced from the data of the Incident through 1978. Altogether only 4 deaths were observed by the end of the follow-up vs. 7.8 expected. Of these, 3 were cancer deaths vs. 1.6 expected. The remaining death was hypersensitive heart disease vs. 3.8 expected. The histopathologic causes of death of the three cancer victims were 1) fibrosarcoma, 2) glioma with mtastass, and 3) adenocarcinoma. The authors report that all three victims smoked a minimum of one pack of cigar ettes a day for 'many years.* Hot enough Information Is provided by the authors to conclude that any of these four deaths were smoking related. Site of tumor 1s not mentioned 1n the cancer deaths. Cancer mortality was slightly elevated 1n this cohort. This study had low sensitivity and lacked a sufficient latent period. This Increased 01340 VI-29 09/18/84 mortality was not attributable to any particular cause and no deaths were attributable to liver cancer. Additionally, the authors state that only one of the -cancer deaths possessed "documented" evidence of chloracne, although this appears to be at variance with the definition of the cohort, which was reported by the authors to consist of males who reported to the medical department with skin conditions subsequently "diagnosed as chloracne." The authors concluded that the latency period was sufficient to "allow the Identification of a potent human carcinogen," since 1t "exceeded 14 years." Orris (1981) noted that 1n the Hardell and Sandstrom (1979) study the authors stated that the latent period for soft-tissue tumors may be as long as 27 years and for many, over 14 years. In any case, Hueper and Conway (1964) noted that the latent period for the chemical Induction of solid malignant tumors 1n man exceeds 15 years and 1s probably <30 years. Smith et al, (1982b) conducted an Initial case-control study of 102 males Identified from the New Zealand Cancer Registry as having STSs (ICO 171) between 1976 and 1980. For each case, three controls each with another form of cancer were matched by age and year of registration. The selection of cancer controls from the same registry was done to eliminate recall bias or Interviewer bias or both. The distribution of histological types 1n the cases Is given 1n Table VI-4. An Interview to elicit occupational history Information was accomplished by telephone either to the next of kin or the patient himself, If he was well enough, although the Information was not used 1n this preliminary analysis. Comparisons between cases and controls were accomplished by use of occupational groupings according to the Standard Classification System of 01340 VI-30 08/10/84 TABLE VI-4 Distribution of Histological Types of Soft-Tissue Sarcomas'* Cell Type Fibrosarcoma Uposarcoma Rhabdomyosarcoma Leiomyosarcoma Malignant Histiocytoma Other Unspecified Total `Source: Smith et al., 1982b Number of Cases 25 20 9 7 6 22 13 102 Percent 24 20 9 7 6 21 13 100 01340 VI-31 08/10/84 New Zealand focusing on those occupational groups with a potential for exposure to phenoxy herbicides and chlorophenols. Expected cases for each major occupational classification were derived based upon the occupational distribution of the controls. The authors found no unusual excess of cases o? STS 1n any major occupational category. In agriculture, forestry and fishing, 14 cases were observed vs. 14.0 expected. In laborers, production and transport workers, 35 cases were observed vs. 37.0 expected. A further breakdown of these two broad categories Into finer subcategories within the major occupational categories revealed no significant excesses. The study, however, Is not useful In assessing the risk of STS from exposure to phenoxy adds or chlorophenols for several reasons. First, as was pointed out by the authors but subsequently dismissed by them as having not much of an Influence, 1s the possibility that movement from one major occupational category to another over the time period Involved for latent conditions to manifest themselves could Introduce a negative bias Into any estimates of relative risks. The latency for STS was suggested to be a minimum of 15 years (Hueper and Conway, 1964). The finding of no switching from one occupational category to another that was noted In the 'first 20 Interviews' In which a change could be noted 1s not necessarily Indicative of fidelity to the same job over long periods In all 408 cases and controls. Information Identifying a change may be lacking In those cases and controls 1f In fact one did occur possibly for several reasons, (separation of 'the earlier work history from the latter; purging of earlier employment records, etc). Besides, the 'first 20 Interviews' where a change could be noted Is not necessarily representative of the entire cohort 1n any case. 01340 VI-32 09/ 18/84 Furthermore, the authors do not know absolutely that any of their cases and controls were exposed to phenoxy acids or c h l o r o p h e n o l s , since a p p a r ently no effort was made to co nf i r m "potential" exposures. Only differences in o c cupational c l a s s i f i c a t i o n were noted w h er e "potential" cases or c o n trols could have had exposure to the d i ox i n - c o n t a i n i n g herbicides. It was pointed out that the risk estimates noted do not "preclude" the possibility that an association may be found in this study when the cases and controls {or surviving kin) are interviewed for chemical spraying at a later time. The authors themselves conclude that the preliminary study results "should not be taken as substantial evidence against the hypothesis that phenoxy herbicides and chlorophenols may cause human cancer" (Smith et al., lS82b). The d i st r i bu t i on of tumor types differed consid er a b ly in the H a r d e n and Eriksson et al. (1981) study compared to the ,Smith et al. (1982b) study. Leiomyosarcomas, malignant histocytomas, neurogenic sarcomas and myxosarcoma seem to p r edo m ina t e in the H a r d e n and Eriksson (1981) study, whereas f i b r o sarcomas and liposarcomas appear prominently in the Smith et al. (1982b) study. More attention should be devoted to the study of the distributions of STS types in r e gi s t r y data e v e r y w h e r e in order to d e t e r m i n e if such vari a t io n s in the r e po r t i n g of STS types are r a n d o m o c currences. It is possible that the cancer effect of exposure to phenoxy herbicides may be n a rr o w ed to just c e r t a i n types of STSs, the p r ed o m i n a n t ones in the S wedish studies. Smith et al (1983) conducted another case-control study of STSs in males that were reported to the New Zealand Cancer Registry by Public Hospitals between 1976 and 1980. The author matched one cancer control randomly chosen from the registry with each case, initially starting with 112 of 01340 VI-33 04/14/88 each. Controls were matched for year of registration and by date of birth +2 years. Inquiries were made by the authors with the hospital consultant, family doctor, and finally the next-of-kin or patient 1f alive. Telephone Interviews were conducted by only one Interviewer who had no knowledge of the* patients cancer history and were completed on 80 cases and 92 controls. Because some 32 potential cases (14 Ineligible) and 20 controls were excluded or lost from the study for various reasons, It raises a question whether control of confounding by age and year of registration was main tained In the final group of 172 cases and control Included 1n the analysis. ' Presumably the corresponding "matched* case or control to each of the 52 lost members of the total study group were not excluded. However, since the span of registration was only 5 years, not much age confounding could occur. Patients were classified as having had potential exposure to phenoxyacetlc acids 1f they had definite, probable or possible exposure to phenoxyacetlc acid through spraying or hand contact. The actual chemical was Identified only In some Instances. The authors concluded In all remaining situations that 1f the member sprayed "gorse" or "blackberries," this was tantamount to potential exposure to phenoxyacetlc acid. Smith et al. (1983) calculated elevated but nonsignificant relative risks of exposure to phenoxyacetic acid ranging from 1.3 1n those Individuals who were "probably exposed" for a minimum of 5 days not In the previous 10 years before cancer registration to 1.6 In individuals "probably exposed" for a minimum of 1 day not 1n the previous 5 years before cancer registration. When risk ratios were calculated after stratifying by year of birth and whether or not the patient or a relative was Interviewed, the rates Increased to 1.7 (from 1.6) 01340 VI-34 08/12/84 1n the latter and 1.4 (from 1.3) in the former calculation, although still nonsignificant. If the numbers would allow, H would be of Interest to repeat the above calculations excluding only those with potential exposure occurring only within the 15-year period Just before cancer registration. The^small numbers that remain following the 15-year lapse precludes such an analysis. Furthermore, the categories of exposure "probably or definitely" exposed for >1 day or even 5 days raises a question whether any of the cases or controls could really be said to have ever come 1n contact with enough phenoxyacetlc acid to justify such a designation. It could be that, 1n fact, potentially exposed Individuals In New Zealand have had little or no contact with the herbicide. The authors did conclude that the finding of a relative risk of 1.7 1n Individuals with >1 day exposure not 1n the last 5 years cannot be entirely discounted. But then the authors state that 1f the length of exposure was 5 days or more prior to 10 years before cancer registration they would expect an Increase-, since they do not see an Increase, there Is no evidence of a "real causal link." One might ask whether this 1s a suitable criterion for providing evidence of a causal association. Perhaps a more valid group for study would be one where the potential exposure was considerably longer than "5 days" and >15 years before Initial cancer registration. As a subtle justification for the finding of no significant risk 1n workers exposed 1n phenoxy adds, the authors (Smith et al., 1983) allude to the fact that there are currently 500 full-time workers registered 1n New Zealand who do full time ground spraying and altogether some 2000 workers who were at some time professionally Involved 1n phenoxyacetlc a d d herbicide spraying from the air or ground with exposure "very much greater" than that of patients 1n 01340 VI-35 09/18/84 this study. This kind of argument has appeal if these workers could be shown to have had their exposure sufficiently far In the past that latency considerations could be adequately addressed. However, the real question again remains how much real exposure did those patients In the study have * 10-15 years earlier, and In what numbers. The authors remark that It Is surprising that they found no STS victims who had ever worked full-time 1n phenoxyacetlc acid herbicide spraying. Perhaps they have not yet been observed for a long enough period. However, as was pointed out by the authors, the findings do not support the hypothesis that exposure to phenoxyacetlc a d d herbicides causes STS. But neither do they support a negative finding without better documentation regarding actual exposure and time of actual exposure. The author does not (Smith, 1983), however, state that h1s documentation of exposure to 2,4,5-T (and 2,4-0) 1s at least as good as that In the Harden study, and that although Harden noted higher relative risks of <30 days exposure. Smith (1983) did not. Hence the paradox. Smith does admit the possibility that 2,3,7,8-TCDO contamination might be lower In New Zealand as opposed to 2,3,7,8-TCDO contamination 1n the Swedish studies. Although, there Is no evidence for 1t. Smith (1983) still maintains that his study shows that exposure to phenoxyacetlc acids may not be associated with STS. Pazderova-Vejlupkova et al. (1981) studied 80 workers Involved In the production of 2,4,5-sodium trlchlorophenoxyacetate and butylester of tr1chlorophenoxyacetlc a d d who subsequently became 111 from exposure to 2,3,7,8-TCOO during the period 1965-1968. Only 55 members of this group were followed for 10 years. The remaining 25 either refused participation or moved leaving no forwarding address. Host patients developed chloracne while 11 developed porphyria cutanea tarda. Chief chemical signs were meta- 01340 VI-36 09/18/84 bollc disturbances, pathologically elevated lipids with abnormalities In the lipoprotein spectrum, and pathological* changes In glucose tolerance. Other symptoms noted were biochemical deviations consistent with *a mild liver lesion,' light steatosis, periportal fibrosis or activation of Kupffer I cells, or nervous system focal damage (peripheral neuron lesion 1n lower extremetles). Altogether six patients were reported to be deceased during this 10-year period, 2 from bronchogenic carcinoma, 1 from cirrhosis, 1 atherosclerosis predpue cerebl and 2 1n auto accidents. No STSs or lympho mas were found. Since there was no comparison population with which to estimate relative risk for cancer, the study must be classified at best as clinical with respect to cancer. The six deaths that occurred during the 10-year observation period In the 55 cannot be construed to be associated with exposure to the 2,4,5-T. Because of the small number of cases and the short follow-up period, nothing can be said concerning the association of exposure with cancer, especially specific types of cancer such as STS or non-Hodgkin's lymphoma. R11h1mak1 et al. (1982, 1983) studied a cohort of 1926 herbicide appli cators formed 1n 1972 from personnel records of four Finnish employers (e.g., the forestry Authority, Highway Authority, State Railways and a state-owned electric power company). Chlorinated phenoxyadds had been used since the 1950s 1n Finland for spraying. They constituted 2:1 mixtures of emulsified esters of 2,4-0 and 2,4,5-T dissolved In water. Analyses from old herbicide formulations ddtlng back to the 1960s revealed that these mixtures contained 0.1-0.9 mg of 2,3,7,8-TCDD/kg formulation). This cohort of male workers was exposed a minimum of 2 weeks during at least one growing season from 1955-1971. Follow-up continued 9 years 01340 VI-37 09/18/84 through 1980 for mortality but only until 1978 for morbidity. Fifteen Individuals could not be traced by 1980. Expected deaths were generated based upon cause- and age-specific national Finnish death rates for 1975. Expected cases were similarly calculated based upon national Incidence rates i of 1975. By 1980, 144 deaths had occurred vs. 184.0 expected, a deficit of 22% 1n observed mortality. Only 26 cancer deaths had occurred vs. 36.5 expected, a 29% deficit. The authors separated out 'natural* deaths from the total. The observed residual deaths equaled 39 while the expected deaths equaled 28.7. This excess was of borderline significance. The authors also con sidered 10-year and 15-year latent periods. Even after 15 years, the defi cit of deaths continued to manifest Itself both 1n categories of all causes and total cancers; 35 observed vs. 53.6 expected and 5 observed vs. 11.3 expected, respectively. Similarly, the 7-year follow-up of cancer morbidity revealed 26 cases of cancer vs. 37.2 expected. After 10 years latency, 16 cancer cases were observed vs. 20.1 expected. None of the 26 cancer deaths or 26 cancer cases were of the STS or lymphoma type. (However, only 0.1 STS and 0.5 lymphomas were expected.) In no Instance was cancer of any site significantly elevated. The authors note that this unusual deficit of mortality and morbidity of between 70 and 82% (even after 15 years from Initial exposure) Is probably a consequence of the 'healthy worker effect' In that only able-bodied and healthy Individuals were selected Into the Industry. The fact that the cohort was assembled In 1972 from records of persons who were exposed as early as 1955 (17 years prior) raises the likelihood that In 1972 a 'survi vor* population remained (45 deaths before 1972 were eliminated from the 01340 VI-38 08/10/84 cohort) that was relatively healthy. Furthermore, the unusually large num ber of not 'natural* expected and observed deaths (probably accidents and external causes) occurring to this cohort Indicate a relatively youthful popIulation was under scrutiny. The leading cause of death to persons under 35 years 1s from accidents, based on national vital statistics. The authors correctly note that, because of limitations 1n the study material, only powerful carcinogenic effects could be detected. Risk ratios higher than 1.5 for all cancers, 4.0 for lymphomas and 10.0 for STS could be excluded based on this data set from the authors own calculations. More follow-up 1s needed 1n order to provide a stable assessment of the relation ship between exposure and cancer. The authors concluded that this study will allow no assessment of STS because 'the number of persons having a suf ficiently long latency period 1s too small." It was suggested that more valid conclusions could be made only with the passage of time (R11h1mak1 et al., 1983). Recently, the Michigan Department of Public Health (1983b), conducted an ecological study of soft and connective tissue cancer mortality rates In Midland and other selected Michigan counties. They found that mortality rates for this cause were 3.8-4.0 times the national average for the periods 1960-1989 and 1970-1978, respectively, for white females 1n Midland. These estimates are based upon 5 deaths and 7 deaths, respectively, and are listed 1n Table VI-5. No excess risk was -reported among white males, however. The Michigan Department of Health concluded that because of the occurrence of these two successive elevated rates. It is unlikely to be a chance happen ing. At the same time the age-adjusted male and female cancer mortality rates for Midland were below that of the State of Michigan for the period 01340 VI-39 08/10/84 TABLE VI-5 Midland County Soft and Connective Tissue Cancer Deaths 1960-1981* 01340 VI-40 Identification Year of Sex Age Death 1961 F 24 1963 1964 1968 1969 F 75 F 51 F 37 F 45 1970 1970 F 59 F 56 1974 1976 F1 F 77 1978 F 64 Type Hemanglosarcoma Llposarcoma Leiomyosarcoma Llposarcoma Fibrosarcoma Leiomyosarcoma Kaposi sarcoma Fibrosarcoma Leiomyosarcoma Rhabdomyosarcoma Llposarcoma Leiomyosarcoma Type of Mallqnancv Primary Site Metastases Month and Year Diagnosed Face Right gluteal Uterus Spine Right thigh Uterus Right leg Right thigh Abdominal wall Inguinal area Right thigh Left knee Skull and upper lobe of lung Unknown Widespread Lungs, pelvis Lung, liver Adrenal gland and skin Lymph nodes Spine Lung Unknown Buttock, lung, rib, lymph nodes Liver, lymph nodes, lung, bone 5-58 Unknown 11-63 1-66 10-68 8-68 1960 1967 8-73 12-74 7-70 04/05/84 TABLE VI-5 (cont.) 01340 VI-41 Identification Year of Sex Age Death Type Type of Malignancy Primary Site Mtastass 1978 1978 1979 1962 1967 1967 1969 1971 1972 1976 F 26 Rhabdomyosarcoma Rectum Lung, neck, Inguinal region F 88 Fibrosarcoma Right cheek Facial area F 27 Leiomyosarcoma Left thigh Lung M 63 Rhabdomyosarcoma Left lower leg Lung and right outer chest wall H 77 Mesothelioma Lung Lung, peritoneum and diaphragm M 20 Rhabdomyosarcoma Pharynx Periorbital area and liver M 32 Llposarcoma Left arm Perineum and buttock M 76 Leiomyosarcoma Small Intestine Liver M 89 Leiomyosarcoma Retro- perl tonal region Hepatic system H 53 Fibrosarcoma Per 1tloneum Lung, liver Source: Michigan Department of Public Health, 1983b Month and Year Diagnosed 6-76 6-78 3-78 8-61 6-67 1-67 6-64 10-69 7-72 3-75 04/05/84 1970-1979. Midland County 1s the home of a major chemical company that pro duced phenoxyacetlc acid herbicides until recently. The authors stated that a detailed review of death certificates, hospital records, residency and occupational histories of the 20 male and female cases revealed no common alities* suggesting a "single causative agent," although a majority or their spouses had worked at this chemical facility. They recommend that a casecontrol study should be Instituted to evaluate possible Influences, such as lifestyle, occupation or location of residence on the risk of STS. In a separate review of the epidemiological evidence for STS from expo sure to 2,4,5-T-conta1n1ng herbicides, the United Kingdom Ministry of Agri culture, Fisheries and Food (1983) concluded that there was no evidence to reconnend altering their earlier conclusion that formulations of phenoxy acid herbicides and related wood preservatives as presently cleared" are safe and may continue to be used. This report too readily discounts the positive studies of Harden and Eriksson (1981) as being biased, and 1t makes no reference to the later validity study by Harden (1981) of his own work utilizing colon cancer controls. In this report Harden effectively answered these early criticisms that were reiterated by the British in their report. At the same time, the British report appears to put undue emphasis on nonpositive studies that do not demonstrate a risk, although most of them have methodological limitations (e.g., low power, Insufficient latency and Inappropriate study method). In short, the British review appears to be overly optimistic about the safety of 2,4,5-T herbicides. In summary, the associations reported In the two Swedish soft-tissue sarcoma studies are great enough to make It unlikely that they have resulted 01340 VI-42 09/18/84 entirely from random variation bias or confounding, even though the pos sibility cannot be excluded. These studies provide a strong suggestion that phenoxyacetlc acid herbicides, chlorophenols or their Impurities are carcinogenic 1n humans. I A separate series of clinical observations at the Department of Oncology In Umea, Sweden (Harden, 1979), led the researchers to conduct a casecontrol study of malignant lymphoma In relation to phenoxyacetlc acid, chlorophenols, and other organic compounds (Harden et al., 1980, 1981). Approximately 33% of the cases In this study were patients with Hodgkin's disease; the remainder of the cases were non-Hodgkin's lymphomas. This study employed essentially the same methods and produced results comparable with those of the STS studies: statistically significant 5-fold to 6-fold relative risks In relation to phenoxyacetlc acids and chloro phenols. In addition, an elevated relative risk was found In connection with exposure to organic solvents, such as benzene, trichloroethylene and styrene. In the published report, the methods and results were Incompletely documented, especially the possibility of confounding by exposure to the organic solvents. In the update of the earlier 1980 study, Hardell et al. (1981), utiliz ing the same basic data source, found that 36.1% of the cases had been exposed to phenoxy herbicides or chlorophenols, while only 9.6% of their controls were so exposed. The estimated relative risk was 6.0 when matching was considered and 5.3 when matching was eliminated. When cases and con trols who were exposed to chlorophenols only were excluded, the relative 01340 VI-43 08/10/84 risk of lymphoma from phenoxy acids alone was 4.8 (95% C.I. 2.9-8.1). On the other hand, if e xposures to phenoxy acids are e x cl u d ed and c o ns i d er a t io n is given to just c h lo r o p h e n o l s (which includes c o mb i n ed e x po s u re to phenoxy acids and c h io r o ph e n ol s ), then the rela t i ve risk equaled 4.3 (95% C.I. 2.7-6.9). The authors further subdivided this group into "low-grade" vs. "high-grade" exposures to c h l o r o p h e n o l s . A continuous exposure of not more than 1 week or repeated intermittent exposures totaling not more than 1 month was classified as low-grade. The relative risk for high-grade e x p o sure was 8.4 (95% C.I. 4.2-16.9), while that for low-grade exposure equaled 2.9 (95% C.I. 1.6-5.2). If e x po s u r e to organic solvents is examined, given that cases and controls expo s e d to only p h e n o x y acids or c h lo r o p h e n o l s or both were e x cl u d e d except for c o m b i n e d e x po s u re to organic solvents, it is found that hlghgrade and low-grade relative risks were 2.8 (95% C.I. 1.6-4.8) and 1.2 (95% C.I. 0.5-2.6), respectively. However, the authors noted that exposure to phenoxy acids and high-grade organic solvents (expo sure to chlorophenols excluded) produced a relative risk of 11.2 (95% C.I. 3.2-39.7) based upon a few cases and controls with exposure to both. The authors conc l u de d that " e xposure to organic solvents, chlo r o ph e n ol s or phenoxy acids constitutes a risk factor for malignant lymphoma." This latter study is still subject to the same m e t h o d o l o g i c a l c r iticisms to w h i c h the earlier study was subjected. Chief a m ong those is the p o s sibility of observational or recall bias creeping into the responses that are elicited from self-administered questionnaires on kind and length of exposure. Secondly, confounding by exposure to potentially carcinogenic organic solvents and other agents could have had an effect on the risk estimate, although the authors assure the reader that they did not (Hardell et a l ., 1981). 01340 VI-44 04/14/88 Other research has tentatively suggested that lumberjacks may be at increased risk of lymphoma (Edling and Granstam, 1979). The Nitro study (Zack and Suskind, 1980) found three deaths from cancers of the lymphatic and hematopoietic system, against only 0.88 expected (p=0.06, one-tailed Poisson test). The lymphoma case- c on t r ol study (Hardell et al., 1980, 1981) is c o n sistent with the two SIS studies discussed. On the other hand, the c o ns i s t e n c y c o uld also reflect an as-yet u n i d e n ti f i ed cons i s te n t flaw in all these studies. The two Swedish case control studies on STSs and a later case control study of m a li g n an t lymphoma (Hardell et al., 1981) were subjected to a validity analysis with respect to the assessment of exposure by Hardell and Eriksson (1981). To answer the question raised regarding the recall of o c cu p a t i o n in a f o r e s t r y / a g r i c u l t u r e job, seco n d ar y to the recall of e x p o sure to phenoxy acids or chlorophenols or both, the cases and controls were divided into three groups: those who work e d their entire time since 1950 in an a g r i c u l t u r e/ f o re s t r y job, those who worked some time in an a g ri c u lt u r e/ f o restry job but not e x cl u s iv e l y, and the remainder who never worked in a forestry/agriculture job. The study found that the risk ratio was still 8.2 for STS in e x c l u s i v e l y a g r i c u l t u r e / f o r e s t r y wo rk e r s w h o were e xposed to phenoxy acids comp a r ed with work e r s found in other o c c u p a ti o n s h a vi n g no apparent exposure to phenoxy acids or c hlorophenols. Even when comparing phenoxy acid or chlorophenol exposed agricultural/forestry workers ex cl u sively with nonexposed agricultural/forestry workers, the risk ratio was still 7.1. This argument seems to effectively answer questions regarding recall of occupation secondary to exposure. 01340 VI-45 04/14/88 On the other hand, the relative risk remains 5.4 when comparing phenoxy acid or chlor o ph e n ol e xposed work e r s e x c l u s i v e l y in o c c u p a ti o n s other than a g r i c u l t u r e / f o r e s t r y w i th n o ne x p os e d workers in those same o c cu p a ti o n s, thus suggesting the presence of either recall bias or still another occupation with potential expos u re to p h enoxy acids or c h lorophenols (Table VI-6). When woodworkers are separated out (possible exposure to chlorophenols in treatment of wood) the risk ratio becomes 9.7 (Table Vl-7). These data suggest the presence of some recall bias. Another focus of this study (Hardell and Eriksson, 1981) was to d e t e r m ine if o b ser v ational bias on the part of the investigators could explain the significantly high risk estimates. To answer the question, the study compared the exposure data derived from the Interviewee's returned question naires only with the combined Information from both the phone interviews and questionnaires. The study found no substantial differences in the frequency of reporting exposure. Still a third consideration of possible bias involves recall of exposure to phenoxy acids or chlorophenols because of subject knowledge of having cancer in the cases vers u s no k n ow l e d g e of cancer in the r e ferent p o p u l a tion. The study chose as a referent group for the 52 STS cases (Hardell and Sandstrom, 1979) and the 169 malignant lymphomas (Hardell et a 1., 1981) a group of 154 colon cancer cases from the same population source and compared their exposure to phenoxy acids or chlorophenols by broad age groupings and by rural versus urban residence. 01340 VI-46 04/14/88 I TABLE VI-6 Other Occupations (Minus Forestry/Agrlculture)* Group Phenoxy Aclds/Chlorophenols Cases Referents 11 5 RR = 5.4 Source: Adapted from Hardell and Erlkkson, 1981 RR = relative risk Non-exposed 68 167 X2 = 11.01 (p<0.01) 01340 VI-47 09/18/84 t TABLE VI-7 Other Occupations (Minus Forestry/Agrlculture/Woodworkers)* Group Phenoxy Adds/Chlorophenols Cases Referents 4 1 RR = 9.7 Source: Adapted from Hardell and Erlkkson, 1981 RR = relative risk Non-exposed 66 160 Xa = 5.98 (p<0.05) 01340 VI-48 09/18/84 Utilizing a Hantel-Haenszel rate ratio, the study found the risk of exposure to phenoxy adds remaining significantly high at 5.5 and to chlorophenols 5.4 In the STS cases compared with the colon cancer controls. Simi larly, with the malignant lymphomas, the Identically derived risk ratios Remain significantly high at 4,5 with respect to phenoxy adds or chlorophenol exposure 1n the cases; hence, the study concludes that, no substantial observational bias' exists. If the study Is assuming that recall bias was and 1s the same as observational bias, then such a conclusion may not be entirely warranted from the comparison. Certainly, 1t appears that no recall bias existed because of subject 'knowledge of having cancer* based on the authors analysis. But U does not rule out the possibility that recall bias can still be present In their data for other reasons. Harden and Eriksson (1981) refer to an intense 'debate about phenoxy adds and their presumptive risk* 1n Sweden at the time the colon cancer study was conducted. But, there 1s no reason to think that colon cancer victims would assume their disease was brought about from exposure to dioxin containing chemicals 1f no connection was suggested. It seems plausible that STS and non-Hodgkin's lymphoma patients would either learn at the time of their diagnosis that exposure to dioxin contain ing chemicals was the likely cause of this rare type of tumor or quickly learn from other sources, such as the news media, that exposure to herbi cides containing dioxin could cause their rare form of cancer. Whereas, colon cancer victims (a rather common form of cancer) would not necessarily be led to believe that exposure to the same dioxin containing chemicals caused their disease. Hence, It 1s not difficult to Imagine that such unusual victims of cancer could better 'remember* exposure to such chemicals than could colon cancer patients. 01340 VI-49 09/18/84 Therefore, although this study by H a r d e n and Eriksson (1981} may explain any biases introduced from secondary recall of occupation, ob serva tional bias introduced from the telephone interviewer and recall bias based on subject k n ow l e dg e of cancer, it does not a d e q u a t e l y answer questions of recall bias introduced through the acquired awareness on the part of the victim of STS or non-Hodgkin's lymphoma that his condition may have been caused by exposure to dioxin cont a i ni n g herbicides. Lynge (1985) c o mp a r e d the incidence of soft tissue sarcomas in two p henoxy h e rb i c id e m a n u f a c t u r i n g plants in Denmark. An increased incidence of soft tissue sarcomas was obse r v ed only in wo rk e r s from the plant that had a history of producing 2,4,5-T. Other studies that have reported an elevated risk of soft tissue sarcomas, as supposedly associated with exposure to 2,3,7,8-TCDD, Include Cantor (1982), Milham (1982), Kogan and Clapp (1985), Fett et al. (1984), Puntoni et al. (1986), and Merlo and Puntoni (1986). Studies of two of the oldest cohorts of workers known to have been exposed to phenoxyacet1c acid herbicides or 2,3,7,8-TCDD or both report stomach cancer mortality rates significantly higher than expected. The results in each study were b ased on small numbers of deaths. In one study (Axelson et al., 1980), 348 Swedish railroad workers with at least 46 days of herbicide exposure between 1955 and 1972 were followed through October 1978. The workers were grouped on the basis of their primary herbicide exposures: those primarily exposed to phenoxyacetic acids (2,4-D and 2,4,5-T) only, to amitrole (aminotriazole) only, and to both types of herbicides. After a 10-year latency was achieved, 3 stomach cancer deaths were observed vs. 0.71 expected (p<Q.Q5). None were attributable to amitrol 01340 VI-50 04/14/88 alone, but two were assigned to phenoxy acids alone while the remaining stomach cancer death occu r r ed in a worker exposed to both amitrol and phenoxy acids in c o mbination. The excess was more p r on o u nc e d (3 observed vs. 0.57 expected, p<0.05) among those with early e xposure (1957-1361) to phenoxy acids or amitrol or both. If persons who were exposed to just amitrol alone are excluded, thus leaving Individuals exposed to phenoxy acid a l one and amitrol in c o mbination, the excess is e n ha n c ed further (3 observed vs. 0.41 expected, p < 0 .01). A x el s o n et al. (1980) also note an excess in total "tumors" after 10 years latency as well (15 observed vs. 6.87 expected, p<0.005). This is pron o u nc e d in those exposed early to ph en o x y acids alone (6 observed vs. 2.60 expected, p<0.01) and ph en o x y acids in co mb i n at i o n with amitrol (5 o b se r v ed vs. 1.34 expected, p<0.05). P r es u mably, "tumors" in Sweden are analogous to malignant neoplasms in the United States. The author states that no specific type of tumor predominates and no breakdown by tumor type 1s provided. . The other study showing increased stomach cancer m o r t a l i t y is the follow-up of 75 workers exposed to 2,3,7,8-TCDD during and after a 1953 run away reaction at a tr ic h l or o p he n o l m a n u f a c t u r i n g faci l i ty in Ludwigshafen, Federal Republic of Germany (Theiss and Frentzel-Beyme, 1977). Two sources were used to calculate expected deaths: national mortality rates for the period 1971-1974, and 1972-1975 -rates for Rhinehessen-Palatinate, the region in which L u d w i g s h a f e n is located.* *The report originally included expected deaths using rates for the city of Ludwigshafen, which were later shown to be inaccurate. 01340 VI-51 04/12/88 The results, shown in lable Vl-8, indicate an increased rate of stomach cancer mortality that also is not likely to have been due to chance alone. Two aspects of the methodology used should be noted that could have influenced these results. First, the available report does not Include an analysis allowing for a m i n i m u m period of cancer induction. All three stomach cancer deaths in the L u dwigshafen cohort occurred more than 10 years after initial exposure. E m p l o y i n g a 10-year r e s t r i ct i o n to follow-up (as In the Swedish cohort study) would result in a higher relative risk estimate by reducing the number of expected deaths. Secondly, national and regional mortality rates from the 1970s were used to generate expected deaths to compare with observed mortality over a much longer period (1953-1977). Stro n g temporal trends In stomach cancer m o r t a l ity in West Germany during the late 1950s and 1960s w ould make these expected figures too Targe. The researchers also used an Internal control group that does not raise the second concern discussed above. This group consisted of 75 men, each matched to study group members by age and date of entry into employment, and selected at random from a list of over 10,000 persons who had been included in previous cohort studies by the same Investigators. No stomach cancer deaths occurred In this control group during the follow-up period. Thus, use of the Internal control groups also indicates an excess of stomach c a n cers In the exposed workers. In an update of this earlier study, Thelss et al. (1982) continued the follow-up of his cohort through 1979 by adding 2 additional years of follow- 01340 VI-52 04/08/88 TABLE VI-8 Analysis of S tomach Cancer M o r t a l i t y in a Group of West German Factory Workers Exposed to 2 , 3 , 7 ,8-TCDD* Source for Expected Deaths Stomach Cancer Deaths Observed Expected Relative Risk Federal Republic of Germany 1971-1974 RhinehessenPalatinate 1972-1975 3 0.559 3 0.495 5.4 6.1 Source: Adapted from Theiss and Frentzel-Beyme, 1977 Signi ficance Level 0.02 0.01 01340 VI-53 04/08/88 up and apparently reducing the size of his cohort from 75 to 74. Altogether 21 deaths (4 m o re than from the earlier study} occurred vs. 18 and 19 deaths in the 2 ma tc h e d (1 to 1) internal c o mp a r i s o n groups. With respect to c a n cer deaths, the numbers were r e s p e c t i v e l y 7, 5 and 5. The first control group was manually matched from the total number of persons (5500 included in the cohort until the end of 1976) and the second, at random, by computer for some 8000 employees. In addition, 19 expected total deaths were e s t i mated based on 1970-1975 mortality statistics of Rhinehessin-Palatinate, 18 expected deaths based on 1970-1975 mortality statistics of Ludw i g sh a f en , and 20 expected deaths based upon 1971-1974 mortality statistics of the Federal R e public of Germany. Just as in the earlier study, the three stomach c a r cinomas noted earlier appear to be significantly elevated regardless of w h ich external c o m p a r i s o n group is used (Table VI-9). On the other hand, one stomach cancer a p pe a r e d in the rand o m iz e d i n t e r nal control group. None appeared in the ma nu a l ly matched internal control. No other elevated risks for any other cause were evident and no STSs appeared. When latency was considered only, the risk of stomach cancer remained significantly elevated after a lapse of 10 years (3 observed, 0.52 expected, p<.016) and then after a lapse of 15 years (2 observed, 0.23 expected, p < .02) based upon death rates of Rhinehessin-Palatinate, 1970-1975. Again, these study conclusions are limited by the small size of the study group and the very few cancer deaths noted at any particular site. Thus, it is I n se n s i t i v e to the d e te c t i o n of a s i g n i f i c a n t l y e l ev a t e d risk for most causes of cancer, especially STS and lymphomas. Although, stomach cancer is e l ev a t e d s i g n i f ic a n tl y , it is based o n ly upon three deaths and since one stomach cancer death has been noted in an Internal control group 01340 VI-54 04/08/88 TABLE VI-9 Rean a l ys l s oF Stomach Cancer M o r t a l i t y In a Group of West German Factory Workers Exposed to 2 , 3 , 1 r8-TCDD* Source for Expected Deaths Stomach Cancer Deaths Observed Expected Federal Republic of Germany 1971-1974 RhinehessinPalatlnate 1970-1975 Ltidwlgsshafen 1970-1975 3 3 3 0.7 0.64 0.61 *Source: Adapted from Theiss et a 1.. 1982 R e l a t 1ve Risk 4.3 4.7 4.9 Significance Level 0.034 0.027 0.024 01340 VI-55 04/08/88 in the updated version, it appears that this finding has been w e ak e n ed s o m e what. Furthermore, as was pointed out earlier, trends in stomach cancer m o rt a l it y during the 1950s, 1960s and 1970s could make the comp a r is o n of stomach cancer mortality with expected deaths less valid based upon 1970-1975 rates. In summary, the e v id e n ce that p h e n o x ya c e ti c acids or 2 , 3, 7 , 8- T C DD or both might increase the risk of stomach cancer consists of two studies, each of w h ic h reports a s t a t i s t i c a l l y significant excess that is b ased on only three stomach cancer deaths. Further follow-up of these and similar cohorts is w a rranted, but firm c o n c l u si o n s cannot yet be made. Four additional cohort studies have reported results that do not show Increased stomach cancer m o r t a l i t y rates in groups of workers exposed to phenoxyacetic acids or 2,3,7,8-TCOD or both. These are studies of 2,4,5-T production workers in Midland, Michigan (Ott et al., 1980), Finnish p h e n o x y acetic acid herbicide applicators (Riihimaki et al., 1978), the Nitro study in w h i c h w o r k e r s were expo s e d to 2 , 3 , 7 , 8 - T C D D (Zack and Suskind, 1980) and trichlorophenol manufacturing workers (Cook et al., 1980). As previously mentioned, the Nitro study Included a single death from STS and a w e ak l y s u gg e s t i v e Increase in lymphatic and h e ma t o po i e ti c system cancer mortality. The Midland study included only one cancer death, a tumor in the respiratory system. In the Finnish study, histologic information on tumor types was not provided; however, there were no deaths from lymphoma. The results p e r t i n e n t to stomach cancer m o r t a l i t y in the three studies are shown in T able VI-10. Neither the M i d l a n d study nor the N i tro study 01340 VI-56 04/08/88 TABLE VI-10 Stomach Cancer M o r t a l i t y in Three Studies of W orkers Exposed to Phenoxyacetic Acid Herbicides and/or 2,3,7,8-TCDQ Stomach Cancer Deaths Observed Expected Relative Risk 95% Confidence Interval Reference 0 0.14a 0 5 6 . 9 a *b 0.7 0-26.3 0.2-1.7 0 0.5b 0 ' 0-7.4 Ott et al., 1980 Ri ihmaki et a l ., 1978 Zack and Suskind, 1980 E s t i m a t e d from total cancer e x pe c t e d deaths (see footnote In t e x t ). ^Entire follow-up period without regard for minimum time for cancer induc tion (Ott et al., 1980 used a 10-year minimum induction period). 01340 VI-57 04/08/88 contradicts the Findings oF the Swedish and West German investigations p r e v iously discussed. This can be shown in two ways. First, the upper 9 5% confidence limits For the relative risk estimates From these two "negative" studies exceed even the highest point estimates oF relative risk (6.1) From the two "positive" studies (see Table VI-8). This indicates that the relative risk estimates From the Midland and Nitro studies, even though equal to zero, are nevertheless not signiFicantly diFferent from the estimates oF 6.1, given the sample sizes, Follow-up p e r iods, age distribution and comparison group rates. In addition, the smal l e st d e t e c t a b l e r e la t i ve risk in the M i d l a n d study (a = 0.05, <p = 0.2 o n e - t a i l e d P o i s s o n test) was 21.4 (3 o b se r v ed deaths, 0.14 e x p e c t ed ) . * S i milarly, the smallest d e t e c t a b l e r e la t i ve risk in the N i tr o study (a = 0.05, = 0.2, o n e- t a i l e d Poisson test) was 10.0 (5 o b se r v ed deaths, 0.5 expected). This c a l c u l a t i o n is based on results For the entire follow-up period. If, as in the Midland study, a m i ni m u m period of cancer induction had been employed, the expected deaths would have been fewer and the smallest reasonably detectable relative risk would have been greater. This analysis of statistical power indicates that the Nitro and * 0 11 et al. (1980) did not report expected deaths from stomach cancers. The figure 0.14 was obtained by multiplying the numbers oF expected deaths from all cancers (2.6, allowing a 10-year minimum induction period) by the p e r c e n t a g e of s tomach cancers a m b n g the e x p e c t e d deaths in the Nitro study (0.5/9.04 = 5.5%). The two studies used United States white male mortality rates and covered similar calendar years in follow-up (1949-1978 in Nitro and 1 9 50- 1 976 in M idland), but a s i milarity in age d i stributions cannot be established from the published reports. 01340 VI-58 04/08/88 Midland studies had very low probabilities of detecting the -6-fold i n creases in risk sugg e s te d by the Swedish and West German investigations. S t a t i s ti c a ll y , the study of Finnish h e rb i c id e a p p l i c at o r s is i n c o n sistent with the results of the Swedish and West German cohort studies. The smallest reasonably detectable relative risk (a - 0.05, = 0.2, o n e tailed Poisson test) was only 3.1 (11 observed deaths, 3.6 expected).* The study, therefore, appears powerful enough to detect relative risks even smaller than those seen in the Swedish and West German studies. A partial explanation for this apparent inconsistency could lie in the fact that the Finnish study set the m i n i m u m period of h e r b i c i d e e x po s u r e for m e m b e r s h i p in the cohort at 10 days (2 w o rk i n g weeks) and noted that the "total strength of e x po s u re has, in most cases, been a few weeks only." The Swedish study of h e rb i c i d e a p pl i c a t o r s set the m i n i m u m e x po s u r e at 46 days (>1 spraying season). T h ere are also c e r t a i n I n c o n s is t e nc i e s In the data from the Finnish study that the authors note but find difficult to explain. In particular, no cancer deaths occurred during the latter part of the study period among F o re s t ry A u t h o r i t y w o r k e r s (1 of 4 groups included in the cohort), even though 9.0 deaths were expected. This finding strongly suggests some defi cien c y in foll o w -u p or in the source records from w h ic h vital status was determined. *The e x pe c t ed s tomach cancer deaths w e re e s ti m a te d 1n the same m a nn e r as for the Midland study. A proportion of 20% of all cancer deaths was applied because Finnish male mortality rates are known to be very high. 01340 VI-59 04/08/88 In summary, four cohort studies of workers exposed to p h e n o x y a c e t ic acid herbicides or 2,3, 7 , 8-T C DD or both do not report increased risks of stomach cancer. Only one of these, however, was s t at i s ti c a ll y powerful enough to be inconsistent with the two studies that t e nt a tively suggest an increase in stomach cancer risk. The available report of this study of Finnish h e rb i cide applicators contains methodologic questions that require clarification. By adding together the number of workers exposed to phenoxy acids or c h l o r o ph e n ol s or both from all case studies, an unusually high number of STSs is shown, c o n s i d e r i n g the rarity of the disease. This excess is suggestive of an a s so c i at i o n of cancer with exposure to phenoxy acids or c h l o r o p h e n o l s or both, and cons e q ue n t ly , with the impurities found in these herbicides, including 2,3,7,8-TCDD. Two Swedish case-control studies report highly significant association of STS with exposure to phenoxy acid or chlorophenols or both. They do not pinpoint the risk to the dioxin c o nt a m in a n ts , however. In fact, in one study, the risk was found to extend to phenoxy acids free of dioxin impur ities. In that study, the risk increases to 17 when phenoxy acids known to contain dioxin impurities (polychlorinated dibenzodioxlhs and dibenzofurans) are considered. The extent of possible observer bias and recall bias intro duced into these studies by using s e l f - a d m i n i s t e r e d q u es t i on n a ir e s is not of s u fficient m a g n i t u d e to have p r od u c ed the highly si gn i f ic a n t risks found in the studies. Later studies did not reveal a significant excess risk of STS. However, m e t h o d o l o g y problems make these latter studies l imited with respect to 01340 VI-60 04/08/88 evaluating the risk of STSs from exposure to phenoxy acids or chlorophenols or both and, consequently, 2,3,7,8-TCDD. The Swedish case-control studies provide limited evidence for the c a r c i n o g e n i c i t y of p h e n o x y acids or c h i o r o p h e n o l s or both in humans. However, with respect to the dioxin impurities contained therein, the evidence for the human carcinogenicity for 2,3,7,8-TCDD based on the e p i d e m i o l og i c studies is only s u g g e s ti v e b ecause of the d i f f i c u l t y of e v a l u a t i n g the risk of 2 , 3 , 7 , 8 - TC D D e x po s u r e In the p r es e n c e of the c o n founding effects of phenoxy acids and/or chlorophenol. There is less e v id e n c e i n c r i m i n a t i n g 2,4,5-T or 2 , 3 , 7 , 8 - T C D D or both as the cause of malignant lymphoma and stomach cancer in humans. Mutagenicity Studies Czeizel and K i ra l y {1976) r e po r t ed an i ncreased inci d e nc e {p<0.001) of c h r o m a t i d - t y p e and u n st a b le c h r o m o s o m e a b e r r a t i o n s in the p e ripheral l y m p h o cytes of workers exposed to the herbicides 2,4,5-trichlorophenoxyethanol (2,4,5-TCPE) and Buminol. The 2,3,7 , 8- T C DD levels in the final product were <0.1 mg/kg; however, the exposure levels for individual workers were not available. Mulcahy (1980) reported no increased incidences of chromosomal aberra tions in the lymphocytes of 15 soldiers exposed to Agent Orange. The e x p o sure was for 6-15 months and all subjects complained of symptoms, including skin eruptions, which they associated with Agent Orange. The analyses were performed with lymphocytes obtained -10 years after the last exposure, and comparisons were made with eight subjects who had no history of exposure to 01340 VI-61 04/14/88 2,3,7,8 - TC D D . Neither sister chromatid exchange nor structural aberrations including both gaps and breaks were increased. The authors note that the long time between exposure and analysis may have accounted for the negative results. Also, both Reggiani (1980) and Mottura et a 1. (1981) have studied i n h a b itants in Seveso, Italy, e xposed to 2 , 3 , 7 , 8-TC D D from an acci d e nt in a trichlorophenol manufacturing plant. Reggiani (1980) examined 4 adults and 13 children (3-13 years) for chromosomal aberrations within 2 weeks of the accident. These 17 individuals were examined to support claims of and dete r m in e extent of injury. A l th o u g h b u rn - l ik e skin lesions in these 17 individuals indicated chemical exposure, no increase in chromosomal a b e r r a tions was detected. The methods of performing the analyses and the actual number of aberrations detected were not described. Similar negative results were reported in an a b st r a c t by M ottura et al. (1981). In this study, s u b jects were chosen from the area of heavy contamination following the a c c i dent (acute high level exposure), from the working population of the plant (chronic low level exposure) and a nonexposed control population. The num ber of subjects in each g roup was not provided. The specimens w e re examined by three i n de p endent l a bo r a to r i es and no l a bo r a t o r y repo r t ed an increase in chromosomal a b er r a ti o n s, a l t h o u g h there was a s i g n i f ic a n t d i f f e r e n c e in the reported scores be tw e e n laboratories. T h ere was no information in this abstract on the extent of Individual exposure or the length of time that elapsed between the accident and obtaining samples for analyses of chromo so mal aberrations. DiLernia et al. (1982) conducted additional studies on lymphocytes prep a r ed in 1976 and 1979 f r om eight persons c o n s i d e r e d a c u t e l y exposed to 01340 VI-62 04/14/88 2 , 3 , 7 ,8-TCQD in the Seveso accident, eight ICMESA factory workers ( c o n s i d e r ed chronically exposed), and 14 control subjects (eight had chromosome p r e p arations made in 1976 and six in 1979 ). Cells were exam i n ed for a verage number of SAs (evidence for functional rlbosomal genes), both on a cell - basis and for the large a c ro c e n t r i c c h ro m osomes (D group chromosomes), there was no change in the freq u e nc y of SAs on a per cell basis in any of the groups as comp a r ed to control values, nor in D group c h ro mosomes from acutely exposed subjects examined immediately after the accident. There was, however, a d e cr e a s e in the av er a g e frequ e nc y of SAs in group D c h r o m o somes of a c u t e l y exposed subjects examined in 1977 and in ICMESA w o r k e r s at both the 1976 and 1979 examinations. Although the biologic relevance of these observations has not yet been confirmed, DiLernia et al. (1982) o b s e r v e d a similar d e cr e a s e in SAs after e x po s u re of lymphocytes to x-ir r a di a t io n . It was c o nc l u d e d that the decr e a se in SAs may have resulted from mutagenic damage to functional nucleolar organizing regions. High Risk Subpopulations Little information was found in the a v ailable literature to indicate that specific human subpopulations may be unusually susceptible to the toxic effects of 2 , 3 , 7 ,8-TCOD. Limited evidence from the Seveso area and Eastern Missouri indicates that children may be more sensitive to the immunological effects of 2,3,7,8-TCDD. Tognonl and Bonaccorsi (1982) found elevated peripheral blood lymphocytes, lymphocyte blastognie response, and serum complement a c t i v i t y in exposed children; however, no immunological effects were detected in adults (Reggiani, 1980; May, 1982). Among adults and c h il d r en exposed to c o n t a m i n a t e d soil in horse arenas in Eastern M issouri, the only adverse health effects reported were In children (Kimbrough et al., 1977). Children playing in the arena were prob a b ly in more intimate contact 01340 VI-63 04/14/88 with the contaminated soil, and thus subject to higher exposures, than were the adults. Summary By adding together the number of workers exposed to phenoxy acids and/or c h l o r o ph e n ol s from all case studies, an u n us u a ll y high number of STSs is shown, c o ns i d e r i n g the rarity of the disease. This excess is s u gg e s ti v e of an association of cancer with exposure to phenoxy acids and/or chlorophenols, and c o ns e q ue n t ly , with the impurities found in these herbicides, including 2,3,7,8-TCDD. Two Swedish case-control studies report highly significant association of STS with exposure to phenoxy acid and/or chlorophenols. They do not pinpoint the risk to the dioxin contaminants, however. In fact, in one study, the risk was found to extend to phenoxy acids free of dioxin impurities. In that study, the risk Increases to 17 when phenoxy acids known to contain dioxin impurities (polychlorinated dibenzodioxins and dibenzofurans) are considered. The extent of possible observer bias and recall bias introduced into these studies by using self-adminlstered q u e s t i o n n a i r e s is not of s u f f i c i e n t m a g n i t u d e to have p r od u c ed the highly significant risks found in the studies. Later studies did not reveal a signficant excess of risk of STS. However, methodology problems make these latter studies limited with respect to evaluating the risk of STSs from exposure to phenoxy acids and/or chlorophenols and, consequently, 2,3,7,8-TCDD. Epidemiological studies on more recently exposed populations, where dioxin exposure can be more accur ately assessed, and subsequent studies, including a higher number of deaths, 01340 VI-64 04/14/88 on populations previously studied will increase the pool of data on dioxin and help e v al u a t e its c a rc i n og e n ic risk in humans. Either acute or chronic exposure to 2,3,7,8-TCDD may result in chloracne, altered liver function, hematological pathologies, porphyria cutanea tarda, h y p e r p i g m e n t a t i o n , h i r s u t i s m and neural d e g e n e r a t i o n in the e x t r e m i t ies. Stevens (1981) has e stimated that the m i n i m u m c u mu l a ti v e toxic dose of 2 . 3 . 7 . 8 - TCOO in humans is 0.1 yg/kg. The toxic effects of exposure to 2,3,7,8-TCDD may persist for many years, though some effects s e em to be r e ve r s i b l e in some cases. Even though 2 . 3 . 7 . 8- TCDD has been found to be fetotoxic and/or teratogenic in all animal species tested (see the T e r a t o g e n i c i t y and R e p r o d u c t i v e T o x i c i t y S ection in Chapter V), epidemiological studies have failed to demonstrate a convincing c o nnection b e tween 2 , 3 , 7 , 8- T C DD exposure and spontaneous abortions or m a l f o r m a t i o n s in humans. T h es e studies are difficult to Interpret, since quantitative exposure data are not available. Some evidence of c y to genetic damage has been reported in humans exposed to chemicals contaminated with 2,3,7,8-TCDD, but negative results have also been reported; exposures were not quantitated; and the other chemicals cannot be ruled out as c a u s a tive agents. 01340 VI-65 04/14/88 I VII. MECHANISM OF TOXICITY A number of studies have attempted to determine the mechanism of toxic ity of 2,3,7,8-TCDO. The ultimate purpose 1s to provide a better estimate of man's relative sensitivity to 2,3,7,8-TCOD and other compounds having a similar mode of action. Specifically, these studies may be able to explain the reason for the marked interspecies differences In 2,3,7,8-TCOO toxicity and, thus, help determine If humans possess factors that are associated with sensitivity to 2,3,7,8-TCDD toxicity. Receptor-Mediated Toxicity Pharmacogenetlc studies have played an Important role 1n understanding the biologic and toxic effects of drugs and xenoblotlcs. Nebert and coworkers have shown that carcinogenic polycyclic aromatic hydrocarbons (PAHs) Induce the cytochrome P-450-dependent monooxygenase AHH 1n certain responsive strains of mice (e.g., C57B1/6J, BALBc, C3HF/He) whereas this PAH Induction activity 1s minimal or nonexistent 1n nonresponslve strains (DBA/2J) (Nebert, 1979, 1982; Nebert and Glelen, 1972; Nebert and Jensen, 1979; Nebert et al., 1972, 1981, 1983). The gene complex responsible for the Induction of AHH and several other enzymes has been designated the Ah locus which comprises regulatory, structural and possible temporal genes. Extensive studies on genetically Inbred responsive and nonresponslve mice (and their backcrosses) Indicate that these differences are related to the Ah regulatory gene and Its gene product, the Ah cytosolic receptor protein. This receptor protein Interacts with PAH ligands and the resultant PAH:Ah receptor complex translocates Into the nucleus and presumably Initiates the Induction of AHH via a process comparable to that proposed for the steroid hormones. 01350 VII-1 08/13/84 Since the carcinogenic and toxic effects of PAHs are dependent on their oxidative metabolism to reactive electrophilic forms, 1t 1s not surprising that the Ah receptor plays an Important role In mediating their toxldty and carc1nogen1c1ty (Kourl, 1976; Kourl et al., 1974; 8ened1ct et al., 1973; k Shum et al., 1979; Thomas et al., 1973; Legraverend et al., 1980; DuranReynolds et al., 1978; Robinson et al., 1975; Hattlson and Thorgelrsson, 1979). Responsive mice are more susceptible to the toxic {Inflammation, fetotoxlclty, primordial oocyte depletion) and carcinogenic effects of PAH at organs/tlssues 1n direct contact with the applied chemical; In contrast, nonresponslve mice are more susceptible to the tumorlgenlc effects of PAHs at ttssue/organ sites remote from the Initial site of exposure to the PAHs. These differences in susceptibility are due to several factors Including AHH-medlated toxlcatlon and detoxication. 2.3.7.8-TC00; Segregation of Activity with the Ah Locus. Genetic studies also support the role of the Ah receptor 1n mediating the toxic and biologic effects of 2,3,7,8-TCOO. Initial studies by Poland and coworkers (Poland et al., 1974, 1983; Poland and Glover, 1975; Nebert et al., 1975) demonstrated that the microsomal AHH-1nduc1ng activity of 2,3,7,8-TCDD and 3-MC 1n several genetically Inbred mice strains were similar. Like 3-MC and related PAHs, 2,3,7,8-TCOO Induced AHH 1n several responsive mouse strains (1.e., C57B1/6J). In contrast to 3-MC, 2,3,7,8-TCDD Induced microsomal AHH 1n the 0BA/2J nonresponsl ve rake; however, the E05) for this biologic response was significantly higher than values reported for the responsive mice. In genetic crosses between responsive C5781/6 and nonresponslve D8A/2 mice tt was also shown for both 3-MC and 2,3,7,8-TCOO that the trait of responsiveness Is Inherited In a simple autosomal dominant mode (Poland and 01350 V I 1-2 09/18/84 Knutson, 1982). It has been suggested that the observed differences In the activities of 3-MC and 2,3,7,8-TCDD are related to their relative. Ah receptor affinities (Poland and Knutson, 1982) and the pharmacokinetic and metabolic factors which would more rapidly diminish the 'available" concen t tratlons of 3-MC caused by metabolism and excretion. Several studies with 2,3,7,8-TCDD In genetically Inbred mice support the receptor mediated hypothesis. The Induction of UOP-glucuranosyl transfer ase, DT dlaphorase, -aminolevulinic acid, glutath1one-S-transferase 8 , T-aldehyde dehydrogenase and chollneklnase by 2,3,7,8-TCDD or 3-MC 1n genetically Inbred mice have also been shown to segregate with the Ah locus (Beatty and Neal, 1976b; Owens, 1977; Klrsch et al., 1975; Dietrich et al., 1978; IsMdate et al., 1900; Poland and Glover, 1973a). Toxicology studies with genetlcally-1nbred mice confirm the role of the Ah locus 1n mediating several toxic effects Including porphyria, Immunotoxlclty (a wasting syn drome), thymic atrophy and cleft palate formation (Jones and Sweeney, 1980; Poland and Glover, 1980; Courtney and Moore, 1971; Vecchl et al., 1980, 1983). Poland et al. (1982) have also linked the tumor-promoting activity of 2,3,7,8-TCDD 1n hairless mice to the cytosolic receptor. In vitro studies with XB cells 1n culture also support the role of receptor 1n mediating a dose-related cell keratlnlzatlon by 2,3,7,8-TCDD which resembles some of the characteristics of chloracne (Knutson and Poland, 1980). This cell line 1s also responsive to ANH Induction and contains a cytosolic receptor binding protein. Although the murine Ah receptor has not been characterized, several studies confirm that a protein with high affinity for 3-MC and 2,3,7,8-TCDD Is present In low concentrations In the hepatic (-30-50 fmolar) and extrahepatlc tissues of responsive C57B1/6J mice 01350 VI1-3 09/ 18/84 (Greenlee and Poland, 1979; Okey et al., 1979, 1980; Poland et al., 1976; Mason and Okey, 1982; Gaslewlcz and Neal, 1982; Okey and Vella, 1982; Okey, 1983; Nebert et al., 1983). In responsive C57B1/6J mice and Sprague-Dawley rats, but not In nonresponslve DBA/2J mice, the Ah receptor can be Induced by pretreatment with phnobarbital which Is the only known agent at present that has been demonstrated to affect tissue concentrations of the receptor (Okey and Vella, 1984). Although the Ah receptor has not been detected In the cytosol of 08A/2J mice, after the administration of radiolabeled 2,3,7,8-TCDD to these mice, some of the radlolabel 1s detected in the nuclei of the nonresponslve mice. Moreover, the sedimentation characteristics of the [3H]-2,3,7,8-TCDD:nuclear protein complex In 0BA/2J mice are similar to those observed with the bound Ah cytosolic receptor protein 1n C57B1/6J mice using a sucrose density gradient centrifugation separation technique {Okey, 1983). Several reports have also demonstrated that the cytosolic Ah receptor protein migrates Into the nucleus of the cell only after binding with 2,3,7,8-TCDD (Greenlee and Poland, 1979; Okey et al., 1979, 1980) and this parallels the observations noted for the Interactions between steroids and their receptor proteins. ,, 2,3,7.8-TCDD and Related Toxic Halogenated Aryl Hydrocarbons: Structure-Activity Correlations. The evidence for a receptor mediated mechanism of action for 2,3,7,8-TCDD 1s supported by data reported for the effects of other halogenated aryl hydrocarbons in genetically Inbred mice and other diverse animal species. A number of reviews and comparative studies (Allen et al.f 1979; Allen and Norback, 1977; Kimbrough, 1974; Kimbrough et al., 1978; McConnell and Moore, 1979; Taylor, 1979) clearly indicate that the toxic halogenated mixtures and Individual compounds 01350 VII-4 08/12/84 (Including the PCDOs, PCDFs, PCBs and PBBs) elicit similar toxic and biologic responses which Include 1 ) a wasting syndrome which 1s manifested by a progressive weight loss and decreased food consumption by the treated animals; 2 ) skin disorders Including acneform eruptions or chloracne, fc alopecia, edema, hyperkeratosis, and hypertrophy of the Meibomian glands; 3) lymphoid Involution and atrophy; 4) porphyria (resembling porphyria cutanea tarda); 5) endocrine and reproductive disorders; 6 ) modulation of chemical carcinogenesis; and 7) the Induction of numerous enzymes Including the cytochrome P-448 (or P-450c) dependent monooxygenases. It 1s apparent that the effects of these compounds are not manifested In all the animal species tested. McConnell and Moore (1979) summarized the pathologic findings observed 1n several animal species after pretreatment with PCDOs, PCDFs, PCBs and PBBs and these data Illustrate the different species and organ/tlssue susceptibilities to these compounds. It 1s also evident that for most of these effects, all the toxic halogenated aromatics elicit similar effects 1n these species which also contain the cytosolic receptor protein (Carlstedt-Duke, 1979; Carlstedt-Duke et al., 1979, 1981; Okey, 1983; Okey and Vella, 1982; Mason and Okey, 1982). These observations support a conmon mechanism of action for all the toxic halogenated aryl hydrocarbons (Poland and Knutson, 1982; Safe et al., 1982; McConnell and Moore, 1979). Several reports have demonstrated the effects of structure on the activ ity of PCDOs. The most active member of this group Is substituted In the lateral 2, 3, 7 and 8 positions; activity 1s decreased with 1) decreasing lateral substituents, and 2) Increasing Cl substitution. Moreover, for several PCDDs, there 1s an excellent correlation between the toxicity of 01350 vn-5 09/18/84 Individual PCOO congeners In guinea pigs and mice (McConnell et al., 1978b) and their AHH Induction potencies 1n chick embryos and rat hepatoma H-4-II-E cells In culture and their binding affinities for the C5781/6J mouse hepatic cytosolic receptor protein (Poland et al., 1976, 1979; Bradlaw et al., 1980; Bradlau and Casterllne, 1979). Comparable structure-activity correlations have been reported for the PCDFs In which the most active compound, 2,3,7,8TCDF, 1s an approximate Isostereomer of 2,3,7,8-TCDD (Poland et al., 1979; Poland and Knutson, 1982). Moreover, like the PCDDs, there was an excellent correlation between the toxicity of several Individual PCDFs (Yoshlhara et al., 1981), their AHH Induction potencies In rat H-4-U-E hepatoma cells and binding affinities to male Wlstar rat hepatic cytosolic receptor protein (Bandlera et al., 1983). The most active PC8 congeners, 3,4,4',5-tetra-, 3,3',4,4'-tetra-, 3,3',4,4',5-penta- and 3,3',4,4' ,5,5'-hexachloroblpheny 1, are substituted at both para and at two or more meta positions. The four coplanar PCBs Induce rat hepatic microsomal AHH and cytochromes P-450a, P-450c and P-450d and resemble 3-MC and 2,3,7,8-TCDD In their mode of Induction of the cytochrome P-450 Isozymes (34) (Parkinson et al., I980a,b, 1983; Safe et al., 1982; Sawyer and Safe, 1982; Poland and Glover, 1977; Goldstein et al., 1977). Like Aroclor 1254, all the monoortho and at least eight dlortho-chloro analogs of the coplanar PCBs exhibited a mixed-type* Induction pattern and Induced microsomal AHH, DMAP N-demethylase and cytochromes P-450a to P-450e (Parkinson et al., 1980a,c, 1983). Quantitative structure-activity rela tionships (QSARs) within this series of PCBs were determined by comparing their AHH Induction potencies (EC^q ) In rat hepatoma H-4-I1-E cells and their binding affinities (EDgQ ) for the 2,3,7,8-TCDD rat cytosolic recep tor protein (Sawyer and Safe, 1982; Bandlera et al., 1983). The results 01350 V I 1-6 09/18/84 showed that there was an excellent correlation between AHH Induction potencies and receptor binding avidities of these compounds and the order or activity was coplanar PCBs (3,3',4,4'-tetra-, 3,3',4.41,5-penta- and 3,j',4.4',5,5'-hexachlorobiphenyls) > 3,4,4',5-tetrachloroblphenyl > mono ortho coplanar PCBs > diortho coplanar PCBs. It was also apparent that the relative toxlcitles of this group of PCBs paralleled their biological potencies (Blocca et al., 1981; Yoshlhara et al,, 1979; Marks et al., 1981; McKinney et al., 1976; Yamamoto et al., 1976; Ax and Hansen, 1975; KurokV and Masuda, 1977). The coplanar and monoortho coplanar PCBs also exhibit differential effects 1n the Inbred C57B1/6J and DBA/2J mice. These compounds Induce AHH and cause thymic atrophy In the former "responsive" mice whereas at compar able or higher doses none of these effects are observed In the nonresponslve DBA/2J mice (Parkinson et al., 1982; Robertson et al., 1984). The results obtained for structurally diverse PCDDs, PCBs and PCDFs clearly support the role of the receptor protein In Initiating the broad spectrum of biologic and toxic effects elicited by these chemicals. Bandlera et al. (1983) have demonstrated that the 2,3,7,8-TCDD receptor protein 1s not only susceptible to halogen substitution patterns but also the structure of the substituent. The cytosol receptor binding avidities and AHH Induction potencies In rat hepatoma H-4-II-E cells for several 4'-X-2,3,4,5-tetrachlorob1phenyls were remarkably dependent on the structure of the X substituent. The binding data for 13 different substituents was subjected to multlparameter regres sion analysis to correlate binding avidities with the physical chemical characteristics of the critical lateral X substituents. The equation log (1) = 1.53a 1.47 1 + 1.09 HB + 4.08 01350 V11 -7 08/10/84 showed that ligand binding was dependent on substituent electronegativity (c), 1IpophlI1c1ty (1 ) and hydrogen binding (H8 ) with a correlation coefficient (r) equal to 0.978 for 13 different substituents. ' The receptor mediated hypothesis for the mechanism of action of 2,3,7,8- TCDD still requires further confirmation and numerous problems must be clarified. For example: 1. Several cell culture lines which appear to have the Ah recep tor are highly resistant to the toxicity of 2,3,7,8-TC00; the nonresponslve HTC and responsive H-4-II-E cell lines (1.e., for AHH InducIbllUy by 2,3,7,8-TCDO) do not possess cytosolic receptor; however, the nonresponslve HTC cells possess more nuclear receptor binding protein than the responsive H-4-II-E cells (Okey, 1983; Okey et al., 1980). 2. Hepatic cytosolic receptor levels In rats (Wlstar and SpragueOawley), C5781/6J mice, hamsters and guinea pigs are compar able (Gaslewlcz et al., 1983b); however, their susceptibility to the biologic and toxic effects of 2,3,7,B-TCOO are highly variable: guinea pigs are highly susceptible to the lethal effects of 2,3,7,8-TCDO <LO5Q = 1-2 yg/kg) whereas the susceptibility of the other species follows the order rat > C57B1/6J mice > DBA/2J mice > hamster (Neal et al., 1982). Metabolism The metabolism of 2,3,7,8-TCDO has been examined 1n the guinea pig, rat, mouse and hamster. Urine and bile from lC-TCDD-treated animals were found to be free of unmetaboll zed 2,3,7,8-TCDO, demonstrating that metabol ism was required for elimination through these routes (Olson et al., 1983). The direct Intestinal elimination of unchanged 2,3,7,8-TCDD 1n feces suggests, however, that some`routes of excretion may not be dependent on prior metabolism of the toxin (Olson et al., 1983). Thus, 1t 1s not possible to directly correlate the ha 1f-11fe for elimination of 2,3,7,8-TCDO with Its In vivo rate of metabolism In a given species. The relative per sistence of 2,3,7,8-TCDO In a given species may be related to the Vn vivo 01350 V I I -8 09/18/84 rate of 2,3,7,8-TCDD metabolism, excretion of the toxin not dependent upon metabolism (direct Intestinal elimination, lactation, sebum), and the rela tive tissue distribution of 2,3,7,8-TCDD, particularly to adipose stores. Qualitative and quantitative differences 1n the metabolism and disposition of 2,3,7,8-TCDD have been observed between various species, and these may In part be related to the remarkable 1nterspec1es differences 1n sensitivity to 2,3,7,8-TCDD toxicity (Olson et al., 1983). Polger et al. (1982a) suggest that 2,3,7,8-TCDD metabolism represents detoxification, since they observed relatively little toxicity In guinea pigs given extracts of dog bile containing 2,3,7,8-TCDO metabolites. How ever, a recent study proposes that metabolites of 2,3,7,8-TCDD may Inhibit uroporphyrinogen decarboxylase activity and lead to 2,3,7,8-TCDD-1nduced porphyria (DeVerneull et al., 1983). Current data on the structural Identi fication of 2,3,7,8-TCDD metabolites suggest that reactive epoxide Inter mediates may be formed during metabolism (Polger et al., 1982b; Sawahata et al., 1982). Poland and Glover (1979) reported that the maximum possible l_n vivo covalent binding of 1,6-1H-2,3,7,8-TCDD derived radioactivity to hepatic DNA was 4 orders of magnitude less than the levels of binding observed with other chemical carcinogens. The study did find much higher levels of 2,3,7,8-TCDO derived radioactivity bound to hepatic protein of the rat. No data 1s available, however, on the degree 2,3,7,8-TCDD derived radioactivity 1s bound to tissues of various species of laboratory animals, which have demonstrated remarkable variability 1n sensitivity to 2 ,3,7,8- TCOD. While biliary excretion products may represent detoxified, polar metabolites of 2,3,7,8-TCDO, 1t remains to be shown whether unexcreted reactive metabolites Initiate some of the toxic responses associated with exposure to this toxin. 01350 VII-9 08/10/84 Vitamin A Depletion Many of the toxic effects of 2,3,7,8-TCDD resemble the effects of vitamin A deficiency, such as epithelial lesions, keratosis and Immuno suppression, The administration of a single oral dose of 0.1, 1.0 or 10 wg 2,3,7,8-TCDD/kg bw produces a dose-related decrease 1n the hepatic storage of retinol In Sprague-Dauley rats (Thunburg et a 1.. 1979, 1930). The authors suggested, but did not demonstrate, that the low storage of retinol 1n the 2,3,7,8- TCOD-treated animals 1s the result of an Increased turnover of retinol. These results suggest that an Induced vitamin A defi ciency may be responsible for some, but not all, of the toxic effects pro duced by 2,3,7,8-TCDD. At the highest dose of 2,3,7,8-TCDD, dietary retinol supplements could not fully compensate for the 2,3,7,8-TCDD-produced decrease 1n hepatic retinol content. Lipid Peroxidation Increased Upld peroxidation has been suggested as a possible mechanism of 2,3,7,8-TC0D-1nduced toxicity (Sweeney and Jones, 1983). This hypothesis Is based on the following limited pieces of evidence. First, Iron defi ciency Inhibits 1_n vitro Upld peroxidation (Bus and Gibson, 1979; Sweeney et al., 1979) and reduces the hepatotoxlc effects of 2,3,7,8-TCDD (Sweeney et al., 1979). Secondly, llpofuscln pigments, by-products of Upld peroxi dation, are Increased 1n the heart muscle of rats treated with 2,3,7,8-TCDD (Albro et al., 1978). Thirdly, Sweeney and Jones (1983) reported that administration of the antioxidant butylated hydroxyanlsole (BHA) at a level of 0.75% 1n the diet provided some protection from 2,3,7,8-TCDD-1nduced prophyrla and neutral lipid accumulation. At this dose level of BHA, 4 of the 6 mice (sex not specified) tested were protected; however, at a lower 01350 VII-10 09/18/84 dose (G.25X), all animals were protected from these toxic effects. No beneficial effects were observed when the antioxidant vitamin E (0.01X) was Included 1n the diet. * Recently, Stohs et al. (1983) obtained direct evidence that 2,3,7,8-TCDD accelerates lipid peroxidation 1n Sprague-Oawley rats. Groups of 4-8 female rats were treated for 3 days with 2,3,7,8-TCDO at doses of 0, 10, 20 or 40 yg/kg by gavage (In a corn oil vehicle). At days 1, 6 and 11 after the last treatment the animals were sacrificed and H p 1d peroxidation was deter mined In Isolated liver mlcrosomes by the reaction of formed malondlaldehyde with thlobarblturic acid. At all sacrifice periods, Increased I1p1d peroxi dation was observed and the Increase was dose-related. The maximal Increase detected on day 6 after the last treatment was 5- to 6-fold greater than 1n the controls. In addition, these workers measured lipid peroxidation in vivo by the determination of conjugated dienes 1n rats receiving 2,3,7,8TCDO at 40 yg/kg. Using this latter method, similar Increases In Hp1d peroxidation were detected, although the maximal Increase of 2.35-fold was observed at day 1 postexposure rather than day 6 . The authors suggested that the in vivo formation of reactive free radicals during lipid peroxida tion could account for the nonspecific nature of 2,3,7,8-TCDD toxicity. Endocrine Imbalance ' Some of the toxic response to 2,3,7,8-TCOD, Including hirsutism and diminishing libido, Indicate that 2,3,7,8-TCDD may produce some of Its toxicity through endocrine disturbances (Oliver, 1975). Nlenstedt et al. (1979) reported that a single oral dose of 20 yg 2,3,7,8-TCDD/kg bw significantly reduced testosterone catabolism. Catabolism of exogenous estrogen In ovarlectomlzed rats 1s also decreased by 2,3,7,8-TCDD pre 01350 VII-11 09/18/84 treatment (Shlverlck and Huther, 1982). In this study, there was a 57% Increase in serum estrone concentrations following administration of 10 mg estrone/100 g bu/day for 4 days to either control or 2,3,7,8-TCDD pretreated ovairlectomlzed rats. No differences were observed 1n the Increase in uterine wet weight following estrone administration in control and 2,3,7,8TCDD pretreated rats. Thus, the uterotrophic response was not altered by any 2,3,7,8-TCDD-mediated change in estrone disposition. Shiverick and Huther (1983) also measured estradiol metabolism In female Holtzman rats given 2,3,7,8-TCDD at a dose of 1 yg/kg bw on days 4-19 of gestation. At this fetal toxic dose, the catechol estrogen formation abil ity of isolated liver microsomes from the dams was decreased 50% when mea sured on day 20 of gestation. These microsome preparations had a 4-fold Increase in the 7a-hydroxylation of testosterone, while there was no change in the 16a- or 6B-hydroxylase activity. Although steroid metabo lism was altered in microsomes Isolated from 2,3,7,8-TCDD-treated pregnant rats, similar exposure of pregnant rats on days 4-15 of gestation did not result in any change in circulating levels of serum l7B-estrad1ol. The authors suggested that other mechanisms besides liver metabolism of steroids may be Involved 1n the fetotoxlc effect of 2,3,7,8-TCDO. Gustafsson and Ingelman-Sundberg (1979) observed that 2,3,7,8-TCDD pro duced greater change in steroid metabolism in female Sprague-Dawley rats than in male rats of the same strain, resulting in a liver enzyme pattern displaying less sex differentiation than in uninduced rats. Based on this result, they propose that some of the effects of 2,3,7,8-TCDD result from an interaction with the hypothalamo-pitultary axis, rather than from a direct effect on steroid metabolism. 01350 VII-12 08/12/84 Since glucocorticoid hormones are known to have a catabolic effect on lymphoid tissues, such as the thymus and spleen, and these tissues degener ate after exposure of rats to 2 , 3 , 7 ,8-TCDD, Neal et al. (1979) investigated the ability of 2,3,7,8-TCDD to either stimulate the production or mimic the effects of these hormones. In male Sprague-Dawley rats treated by gavage with 2,3,7,8-TCDD at a dose of 50 ug/kg (the -LD^g), there was a slight, d e pr e s si o n in blood g l u c o c o r t i c o i d s during p o st - t re a t me n t days 1-4, followed by an ~2.5 - f ol d increase on p o s t - t r e a t m e n t days 7 and 14. W hile in c o m p e t i tive binding assays between 2,3,7,8-TCDD and a synthetic hormone, dexamethasone, 2,3,7,8-TCDD had no affinity For the hormone receptor. Thus, 2,3,7,8TCDD may stimulate glucocorticoid production, but was not able to mimic the action of these hormones by bi nd i n g to the g l uc o c o r t i c o i d receptor. It was determined, however, that the increase in g l u c o c o r t i c o i d s was likely not to p a rt i c i p a t e in the t o xi c i ty of 2 , 3 , 7 , 8 - T C D D through adrenal hyper f un c t io n , since prior adrenalectomy did not provide any protection from the lethal effects of 2 , 3 , 7 , 8 - T C D D in rats. H y pe r t h y r o i d i s m , o b se r v ed in aminals exposed to 2,3,7,8-TCDD, is suggested to be involved in the ma ni f e st a t io n of the pathological conditions associated with 2,3,7,8-TCDD exposure (Bastomsky, 1977). A partial protec tion from 2,3,7,8-TCDD Induced wasting syndrome and Immunotoxlcity as a result of thyroidectomy has been observed (Rozman et al., 1984; Pazdernik and Rozman, 1905) although this process does not appear to involve any change in the liver Ah-receptor regulated precesses (Henry and Gasiewicz, 1986). The data on the concen tr a t io n of the Ah-receptor in various human tissues are limited (Roberts et al., 1985) and the toxic r e sponse in humans has not yet been correlated with enzyme induction mediated by the Ah-receptor. 01350 VII-13 04/12/88 Hakansson and Ahlborg (1985) pretreated male Sprague-Dawley rats with 2,3,7,8-TCDD at 10 pg/kg bw 4 days before the oral adminstration of 1200 IU/kg of retinyl acetate. One hundred n i ne t y -t w o hours p o st a d mi n s t r a t i o n of retinyl a c e t a t e the 2 , 3 , 7 , 8 - T C D D- p r et r e at e d rats e x cr e t ed 41% if the retinyl acetate compared to the control, excreting only 30%. After 2,3,7,8,-TCDD treatment the decr e a se in v itamin A content was 39-53, 19-67 and 1 8 -4 4 % in the liver, Intestine and epididymis, respectively. 2,3,7,8-TCDD treatment also Influenced vitamin A content in the thymus, Initially Increasing by 42% in 6 hours and the d e cr e a s i n g by 4 0 % in 192 hours as comp a r ed to the controls. 2,3,7,8-TCDD pretreatment increased the vitamin A content in the kidn e y 3-30 times that of the control. It is important to note that the kidney becomes the pr im a r y v itamin A storage organ in vitamin A d eficient animals (Johnson and Baumann, 1947; Moore and Sharman, 1950). In a similar study Thunberg and Hakansson (1983) has also found an increase of vitamin A stor a g e in the kidney after a single oral dose of 2 , 3 , 7 , 8 - T C D D in male Sprague-Dawley rats. Result from these observations suggest strongly that pretreatment with a single oral dose of 2,3,7,8-TCDD can affect both storage and e x c r e t i o n of retinyl acet a t e as well as the vita m i n A storage in several tissues. Furthermore, s ince the u n de r l y i n g cause of the w a s t i n g syndrome is not c l e a r l y u n de r stood, it remains to be d e t e r m i n e d if the severe body weight loss a s s o c i a t e d with 2 , 3 , 7 , 8 , -TCDD toxicity is the cause of the death of the animal, or is an effect w h ic h can be s eparated from lethality. Aust (1984) suggested that 2,3,7,8-TCDD activated thyrotropin releasing hormone, which has an anorectic action, and in c o njunction with 2 , 3 , 7 ,8-TCDD-induced v itamin A d e pl e t io n results in loss of body weight. Rega r d le s s of the 01350 V I I -14 04/12/88 mec hanism or length of exposure, the wasting sydrome appears to be an indi cation of impending death rather than an early sign of toxicity. Summary Carci n og e n ic PAHs induce c y to c h ro m e P - 4 5 0 - d e p e n d e n t m o n o o x g e n a s e AHH in genetically responsive strains of mice, possibly by binding to a cytosolic receptor protein that translocates to the nucleus to initiate induction of AHH (Nebert, 1979, 1982; Nebert and Gielen, 1972; Nebert and Jensen, 1979; Nebert et al., 1972, 1981, 1983). Since the carcinogenic and toxic effects of PAHs require o x idation to reactive electrophi 1s , it is likely that the Ah receptor and AHH induction play an important role in mediating theirtoxi city and carcinogenicity. Several studies with 2 , 3 , 7 , 8 - T C D D in inbred mice support the Ah receptor mediated hypothesis and have shown that effects associated with the Ah receptor (porphyria, immunotoxicity, thymic atrophy, cleft palate) segregate with the Ah locus (Beatty and Neal, 1976b; Poland and Glover, 1973a, 1980; Jones and Sweeney, 1980). In responsive mice and Sprague-Dawley rats, the Ah receptor can be Induced with phnobarbital, which has been demonstrated to increase the concentration of the receptor. A number of reviews and comparative studies (Allen et al., 1979; Allen and Norback, 1977; Kimbrough, 1974; Kimbrough et al., 1978; McConnell and Moore, 1979; Taylor, 1979) indicate that the toxic halogenated compounds (PCDDs, PCDFs, PCBs and PBBs) all elicit similar toxic resp o n se s (a w a s t i n g syndrome, skin disorder, lymphoid atrophy and immunodeficiency, porphyria, endocrine and reproductive disorders, modification of chemical carcinogene sis and hepatic enzyme induction) in several different species. A common 01350 V I I -15 04/08/88 cyto s o li c receptor protein (A h - r e c e p t o r ) appears to be present in species that respond similarly to these compounds, indicating a common m e c h a n i s m of action (Safe, 1982; McConnell and Moore, 1979; Poland and Knutson, 1982). Structure acti v i ty studies have d e termined that the 2 , 3 , 7 ,8-(lateral ly s u bstituted) member is the most active of the PCDDs (McConnell et al., 1978b). Of the PCDFs, 2 , 3 , 7 , 8-TCDF is the most acti v e (Poland et al., 1979). Activity of the toxic halogenated aryl hydrocarbons correlates well with their a b i l i t y to I n d u c e . A H H in chick embryos (McConnell et al., 1978b) and rat hepatomas H - 4 - 1 1-E cells (McConnell et al., 1978b; Yoshihara et al., 1981). The activity of the various PCDD congeners has also been correlated w i th their b i n d i n g a f f i n i t y w i th c ytosolic receptor p rotein in m ouse (Poland et al., 1976, 1979; B r ad l o w et al., 1980; B r adlow and Casterline, 1979) or rat (Bandiera et al., 1983) hepatic cytosolic receptor protein. Activity of P C DD c ongeners is d i m i n i s h e d by d e cr e a s i n g lateral s u b s t i tu t i on or i n c r e a s ing Cl s u b s t i tu t i on in the other posi t i on s (McConnell et al., 1978b). Slight t o xicity was o b served in guinea pigs a d m i n i s t e r e d extracts of the bile of dogs treated with 2,3,7,8-TCDD led Poiger et al. (1982a) to conclude that metab o li s m represents detoxification. The structural identification of 2,3,7,8-TCDD metabolites, however, has led investigators (Poiger et al., 1982b; Sawahata et al., 1982) to hypothesize that reactive epoxide inter mediates may form that account for a substantial amount of the toxicity associated with 2,3,7,8-TCDD. Compared to other chemical carcinogens, h o w ever, the binding of 1,6-3H -2,3,7,8-TCDD derived radioactivity to hepatic DNA was less by about four-orders of magnitude than the carcinogen DMN (Poland and Glover, 1979). Much higher levels of radioactivity, however, were found bound to cytosolic protein. 01350 V I I -16 04/08/88 Since some of the effects of 2,3,7,8-TCDD toxicity resemble vitamin A deficiency (epithelial keratosis, immunosuppression) Thunburg et al. (1979, 1980; Hakansson and Ahlborg, 1985) investigated the ability of single low oral doses of 2 , 3 , 7 , 8- T C DD to reduce hepatic storage of retinol in rats. A dose- r el a t ed decr e a se in hepatic retinol was d e mo n strated. At the highest dose of 2,3,7,8-TCDD, dietary retinol supplements could not fully compensate for the 2,3,7,8-TCDD- Induced hepatic losses of retinol. Increased lipid peroxidation has been suggested as a possible mechanism for 2 , 3 , 7 ,8 -T C D D- i n du c e d toxicity (Sweeney and Jones, 1983). It was d e m o n strated that iron deficiency, which reduces lipid peroxidation, reduced 2.3.7.8- TCDD-induced hepatotoxicity (Bus and Gibson, 1979; Sweeney et al., 1979). Lipofuscin pigments, by-products of lipid peroxidation, accumulate in the cardiac m uscle of 2 , 3,7,8-TCDD treated rats (Albro et al., 1978). Butylated h y d r o x y a n i s o l e , a known antioxidant, provided some protection to mice treated with 2,3,7,8-TCDD (Sweeney and Jones, 1983). Dietary vitamin E was not p r ot e c t i v e in this study. Recently, a d o s e - r e l a t e d increase in liver m i crosomes was d e monstrated in rats treated with oral doses of 2.3.7.8- TCDD (Stohs et al., 1983). Some of the effects associated with 2,3,7,8-TCDD (hirsutism, diminished libido) suggest that the compound may Induce some of its toxicity through endocrine disturbances (Oliver, 1975). 2,3,7,8-TCDD has been shown to retard catabolism of testosterpne (Nienstedt et al., 1979) and estrone (Shiverick and Huther, 1982) and estradiol (Shiverick and Muther, 1983). Since glucocorticoids are known to have a lytic effect on lymphoid tis sues and lymphoid a t r o p h y is part of the p i c t u r e of 2 , 3 , 7 , 8 - T C D D toxicity, 01350 VII-17 04/08/88 Neal et al. (1979) investigated the ability of the compound to stimulate corticoid production or mimic its effect by c o mp etitively binding to r e c e p tor sites. Rats treated with a single dose of 2,3,7,8-TCDD responded with slightly decreased blood corticoids for ~4 days followed by a substantial (~2.5-fold) increase on post-treatment days 7-14. 2,3,7,8-TCDD did not, however, bind to receptor sites to.which dexamethasone did bind. Gustafsson and Ingelman-Sundberg (1979) proposed a hypothalamic-pituitary axis for the action of 2,3,7,8-TCDD on steroid levels rather than a direct effect on steroid metabolism. 01350 V I I -18 0 4 /0 8 /8 8 VIII. QUANTIFI C A TI O N OF TOXICOLOGICAL EFFECTS Introduction The quantification of toxicological effects of a chemical consists of separate a s s e s s me n t s of n o n c a r c 1nogen 1c and c a rc i n og e n ic health effects. Chemicals that do not produce carcinogenic effects are believed to have a threshold dose below which no adverse, noncarcinogenic health effects occur, while carcinogens are assumed to act without a threshold. In the quantification of noncarcinogenlc effects, a Reference Dose (R fD }, [ formerly termed the A c c e p t a b l e Daily Intake (ADI)] is calculated, lhe RfD is an e s ti m a t e (with u n ce r t a i n t y s p anning perhaps an order m a g n i tude) of a dally exposure to the human population (Including sensitive subgroups) that is likely to be with o u t an a p p r e c i a b l e risk of d e le t erious health effects duri n g a lifetime. The RfD Is d erived from a n o -o b s er v e dadverse-effect level (NOAEL), or lowest-observed-adverse-effect level (LOAEL), Identified from a subchronic or chronic study, and divided by an u n c e r t a i n t y factor(s) times a m o d i f y i n g factor. The RfD Is calc u l at e d as follows: RfD . ------------- (NOAEL.or. LOAEIJ --------------- ----- mg/kg bw/lJay [Uncertainty Factor(s) x Modifying Factor] S e le c t io n of the u n c e r t a i n t y factor to be empl o y ed In the c a l c u l a t i o n of the RfD is based upon p r of e s si o n al judgment, w h il e c o n s i d e r i n g the entire data base of toxicological effects for the chemical. In order to ensure that u n ce r t a i n t y factors are selected and ap pl i e d in a c o ns i s te n t manner, 01360 V I I I -1 07/22/87 the U.S. EPA (198^f) employs a modification to the guidelines proposed by the National Academy of Sciences (NAS, 1977, 1980) as follows: Standard Uncertainty Factors (UFs) * Use a 10-fold factor when extrapolating from valid experimental results from studies using prolonged exposure to average healthy humans. This factor is intended to a ccount for the v ariation in sens i t iv i t y a m ong the m embers of the human population. [1 O H ] Use an additional 10-fold factor when extrapolating from valid results of long-term studies on experimental animals when results of studies of human exposure are not available or are inadequate. This factor is intended to a ccount for the u n c e r tainty in e x t r a p o l a t i n g animal data to the case of humans. [10A] Use an additional 10-fold factor when extrapolating from less than chronic results on exper i me n t al a nimals when there is no useful l o n g - t e r m human data. This factor is intended to account for the u n certainty in ex tr a p ol a t in g from less than chronic NOAELs to chronic NOAEls. [10S] * Use an additional 10-fold factor when deriving an RfD from a ' L O AEL instead of a NQAEL. This factor is intended to account for the uncer t ain t y in extrap ol a t in g from LOAELs to NOAELs. [10L] Modifying Factor (MF) Use professional judgmentto determine another uncertainty factor (MF) that is greater than zero and less than or equal to 10. The m agnitude of the MF depends upon the professional assessment of scientific uncertainties of the study and data base not explicitly treated above, e.g., the completeness of the overall data base and the number of species tested. The default v alue for the MF is 1. The u n c e r t ai n t y factor used for a specific risk a s se s s m e n t is based principally upon scientific judgment rather than scientific fact and accounts for possible intra- and interspecies differences. Additional c o n s i d er a t io n s not inco r p or a t ed in the N A S/ODW guid e l in e s for selection of an uncertainty factor Include the use of a less than lifetime study for deriving an RfD, the significance of the adverse health effects and the counterbalancing of beneficial effects. 01360 V I 11-2 04/14/88 From the RfD, a Drinking Water Equivalent Level (DWEL) can be c a l c u lated. The DWEL represents a medium specific (i.e.p drinking water) lifetime exposure at which adverse, noncarcinogenic health effects are not anticipated to occur. The DWEL assumes 100% exposure from drinking water. The DWEL provides the noncarcinogenic health effects basis for establishing a d r i n k i n g water standard. For i ngestion data, the DWEL is derived as f o l 1o w s : DWEL (RfD) x (Body weight in kg) D r in k i n g Water Volu m e in 1/day mg/l where: Body weight = assumed to be 70 kg for an adult Drinking water volume = assumed to be 2 i/day for an adult In addition to the RfD and the DWEL, Health Advisories (HAs) for e x p o sures of shorter duration (1-day, 10-day and longer-term) are determined. The HA values are used as Informal guidance to muni c i pa l i ti e s and other organizations when emergency spills or contamination situations occur, lhe HAs are calculated using an equation similar to the RfD and DWEL; however, the NOAELs or LOAELs are identified from acute or subchronic studies. The HAs are derived as follows: HA _ (NOAEL or L D A E L ) x (bw) " (UF) x (__ . */day) mg/i Using the above equation, the following drinking water HAs are developed for noncarcinogenic effects: 1. 1-day HA for a 10 kg child i ngesting 1 it water per day. 2. 10-day HA for a 10 kg child ingesting 1 i. water per day. 3. A. Longer-term Longer-term HA HA for for a a 10 70 kg kg child adult ingesting ingesting 1 2 lit water water per per day. day. 01360 V I I 1-3 07/22/87 The 1 -day HA calculated for a 10 kg child assumes a single acute e x po s u re to the chemical and is gener a ll y derived from a study of <7 days duration. The 10-day HA assumes a limited exposure period of 1-2 weeks and is g e ne r a ll y derived from a study of <30 days duration. The l o n g e r - t e r m HA is derived for both the 10 kg child and a 70 kg adult and assumes an e x po s u re period of ~7 years (or 10% of an Individual's lifetime). The l o ng e r - t e r m HA is g e n e r a l l y d erived from a study of subchronic duration (exposure for 10% of animal's lifetime). The U.S. EPA categorizes the carcinogenic potential of a chemical, based on the overall weight-of-evidence, according to the following scheme: Group A: Human C a r c i n o g e n . Sufficient evidence exists from epidemiology studies to support a causal association between exposure to the chemical and human cancer. G roup B: P r ob a b le H u man C a r c i n o g e n . Suff i c ie n t e v id e n ce of c a rc i n o g e n i c i t y in animals with limited (Group B 1 ) or i nade q uate (Group B2) e v id e n ce in humans. Group C: Possi b le Human C a r c i n o g e n . Limited evidence of c a r c i n o g e n i c i t y in animals in the a b s e n c e of human data. Group D: Not C l as s i f i e d as to Human C a r c i n o g e n i c i t y . I n a d e quate human and animal evidence of carcinogenicity or for which no data are available. G r oup E: E v id e n c e of N o n c a r c i n o g e n l c i t y for H u m a n s . No e v id e n ce of c a r c i n o g e n i c i t y in at least two a d eq u a te animal tests 1n different species or in both adequate epidemiologic and animal studies. If toxicological e v i d e n c e leads to the class i fi c a ti o n of the contaminant as a known, probable or possibld human carcinogen, mathematical models are used to calculate the estimated excess cancer risk associated with the ingestion of the c o n t a m i n a n t in d r i n k i n g water. The data used in these 01360 V I 1 1 -4 07/22/87 estimates usually come from lifetime exposure studies using animals. In order to predict the risk for humans From animal data, animal doses must be converted to equivalent human doses. This conversion includes correction for noncontinuous exposure, less than lifetime studies and for differences in size. The factor that comp e n sa t e s for the size d i ff e r e n c e is the cube root of the ratio of the animal and human body weights. It is assu m e d that the aver a g e adult human body weig h t is 70 kg and that the av er a g e water c o n s u m p t i o n of an adult human is 2 l of water per day. For contaminants with a carcinogenic potential, chemical levels are correlated with a carcinogenic risk estimate by employing a cancer potency (unit risk) value together with the assumption for lifetime exposure from ingestion of water. The cancer unit risk is u s u a l l y d erived from a l i n e a r ized multistage model with a 95% upper confidence limit providing a low dose estimate; that is, the true risk to humans, w h il e not identifiable, is not likely to exceed the upper limit e s ti m a t e and, in fact, may be lower. Excess cancer risk estimates may also be calculated using other models such as the one-hit, Welb u l l, logit and probit. T here Is little basis In the c urrent u n d e r s t a n d i n g of the b i ological m e c h a n i s m s Involved in cancer to suggest that any one of these mode l s Is able to predict risk more a c cu r a t e l y than any other. Be ca u s e each model 1s based upon d i f f e r i n g assum p ti o n s, the estimates derived for each model can differ by several orders of magnitude. The scie n t if i c data base used to c a l c u l a t e and support the setting of cancer risk rate levels has an inherent u n c e r t a i n t y that is due to the systematic and random errors in scientific measurement. In most cases, only studies using e x pe r i me n t al a nimals have been performed.. Thus, there Is 01360 V I I 1-5 07/22/87 uncertainty when the data are extrapolated to humans. When developing cancer risk rate levels, several other areas of uncertainty exist, such as the incomplete k n ow l e d g e c o n c e r n i n g the health effects of c o ntaminants in drinking water, the impact of the experimental animal's age, sex and species, the nature of the target organ system(s) examined and the actual rate of exposure of the internal targets in experimental animals or humans. Dose-response data usually are available only for high levels of exposure and not for the lower levels of exposure closer to where a standard may be set. When there is e x po s u re to m o re than one c o nt a m in a n t, additional u n ce r t ai n t y results from a lack of information about possible synergistic or antagonistic effects. Noncarcinoqenic Effects The characteristic efFects of exposure to 2,3,7,8-TCDD Include thymic atrophy and weight loss (see the General T o xicity Section in Chapter V). In rats and rabbits, and to a lesser extent in guinea pigs and monkeys, liver dama g e is a major pathol og i c al symptom. Death is c o mmonly preceded by a prolonged period of weight loss, during which time severe deterioration of the anim a l s is observed; however, no specific lesion has been identified as the cause of death. Death generally occurs 1-7 weeks following an acute exposure. This unusual characteristic of 2,3,7,8-TCDD toxicity has resulted in m a ny s h o r t - t e r m studies that report only minor effects at doses near, or sometimes m a n y - f o l d greater that the L D ^ q . 2 , 3 , 7 , 8 - T C D D is also an Immunosuppres s ant in mice, rats and guinea pigs. The acute toxicity of 2,3,7,8-TCDD varies among species tested. Acute oral L Q ^ s ranging from 0.6 ^g/kg bw for male guinea pigs to 5051 v g /kg bw for h a ms t e rs h a ve been repo r t ed (see T a b l e V -.1}. The r e la t i ve 01360 V II1-6 09/23/87 sensitivity of man to 2,3,7,8-TCDD toxicity, compared with other species, cannot be determined from the existing data. S h o r t - T e r m E x p o s u r e . The data used for the d e t e r m i n a t i o n of a 1-day H A ' are summ a r iz e d in T a ble V1II-1. L D ^ data, w h i l e not useful in the derivation of an HA, are a useful way to compare species susceptibility and are, therefore, included in the table for c o mp a r is o n purposes. Four studies were found that identified NOAELs or LOAELs, which could be useful in the derivation of an HA (Harris et al., 1973; Madge, 1977; Smith et al., 1981; Turner and Collins, 1983). Harris et al. (1973) a d m i n i s t e r e d a single oral dose of 2 , 3 , 7 , 8 - T C D D in acetone:corn oil to groups of CD rats of mixed sex. Weights were determined at least once each week. Rats given BO or 100 yg/kg bw demonstrated a decreased weight gain and increased mortality. In the h i gh-dose group, mortality approached 50% with a mean time interval until death of 18.3 days. A dose of 25 y g /kg bw, the LOAEL in this study, resulted in a decreased body w e igh t in females at 1 week postdosing and a decreased rate of weig h t gain in males for 2 weeks postdosing. After 2 weeks, both male and female rats gained weight at the same rate as the controls. Doses of 1 or 5 yg/kg bw had no effect on body weight. Madge (1977) investigated the effect of 2,3,7,8-TCDD on intestinal absorption in CD-I mice. Mice were given single oral doses of 10, 25, 75, 150, 200 or 300 yg 2 , 3 , 7 ,8-TCDD/kg bw. Absorption of D-glucose, D-galactose, L-argenine and L-histidine was measured 7 days later, using the everted intestinal sac technique. Absorption of D-glucose was decreased at all dose levels; however, absorption of the other compounds was not affected 01360 VI11-7 04/14/88 TABLE VIII-1 > Acute Toxicity of 2,3,7,,8-TCDD 1360 Species Route of Exposure Dose pg/kg/day Duration of Exposure Duration of Experiment Effect Level Endpoints Reference Rat Rat (male) Rat (female) Rat House oral oral oral l.p. I.p. 25 22.0 45.0 2.5 1 House < H-4 House 010 House oral oral oral House oral House oral Guinea pig (female) oral 10 50 15 284 114 0.1 Guinea pig (male) oral 0.6 Guinea pig (female) oral 2.1 Guinea pig (male) oral 2.0 Rabbit oral 115 Rabbit ZD S > Hamster N) Hamster -J Hamster dermal oral oral 1.p. 275 5051 1157 >3000 Monkey oral <70 l.p. . Intraperitoneal 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day 1 day B-9 weeks 2-8 weeks 2-8 weeks 8 weeks NA LOAEL ld50 ld50 LOEL LOAEL 7 days LOAEL 12 weeks 12 weeks 30 days 2 months 42 days LOAEL NOAEL lo50 ld50 LOAEL 2-8 weeks 2-8 weeks 30 days 2-8 weeks 3 weeks 55-71 days 50 days 50 days >35 days ld50 ld50 ld50 ld50 ld50 ld50 ld50 L050 LD50 Decreased body weight Increased serum triglycerides Decreased macrophage and natural killer cell number Decreased Intestinal absorption of d-glucose Porphyria Porphyria Hi Id histopathologic effects on liver Harris et al., 1973 Schwetz et al., 1973 Schwetz et al., 1973 Poli et al., 1980 Han tovani et al., 1980 Hadge, 1977 Smith et al.. 1981 Smith et al.. 1981 HcConnell et al., 1978a Vos et al.. 1974 Turner and Collins, 1983 Schwetz et al.. 1973 Schwetz et al., 1973 McConnell et al., 1978a Schwetz et al.. 1973 Schwetz et al., 1973 Henck et al., 1981 Olson et al., 19B0b Olson et al., 1980b McConnell et al., 1978b by any of the treatment. The d e cr e a se in D - gl u c os e a b so r p ti o n was doserelated over the range of 0-75 pg/kg bw. . In this study, 10 yg/kg bw consti tuted a LOAEL . Smith et al. (1981) investigated the effect of 2,3,7,8-TCDD on hepatic p o rp h y ri n levels in C 5 7B 1 / 1Q and D BA/2 mice. A single oral dose was a d m i n istered in arachis oil (0, 5, 15, 50, 75, 150, 300, 600 or 1 200 yg/kg bw) and hepatic porphyrin levels were determined at intervals for <12 weeks. T here w e re large strain d i f f e r en c e s in s u s c e p t i b i l i t y to porphyria induction, with the C57B1/1Q strain being ~20 times as sensitive as the DBA/2 strain. In this study, the lowest dose that induced porphyria was 50 yg/kg bw. Thus, 50 yg/kg bw was a LOAEL and 15 yg/kg bw represented a N0AEL. ' Turner and Collins (1983) administered single oral doses of 0.1, 0.5, 2.5, 12.5 or 20 y g/kg bw of 2 , 3 , 7 , 8 - T C D D in aqueousmethyl c e ll u l os e to groups of 4-6 female guinea pigs. Survivors were killed 42 days after d o s ing and examined for histopat h o lo g i c changes in the liver. Four of the 6 animals in the highest dose group and 1/5 in the 12.5 y g/kg group died before the end of the observation period. Mild histopathologic changes including steatosis (fatty change), focal necrosis and cytoplasmic degener a tion w e re noted in anim a l s f r om all treated groups, but not in controls. The authors indicated that qualitative differences among the dosage groups were not detectable by light microscopy. All of the LOAELs and NOAELs determined for rats and mice are above the LD(.g for guinea pigs (0.6-2.1 yg/kg). A l t h o u g h no NOEL or N0AEL is 01360 V I 1 1 -9 04/14/88 available for guinea pigs, a LOAEl of 0.1 yg/kg can be derived from the study of Turner and Collins (1983). Studies of appropriate duration for determining a 10-day HA are identified in Table VI1I-2. ' L o n g e r - T e r m E x p o s u r e , The health effects of l o n g - t e r m expo s u re to 2 , 3 , 7 ,8-TCDD. are summ a r iz e d in Table V1II-3. Most of the long - t er m studies have been performed using rats. There is, therefore, only limited i n f o r m a tion on the chronic toxicity of 2 , 3 , 7 , 8-TCDD in other species. Of the studies included in T able VIII-3, only those by Kocba et al. ( 1 9 7 8 a , b, 1979) and Murray et al. (1979) were done with administration of 2,3,7,8-TCDD in the diet on a daily basis. The U.S. ERA developed an ADI based on noncarcinogenic effects as Indicated in U.S. ERA (1984). For c o ns i s te n c y, the ratio n al e used by U.S. EPA for the ADI c a l c u l a t i o n in U.S. E PA (1 984 ) is used for the RfD c a l c u l a t i o n herein. The r a ti o n al e as pres e n te d In U.S. EPA (1984) is as follows: 2,3,7,8-TCDD displays an unusually high degree of reproductive toxicity. It is teratogenic, fetotoxic and reduces fertility. In a 3-generation reproductive study, Murray et al. (1979) reported a 2r eduction in f e rtility after dally dosing at 0.1 or 0.01 yg 2 , 3 , 7 ,8-TCDD/kg in the Fg and F ge ne r a ti o n s of S p r a g u e - D a w l e y rats. Although Murray et al. (1979) considered the lowest dose tested, 0.001 yg/kg, to be a no-observed-effect level (NOEL), a re-evaluation of these data, by Nlsbet and Paxton (1982), using different statistical methods, indicated that there was a reduction in the g e st a t i o n index, d e cr e a s e d fetal weight, I ncreased liver to body weight ratio, and increased incidence of dilated renal pelvis at the 0.001 yg/kg dose. The re-evaluated data would suggest that equivocal adverse effects were seen at the lowest dose (0.001 yg/kg/day) and that this dose should, therefore, represent a lowest-observed-adverse-effect level (LOAEL). Schantz et al. (1979) found redu c t io n s in fert i l it y and various other toxic 01360 VIII-1Q 04/14/88 TABLE V I 1 1-2 Effects of 4-13 Weeks Exposure to 2,3,7,8-TCDD 01360 VIII-11 Species Route of Exposure Rat Rat Rat Rat Rat Rat Rat cRat Rat Rat oral oral oral oral oral oral oral oral oral oral House House House House oral oral oral oral House oral House House oVJO \iuC\>D> House oral oral oral Dose ug/kg 0.01 0.1 0.5 1.0 0.1 1.0 0.1 5.0 1.0 0.001 1.0 5.0 25.0 5.0 1.0 1.0 1.5 10 ppb No. of Treatments/ Week 5 5 2 2 7 1 7 1 1 7 2 1 1 1 1 1 1 7 Duration of Exposure Effect Level 13 weeks 13 weeks 13 weeks 13 weeks 30 days 6 weeks 30 days 6 weeks 6 weeks 3 generation N0AEL L0AEL N0AEL L0AEL L0AEL N0AEL L0AEL L0AEL N0AEL L0AEL 13 weeks 4 weeks 4 weeks 4 weeks L0AEL N0AEL L0AEL L0AEL 4 weeks N0AEL 4 weeks 4 weeks 5 weeks L0AEL L0AEL L0AEL Endpoints Reference Decreased body weight Decreased body weight Toxic hepatitis Toxic hepatitis Decreased thymus weight Decreased body weight Thrombocytopenia Decreased body weight and thymus weight Decreased body weight and thymus weight Decreased body weight, decreased fertility, decreased fetal survival Toxic hepatitis Porphyria Porphyria Decreased thymus weight and graft-versus-host response Decreased thymus weight and graft-versus-host response Decreased resistance to Salmonella Increased endotoxlc (E. coll) susceptibility Decreased tetanus response, antigenic RBC response, sensitization to DNFB, resistance to Salmonella Infection, resistance to Listeria Infection Koclba et al.. 1976 Kodba et al.. 1976 NTP, 1980a NTP, 1980a Harris et al., 1973 Harris et al., 1973 Zlnkl et al., 1973 Vos et al.. 1973 Vos et al.. 1973 Hurray et al., 1979 NTP, 1980a Goldstein et al.. 1978 Goldstein et al.. 1978 Vos et al., 1973 Vos et al.. 1973 Thigpen et al.. 1975 Vos et al.. 1978a Hlnsdl11 et al., 1980 09/23/8 07360 -- J_________ u> Oo' Species Route of Exposure Dose ug/kg House Guinea pig Guinea pig l.p. oral oral 0.4 0.008 0.04 I.p. = Intraper Honeal No. of Treatments/ Ueek 1 1 1 Duration of Exposure 4 weeks 8 weeks 8 weeks TA BL E VI 1 1-2 (con i. ) Effect Level L0AEL N0AEL L0AEL Endpoints Decreased cytotoxic T-cell response Thymus weight and tuberculin hypersensitivity Thymus weight and tuberculin hypersensitivity Reference Clark et al.. 1981 Vos et al.. 1973 Vos et al., 1973 TABLE V I 11-3 Effects of Long-Term Oral Exposure to 2,3,7,0-TCDD Specles Dose MAg) T rNeWao.temekoefn t s / ofDuErxaptioosnure ELefvfeecl t Endpoints Reference Rat 0.001 Rat 0.01 Rat 0.1 Rat 0.1 Rat 0.001 Rat 0.01 Rat 0.01 Rat O.OS Rat 1.0 Rat 0.01 House 0.01 house 0.007 7 3 generation L0AEL dDeeccrreeaasseedd bfoedtayl wseuigrvhitv, aldecreased f e r t i l i t y , Hurray et a l .. 1979; 1 16 week s N0AEL Elevated porphyrin levels Goldstein et a l ., 1982b 1 16 weeks L0AEL Elevated porphyrin levels Goldstein et a l . , 1982b 2 28 weeks L0AEL Fatty changes In l iv e r , decreased body weight King and Roesler, 1974 7 104 weeks N0AEL Degenerative and necrotic changes In the liver K19o7c9Iba et a l ., 1978a,b. 7 104 weeks L0AEL Degenerative and necrotic changes In the liver K19o7c9lba et a l . , 1978a,b, 2 104 weeks N0AEL Toxic h ep a titis NTP, 1982a 2 104 weeks L0AEI Toxic h ep atitis NTP, 1982a 1 45 weeks L0AEL Porphyria Cantonl et a l .. 1981 1 45 weeks L0EL Hepatic enzyme Induction, liver weight Cantonl et a l ., 1981 2 104 weeks L0AEL Toxic h ep atitis NTP. 1982a 1 10d542urwwateeieeokknss estxupdoysure, L0AEL Dermatitis and amyloidosis loth el a l . , 1978. 1979 effects in rhesus monkeys fed a 50 ppt 2 , 3, 7 , 8- T C DD diet for 20 months. This corresponds to a calculated daily dose of 0.0015 yg 2 , 3 , 7 ,8-TCDD/kg/day. These results suggest that monkeys may be somewhat more s ensitive than rats, since the effects in monkeys were more severe and not equivocal. Since the data from the limited study by Schantz et al. (1979) are supportive of the findings by Murr a y et al. (1979), it seems reas o n ab l e to d e te r m in e an ADI based on the LOAEL. Quantification of Noncarcinoqenlc Effects Derivation of 1-Day H A . The data on very short-term exposures, 1-14 days, are sufficient for the derivation of a 1-day HA. As previously discussed the available studies establish LOAELs for rats and mice that are greater than the L D ^ for guinea pigs. A N0AEL is not a v a i l a b l e for guinea pigs, but a LOAEL of 0.1 yg/kg can be derived from the study of Turner and Collins (1983). This LOAEL can be used to c a lculate a 1-day HA, using an uncertainty factor (UF) of 1000 for an animal LOAEL. For a 10 kg child: 1-day HA = (0.1 yq/kq bw/day x 10 kg) = 0 . 0 0 1 0 y g / l 1000 x 1 i/day = 1x10 3 yg/s. where: 0.1 yg/kg bw/day 10 kg 1 i/day 1000 = LOAEL from the study of Turner and Collins (1983) =. Assu m e d body weig h t of a child = assumed water consumption by a 10 kg child = uncertainty factor, represents two 10-fold factors to account for both intra and interspecies variability to the toxicity of chemicals, and a 10-fold factor to account for a LOAEL being used Instead of a N0AEL 01360 VI 11-14 04/ 14/88 Thi s HA i s e q u i v a l e n t to 0. 0010 yg/ day or 0. 00010 yg/ kg bw/day. Deri v a ti o n of 10-Day H A . A 10-day HA is c a l c u l at e d by d i viding the 1-day HA by 10 to convert the 1-day HA to a 10-day HA. Therefore, along with using an uncertainty factor of 1000 for an animal L0AEL {i.e., 10-fold for intra- and 10-fold for interspecies v a riability to the toxicity of a chemical in lieu of specific data, and an additional 10-fold because the e s ti m a te is based on a 10AEL rather than a N0AEL), a 10-day HA can be c a l c u lated from the L0AEL of 0.1 yg/kg/day reported by Turner and Collins (1983). For a 10 kg child: 10 day HA = 0.001 y q/kq bw/day x 10 kg = 0.0001 yg/i. 1000 x 1 4,/day x 10 1x10 4 yg/1 where: 0.001 yg/kg bw/day 10 kg 1 i/day 1000 10 = 1-day HA for child assumed body weight of a child assumed water consumption by a 10 kg child uncertainty factor, represents two 10-fold factors to account for both intra and interspecies variability to the toxicity of chemicals, and a 10-fold factor to account for a L0AEL being used Instead of a N0AEL . conversion of 1-day HA to 10-day HA. This HA is e q ui v a l e n t to 0.0001 yg/d a y or 0.00001, y g/kg bw/day. 01360 VIII-15 04/14/88 D e ri v a ti o n of L o n g e r - T e r m H A . The studies included in Table VI 1 1 -3 are c o n s i d e r e d for c a l c u l a t i o n of a l o n g e r - t e r m HA. This is based on a LOAEL of 0.001 yg/kg for r e p r o d uc t i ve effects in the 3 - g e n e r a t i o n r e p r o ductive study in rats by Murray et al. (1979) along the rationale discussed in the L o n g e r - T e r m E x p o s u r e Section. A l t h o u g h D e Ca p r io et al. (1986) found NOELs of 0.61 and 0.68 mg/kg/day, respectively, for male and female guinea pigs in a 90-day ingestion study, this dose is slig h t ly b elow the LOAEL of 1 m g / k g / d a y (0,001 yg/kg/day) observed in the Murray et i. (1979) study. Using the LOAEL of 0.001 yg/kg bw/day, because the Murray et al. (1979 ) study suggested that a dose of 0.001 yg/kg bw/day may be a LOAEL for reproductive effects, and dividing this by an uncertainty factor of 1000 for an animal LOAEL, a longer-term HA can be calculated. For a 10 kg child c o ns u m i n g 1 a. of d r i n k i n g water daily, the l o n g e r -t e r m HA is c a l c u l a t e d as follows: L o n g e r - t e r m HA = l0 -001 yg/k g / da y ) t1 0 .k 9) = o . 00001 yg/l (1000) (1 i/day) = l x l Q ~ 5 yg/i. A c co r d in g l y, for a 70 kg a d ult c o ns u m i n g 2 a, of d r in k i ng water daily, the l o n g e r - t e r m HA for an adult is c a l c u l a t e d as follows: Longer-term HA = ( - 001 bw/da> x 70 ^ = 0.000035 Ug/L 1000 x 2 8,/day = 3 .5xl 0"5 yg/iL 01360 V111 -1 6 04/14/88 This l o n g e r - t e r m HA is identical to the 10-day HA for adults and is e q u i v a lent to an ADI of 70 pg/d a y or 1.0 pg/kg bw/day. This AOI is the same as that esti m a te d in the AWQC d o cument for TCDD for c o m p a r i s o n purposes to the criteria derived from carcinogenicity data (U.S. ERA, 1984). Assessment of Lifetime Exposure and Derivation of a D W E L . 2 , 3,7,8- TCDD may be c l as s i f i e d in Group B: P r obable Human C a rc i nogen, a c c o r d i n g to U.S. EPA's proposed w e i g h t - o f - e v i d e n c e scheme for the c l as s i fi c a ti o n of carcinogenic potential (U.S. EPA, 1986). Because of this, caution must be e x e r c i s e d in m a k i n g a d e c i s i o n on how to deal with p o ss i b le l i fetime e x po s u re to this substance. The risk manager must balance this a s sessment of carcinogenic potential against the likelihood of occurrence of health effects related to n o n c a r c 1nogenic endpoints of toxicity. In order to assist the risk manager in this process, drinking water concentrations a s sociate d with e s timated excess lifetime cancer risks over the range of 1 in 10,000 to 1 in 1,000,000 for the 70 kg adult, drinking 2 i of water per day, are provided in the foll o w in g section. In addition, in this section, a DWEL is derived. A DWEL is defined as the m e d i u m - s p e c i f i c (in this case, d r i n k i n g water) e x po s u r e that is interpreted to be p r ot e c t i v e for n o n c a r c i n o g e n i c endpoints of t o xicity over a l i fe t i me of exposure. The DWEL is d e te r m in e d for the 70 kg a d ult inges t in g 2 i of water per day. Also p r ov i d e d is an esti m a te of the excess cancer risk that w o u l d result if e x po s u re w e re to occur at the DWEL over a lifetime. Neither the risk estimates nor the DWEL take relative source contribu tion into account. The risk manager should do this on a case-by-case basis, considering the circumstances of the specific contamination incident that has occurred. 01360 V1II-17 04/14/88 The U.S. EPA has developed, for comparison with cancer-based criteria, a 1p resumed safe daily intake level based on n o n c a r c nogenic effects as indicated in U.S. EPA (1 984 ). For c o nsistency, the r ationale used by the U.S. EPA for the c a lc u l a t i o n of this value in U.S. EPA (1984) is used here for the DWEL calculation. The r a ti o n al e as p r es e n te d in U.S. EPA (1984) is as Follows: 2,3,7,8-TCDD displays an unusually high degree of reproductive toxicity. It is teratogenic, fetotoxic and reduces fertility. In a 3-generation reproductive study, Murray et al. 8 2r eduction in fertility after daily d o sing at 2 , 3 , 7 , - T CDD/kg in the F-j and F g e n e r a ti o n s (01.917 9 ) orr e p0o. 0r t1e d a yg of Sprague-Dawley rats. Although Murray et al. (1979) considered the lowest dose tested, 0,001 yg/kg, to be a no-observed-effect level (NOEL), a re-evaluation of these data by Nisbet and Paxton (1982), using different statistical methods, indicated that there was a reduction in the ge station index, de creased fetal weight, increased liver-to- body weight ratio, and increased Incidence of dilated renal pelvis at the 0.001 yg/kg dose. The re-evaluated data would suggest 0.001that equivocal a d verse effects were seen at the lowest dose ( yg/kg/day) and that this dose should, therefore, represent a lowest-observed-adverse-effect level (L0AEL). Schantz et al. 1979) found r e ductions in f e rt i l it y and various other toxic effects in rhesus monkeys fed a 50 ppt 2,3,7,8]TCDD diet for 20 months. This corresponds to a calculated daily dose of 0.0015 yg 2,3,7,8-TCDD/ kg/day. These results suggest that monkeys may be somewhat more sens i t iv e than rats, since the effects in monk e y s were more severe and not equivocal. Since the data from the limited study by Schantz et al. (1979 ) are supportive of the findings by Murray et al. (1979), it seems r e a s o n a b l e to d e te r m i n e an RfD based on the L0AEL. From these results, a L0AEL of 0.001 yg/kg was identified. Using this L0AEL, the DWEL is d e r i v e d as follows. Step 1 - RfD Derivation RfD (0. 001 ( 1y0q0/0k) q / d a y ) = 1x 10 6 yg/kg/day 01360 V111 -18 04/14/88 where: 0.001 pg/kg/day = LOAEL from Murray et al. (1979) 1000 = uncertainty factor appropriate for use with a LOAEL from an animal study Step 2 - DUEL Derivation DWEL = (1x10 6 p(2g / kg/day) sVday) (70 kg) = 0 000035 pg/i = 3 . 5 x l 0 ~5 ng/B. where: l x l O ^6 pg/kg/day = RfD 70 kg 2 i/day = weight of protected individual = assumed volume of water ingested by an adult The estimated excess cancer risk associated with lifetime exposure to "5d r in k i ng water c o n t a i n i n g 2 , 3, 7 , 8 - T C D D at 3 . 5 x l 0 pg/i. is ~ 2 x l 0 " 4 . This estimate represents the upper 95% confidence limit from extrapolations prepared by the U.S. EPA's Carcinogen Assessment Group using the linearized, m u l t i s t a g e model. The actual risk is u n l i k e l y to exceed this value, but there is c o n s i d e r a b l e u n c e r t a i n t y as to the a c c u r a c y of risks c a l c u l a t e d by this methodology. Carcinogenic Effects A number of epidemiological studies have attempted to relate 2,3,7,8- TCOD exposure to human health effects (see the Epidemiological Studies Section). These studies are limited by small sample sizes, short follow-up period and exposure to multiple compounds. These epidemiological studies do not unequivocally establish- a relationship between 2,3,7,8-TCOD and the 01360 VI 11-19 04/14/88 de ve l o pm e n t of tumors in humans, though an a s s o c i at i o n has been suggested with s o ft - tissue sarcomas (Harde.ll and Sandstrom, 1 979; E r iksson et a l ., 1979, 1981), lymphomas ( H a r d e n et al., 1980, 1981) and stomach cancer (Axelson et al., 1980; Theiss and F r en t z e l - B e y m e , 1977). In comparison, a number of cancer bioassays have clearly demonstrated the c a r c i n og e n ic potential of 2 , 3 , 7 , 8- T C DD in animals (Kociba et al., 1978a,b; Van Miller et al., 1977a,b; Toth et al., 1979; NTP, 1980a,b). T h es e studies are summ a r iz e d in Table VI11 -4. Oral a d m i n i s t r a t i o n of 2,3, 7 , 8- T C DD , either in the diet or by gavage, results in the p r o d u c t i o n of hepa t o ce l l ul a r carcinomas in female rats and both sexes of mice (Kociba et al., 1978a,b; NTP, 1980a; Toth et a l ., 1979). F o ll i c ul a r - c e l l adenomas of the thyroid have been o b se r v ed in both male rats and female mice (NTP, 1980a). Various squamous cell carcinomas have also been reported in both sexes of rats (Kociba et al., 19 7 8 a ,b ). Toth et al. (1979) administered weekly gavage doses of 0.0, 0.007, 0.7 and 7.0 yg/kg bw to groups of 45 male Swiss mice for 1 year. The r e la tively short duration of e x posure limits the sensitivity of this assay for 8d e te r m i n i n g the c a rc i n o g e n i c potency of 2 , 3 , 7 , -TCDD. Van Miller et a l . (1977a,b) maintained groups of 10 male Sprague-Dawley rats on diets c o n t a i n i n g 0.0, 0.001, 0.005, 0.05, 0.5, 1.0, 5.0, 50, 500 or 1000 ppb 2,3,7,8-TCDD for 78 weeks. Based on the food consumption of two rats from each group, these dietary levels resulted in doses of 0.0, 0.003, 0.001, 0.01, 0.1, 0.4, 2.0, 24, 240 and 500 yg/kg bw/week, respectively. 01360 V I I I -20 09/28/87 TABI.F VI 11- 4 O CcoOr Carcinogenicity Bioassays of ?.3.7,8-tCDD by Oral and Dermal Exposure Species Rat (male) Rat (female) Rat (male) Rat (male) Rat (female) House (male) House (female) House (male) House (male) House (female) Route gavage Dose Range 0-0.5 pg/kg bw/week gavage 0-0.5 pg/kg bw/week diet 0-1000 ppb diet 0-0.1 pg/kg bw/day diet 0-0.1 pg/kg bw/day gavage 0-0.5 pg/kg bw/week gavage 0-0.2 pg/kg bw/week gavage dermal dermal 0-7.0 pg/kg bw/week 0-0.03 pg/wcek 00.015 pg/week of DTurreaattImonent 104 weeks 104 weeks 78 weeks 105 weeks 105 weeks 104 weeks 104 weeks 365 days 104 weeks 104 weeks DofurSattuIodny 105-107 weeks 105-107 weeks 95 weeks 105 weeks 105 weeks 105-107 weeks 105-107 weeks 424-649 days 104 weeks 104 weeks AGnrIomuapls/ 50 50 10 50 50 50 50 45 30 30 Tumor Types Reported fothor yl lcrloacridcuilnaorm- cae ol lf athdeenoma Nhofeepotaphtleoacsletiilvcleurlnaorduclearcoirnoma All tumors Soot hqff euattmhhl oeeonusgha udacerrde,enl laapdlaeclnaacorotmceriatneaoxnmda Hosqfeupatmahteooucsteolnclgeuullelar canacdracriclniuonnmogma a, Hepatocellular carcinoma Hof ofelpltahltceouctlehalyrl-uroclaiedrl l caadrceinnoommasa, Liver tumors FI ni bt er ogsuamr ceonmt aar y ofs y st ht eem FI ni bt er ogsuamr ceonmt aar y o fs yst ht eem Reference NIP, 1982a NTP, 1982a V19an77Ha,ibller et a l . . K19o7c9Iba et a l ., K19o7c9Iba et a l ., NIP, 1982a NTP. 1982a Toth et a l . . 1979 NIP, 1982b NTP, 1982b 04/12/08 The small group sizes (10 rats/group) and relatively short exposure times (78 weeks) limit the usefulness of this study in q u a n t i t a t i v e risk a s s e s s ment . Both the NTP (1982a) study and the Kociba et al. (1978a,b) study contain sufficient a n i m a l s / g r o u p and involved suff i c ie n t ly long dosing schedules to be used for quantitative risk assessment. Kociba et al. (1978a,b) m a i n 0 0tained groups of 50 S p r a g u e - D a w l e y rats on diets prov i d in g doses of . , 0.001, 0.01 or 0.1 pg 2, 3, 7 , 8- T C DD / k g bw/day for 2 years. The high dose resulted in reduced lifespans for the female rats, reduced body weig h t gain in both sexes and signs of tissue toxicity. Statis ti c a ll y significant increases in h e p a t o c e l l u l a r neop l a st i c nodules were o b se r v ed in females at doses of 0.1 and 0.01 pg/kg bw/day. At the high-dose, increases were observed in stratified squamous cell carcinomas of the hard palate and/or nasal turbinates in both sexes, in k e ra t i ni z i ng squamous cell carcinomas of the lungs in females, in squamous cell carc i n om a s of the tongue in males and in hepatocel l ula r carcinomas in females. In the NTP bioa s s ay (NTP, 1982a), groups of 50 Q s borne-Mendel rats or Swiss mice w e re d o se twice w e ek l y by gavage with 2 , 3 , 7 , 8 - T C D D in a 9:1 corn oil;acetone solution. The rats and male mice received TWA doses of 0.0, 0.0014, 0.0071 and 0.071 pg/kg bw/day. The corresponding doses for female mice were 0.0, 0.0057, 0.029 and 0.29 pg/kg bw/day. Dose-related depres sions in mean body weight w e r e . reported for both sexes of rats. In male rats, a dose- d ep e n de n t increase in the Incidence of follicular-cell adenomas or carcinomas of the thyroid was observed. The incidence of subcutaneous tissue fibromas was s i gn i f i c a n t l y increased in the high - d os e group. In female rats, o b s e r v e d increases in the i ncidence of s u bc u t an e o us tissue 01360 VIII -22 04/14/88 fibrosarcomas, adrenal cortical adenomas and hepatocellular carcinomas and neoplastic nodules were s t a t i s ti c a ll y si gn i f ic a n t in the high- d os e group. In mice, s t a t i s t i c a l l y s i gn i ficant increases in tumor incidences were o b served only in the h i gh - d os e group. An increase in h e p a t o c e l l u l a r c a r c i nomas and n e oplastic nodules was noted in the males. In females, increases w e re o b served in h e p a t o c e l l u l a r carcinomas and adenomas, fibrosarcoma, histiocytic lymphoma, thyroid foil icular-cell adenoma and cortical adenoma or carcinoma. Quantlfication of Carcinogenic Effects A summary of 95 % upper-limit human carcinogenic potency estimates for 82 , 3 , 7 , -TCDD derived from the Kociba et al. (1978a,b) and NCI (N T P , 1980a) studies in rats and mice, with two pathologists' findings for the Kociba study, are given in Table VIII-5. These potency estimates have been c a l c u lated using the linearized multistage model by a previously described methodology (Federal Register, 1980). The largest of these potency factors (q^*) comes from data in an i n de p endent p a t h o l o g i s t ' s (Dr. R. Squire) review of the Kociba feeding study of female Sprague-Dawley rats. An a d justment for high early m o r t a l it y in the high dose groups led to a slightly lower estimate. The mean of the two pathologists' estimates after m o r t a l i t y a d j u s t m e n t is as follows: q-|* = [(1.51 x 1 0 s ) x (1.61 x 105 )]"'/2 _ 1.55 x 1 0 s ( m g / k g / d a y ) ~ 1 These potency estimates were derived from the Kociba feeding study. The responses and parameters of the Kociba feeding study in female rats are 6given in Table V I I I - . The number with tumors refers to the number of animals with at least one of liver, lung, hard palate or nasal turbinate tumors. Adjustment For early mortality refers to eliminating those animals 01360 VII1-23 04/14/88 TABLE V I 1 1 -5 8Summary of Human Potency Estimates for 2 , 3 , 7 , - TCDQa Species Sex Pathologist Rat M Kociba Human q-|* P in o tency (mg/k gE/sdtaiym)a't1e 1.47 X 10* Rat Rat Mouse Mouse Squire F Kociba Unadj usted Adjusted for earl y mor tal ity Squire Unadj usted Adjusted for early mortality F NTP-reviewed M NTP-reviewed F NTP-reviewed 1 .73 X 10* 2.52 1.51 X X 1100s5b 41..2615 X X 1100s5b 3,28 X 10* 7.52 X 10* 4.56 X 10* Reference Kociba et a l ., 1978a,b Koc iba et a l ., 1978a,b NIP, 1982a N TP, 1982a NT P , 1982a a Source: U.S. EPA, 1984 1 ' 1^Values used to d e te r m in e the geometric mean of ,.5 6 x 1 0 s ( m g / k g / d a y j 01360 VIII-24 04/14/88 TABLE VI11-6 Responses and Parameters of the Kociba Feeding Study* Dose (mg/kg/day) 0000...001101x x 1x 1010' 0"3~33 le = 720 days Le = 720 days L = 720 days *Source: Kociba et al., 1978a,b No. with Tumors/No. Examined Adjusted for Early Mortality Squire 16/85 8/48 27/48 34/40 Kociba 9/85 3/48 18/48 34/40 W W h0 = = 70 kg 0.450 kg R = 5000 l/kg 01360 V I I I -25 09/23/87 that died during the first year of study. The first tumor appeared in the high-dose group during the thirteenth month. With these parameters, the mean 95% upper-limit carcinogenic potency 05factor for humans, q^*, is 1 . 5 6 x 1 (mg/kg/day l'1 . For a 70 kg human drinking 2 a water/day, the water concentration should be < 2 .2x 10 ' 6 1yg/a. in order to keep the upper-limit individual i fetime cancer risk 1 10< Q ~ 5 . Water c o nc e n t r a t i o n c o r r e s po n d in g to excess cancer risk of "* and 10 6 are, therefore, < 2 .2x10 5 and < 2 .2xl0- 7 , respectively. Existing Guidelines. Recomniendatlons and Standards The U.S. EPA has e s tab l is h e d the limits of 1 . 3 x l 0 " 7 , 1 . 3 x l O " B or ' 3 81 . 3x l 0 ug 2 , 3 , 7 , -TCDD/i in ambient waters, based on an a ssumed daily consumption of 6.5 g of contaminated fish and shellfish and ? l of d r i n k i n g water (U.S'. EPA, 1984). Under these c o nd i tions, 9 4 . 2 % of the total exposure would result from the consumption of aquatic organisms. The recommended levels correspond to estimated human lifetime excess cancer risks of I Q " 5 , 1 0 ~6 or 10" 7 , respectively. These values are c o n s i d e r a b l y lower than the HAs for d r in k i n g water that are r e co m m e n d e d in this document (see the S ummary Section of this chapter), r e flecting the high bioaccumulation of this compound in aquatic species. 8An AOI of K T * pg 2 , 3 , 7 , -TCDO/kg bw/day has p r ev i o us l y been p r o posed by the National Academy pf Sciences Committee on Drinking Water and Health (NAS, 1977). This ADI was based on a 13-week rat feeding study by Kociba et al. (1976) and was proposed before convincing evidence for the carcinogenicity of 2,3,7,8-TCDD had accumulated. 01360 VI 11-26 04/14/88 Agency FDA CDC EPA 1ARC N10SH Advisory The FDA advises that fish c o ntaining >50 ppt of 2,3,7,8-TCDD should not be consumed and those containing >25 ppt, but <50 ppt, should not be consumed more than twice a month (FDA, 1983). This is reflected in a C a na d i an limit of 20 ppt in the Lake Ontario commercial fish imported into the United States (NRCC , 1981). Levels >1 pbb in residential soil are levels of concern (Kimbrough et a l ., 1984) Group B2 - Sufficient animal data to indicate carcinogenicity, plus inadequate human evidence which suggests that 2 , 3 , 7 , 8 - T C D D is prob a b ly a human carcinogen. (U.S. EPA, 1986) IARC Group 28 - Sufficient animal data to indicate carcinogenicity, plus inadequate human evidence which suggests that 2 , 3 , 7 , 8 - T C D D is prob a b ly a human c a rc i nogen. (U.S. EPA, 1986) It is r e co m m e n d e d that 2 , 3 , 7 , 8 - T C D D be c o ns i d er e d a potential occupational carcinogen and exposure should be limited to the fullest extent feasible {NI O S H , 1984). Special Considerations Synergistic Effects. Enzyme Induction -- 2 , 3, 7 , 8- T C DD has been demon s tr a t ed to s i g n i f i cantly alter the toxicity of other toxicants, primarily as a result of enzy m e induction (see the I n t e r a c t i v e Effects S e c t i o n in Chapter VI). These ch an g e s may either i n crease toxicity, if m e t a b o l i s m is p r e d o m i n a n t l y an a c t i v a t i o n pathway, or d e cr e a s e toxi c i ty if m e t a b o l i s m is p r e d o m i n a n t l y a d e t o x i f i c a t i o n m e c h a n i s m . Thus, G r ieg (1972) has d e m o n s t r a t e d a 54/. 2d e c r e a s e in the dura t i on of z o x a z o l a m l n e - i n d u c e d paralysis and a -fold increase in h e x a b a r b ! t o n e s l ee p i gg time. C o c a r c i n o q en e s !s and Promotion -- In addition to being a complete carcinogen, 2,3,7,8-TCDD has been demonstrated to Function as a promoter of DEN-initiated hepatocarcinogenesis (Pitot et al., 1980). Positive results 01360 VII 1-27 04/14/88 For tumor promoting activity have also been obtained when 2,3,7,8-TCDD was tested on the skin of mice homozygous for the "hairless" trait (Poland et a 1., 1982). Another a ttempt to d e m o n s t r a t e the tumor p r om o t i n g a bility of 2,3,7,8-TCDD on mouse skin (Swiss-Webster mice) have produced negative results (NTP, 1980b; Berry et a l ., 1978, 1979 ). 2 , 3 , 7 , 8 - T C D D has a l so been d e m o n s tr a t ed to be c o ca r c i n o g e n i c with 3 - m e t h y l c h o l a n t h r e n e (Kouri et al., 1978). High Risk Subpopulations. The data from human studies are insuffi cient to establish the e x ist e nce of sensitive subpopulations, though there is s u gg e s t i v e e v id e n ce that c h il d r en may be more s e ns i t iv e than are adults (see the High Risk S u b p o p u l a t i o n s Sect i o n in Chapter VI). Summary , The r e co m m e n d e d HAs d e ve l o p e d in this d o cu m e nt are s u mmarized in Table VIII-7. The 1-day HA is b ased on a s i n g l e -d o s e LOAEL in the most s ensitive species, the guinea pig (Turner and Collins, 1983). The 10-day HA is c a l c u lated by dividing the 1-day HA by 10. A DWEL for noncarcinogenic effects from lifetime e x po s u r e is deri v e d from the LOAEL in the 3 - g e n e r a t i o n r e p r o ductive study by Murray et al (1979) along the rationale developed by the U.S. EPA; however, the carcinogenicity risk assessment based on the linearized m u lt i s ta g e model and the c a r c i n og e n ic i t y data in the Kociba et al. (1978a,b, 1979) study indicates lower HAs for lifetime exposure. 01360 V I11 -28 04/14/88 TABLE VI 11-7 Summary oF Calculated Health Advisories For 2.3,7,8-TCDD Health Advisory 1-day HA 10-day HA Longer-term HA LIFetlme DUEL NL00EELL//NL00AAEELL 0.1 pg/kg/day No adequate data3 0.001 pg/kg/dayb SRp oeuctiee s / gp ul gi n/ oe ar a l EFFect reproductive eFFect reproductive eFFect 10'* excess cancer risk 10'* excess cancer risk 10'* excess cancer risk C a l Fc ourl a tSeadFe LEexvpelo s u( pr eg / l Child Adult 1.0x10 l.OxlO" 1.0x10"* 3.5x10* 3.5x10"' 2.2x10* 2.2x10* 2.2xl0"7 dlhe 10-day HA Is derived by dividing the 1-day HA by 10. busing 0.001 pg/kg/day as the LOAEL, the RED Is determined as Follows: RFD = 1000 = 1x10* pg/kg/day where 1000 Is an uncertainty Factor appropriate For use with a LOAEL From an animal study. ReFerence TCuorlnleinr . an1d903 Heturaraly. . 1979 Heturaraly. . 1979 Ke1t9o7c0aIbala.,b. Ke1t9o7ca8lbala,.b, Ke1t9o7ca0lbla.a.b. IX. REFERENCES Abernathy, D.3., W.F. 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